♠♣♦rt❛♥t ❢♦r ❝❡•• ♣♦s✐t✐♦♥✐♥❣ ❬▼❛❝❡ ❛♥❞ ❚✉❣♦r❡s✱ ✷✵✵✹(✱ ❡②❡ ❞❡✈❡•♦♣♠❡♥t ❬❉♦r♦q✉❡③ ❡t ❛•✳✱ ✷✵✵✼❀ ◗✉❡r❡♥❡t ❡t ❛•✳✱ ✷✵✶✺(✱ ❣•✐❛• ❝❡•• ❞❡✈❡•♦♣♠❡♥t ❬❈❤❡♥ ❛♥❞ |❡❜❛②✱ ✷✵✵✵(✱ •✐♣✐❞ ♠❡t❛❜♦•✐s♠ ❬❍❛③❡❣❤ ❡t ❛•✳✱ ✷✵✶✼( ❛♥❞ ✐♥t❡st✐♥❛• st❡♠ ❝❡•• r❡♥❡✇❛• ❬❆♥❞r✐❛ts✐•❛✈♦ ❡t ❛•✳✱ ✷✵✶✽(✳ ❍♦✇ ❙P❊◆ ♠❡❝❤❛♥✐st✐❝❛••② ✐♥t❡r❛❝ts ✇✐t❤ s✉❝❤ s✐❣♥❛•✐♥❣ ♣❛t❤✇❛②s✱ ❛♥❞ ❤♦✇ ✐t r❡❣✉•❛t❡s t❤❡s❡ ❝❡••✉•❛r ♣r♦❝❡ss❡s r❡♠❛✐♥s ✉♥❝•❡❛r ❢r♦♠ t❤❡s❡ st✉❞✐❡s✳ ✷✾ ■♥tr♦❞✉❝t✐♦♥ ❙P❊◆✿ ❉✐s❝♦✈❡r② ❛♥❞ ❢✉♥❝t✐♦♥ ✷✳✷ ❙P❊◆ ✐s ❝♦♥s❡r✈❡❞ ✐♥ ❛♥✐♠❛•s ❈♦♥s❡r✈❛t✐♦♥ ❛♥❛•②s✐s r❡✈❡❛•s ❙♣❡♥ ♦rt❤♦•♦❣s ♦♥•② ❛❝r♦ss t❤❡ ❛♥✐♠❛• ❦✐♥❣❞♦♠✱ ❛•• ♦❢ ✇❤✐❝❤ ❡♥❝♦❞❡ ✈❡r② •❛r❣❡ ♣r♦t❡✐♥s ❝♦♠♣r✐s✐♥❣ t✇♦ t♦ ❢♦✉r ❤✐❣❤•② ❝♦♥s❡r✈❡❞ ◆✲t❡r♠✐♥❛• ||▼s✱ ❛s ✇❡•• ❛s ❛ ❤✐❣❤•② ❝♦♥s❡r✈❡❞ ❈✲t❡r♠✐♥❛• ❞♦♠❛✐♥ ✭❋✐❣✉r❡ ✷✳✶✮✳ ❚❤✐s ❈✲t❡r♠✐♥❛• ❞♦♠❛✐♥✱ ❝❛••❡❞ ❙P❖❈✱ ❢♦r ❙♣❡♥ ♣❛r❛•♦❣ ❛♥❞ ♦rt❤♦•♦❣ ❈ ✲t❡r♠✐♥❛• ❞♦♠❛✐♥ ❬❲✐❡••❡tt❡ ❡t ❛•✳✱ ✶✾✾✾(✱ ✐s ❢♦✉♥❞ ♥♦t ♦♥•② ✐♥ ❙P❊◆✱ ❜✉t ❛•s♦ ✐♥ s♠❛••❡r ❙P❊◆✲ r❡•❛t❡❞ ♣r♦t❡✐♥s✱ ♣r❡s❡♥t ❛❝r♦ss ❢✉♥❣✐✱ ♣•❛♥t❛❡ ❛♥❞ ❛♥✐♠❛•s✱ s✉❣❣❡st✐♥❣

SPOC

SPOC

NCoR/ SMRT HDAC3 SPOC KMT2D [Oswald et al., 2016] ac me me ac ac me me ac facilitates gene activation facilitates gene repression [Shi et al., 2001; Ariyoshi et al., 2003; Mikami et al., 2014] RRM2-RRM3-RRM4 RID [Shi et al., 2001] unliganded NRs

RID RID NR NR

OFF NR target genes RBPID [Oswald et al., 2002; Kuroda et al., 2003; Tsuji et al., 2007; Vanderwielen et al., 201; Yuan et al., 2019] RBPJ/CSL in the absence of Notch In female mouse embryos, one of the two X chromosomes is randomly chosen for inactivation around the time of implantation (Lyon, 1962). This phenomenon is dependent on the coating of the future inactive X chromosome (Xi) by the long non-coding RNA Xist (Penny et al., 1996). The conserved A-repeat region of Xist mediates transcriptional silencing (Wutz et al., 2002), whereas other parts of Xist ensure chromosome coating (Almeida et al., 2017; Wutz et al., 2002). Assays coupling immunofluorescence with RNA fluorescence in situ hybridization (IF/RNA FISH) have revealed that, upon Xist RNA coating, a program of striking chromatin rearrangements ensues. These include rapid loss of histone modifications associated with active promoters (H3K4me3, H4ac, H3K9ac, and H3K27ac) and enhancers (H3K27ac and H3K4me1) (Chaumeil et al., 2002; Jeppesen and Turner, 1993). Furthermore, there is accumulation of H2AK119Ub and H3K27me3, two repressive histone marks dependent on the activity of Polycomb repressive complex (PRC) 1 and 2, respectively (for a review, see Brockdorff, 2017). These progressive alterations of chromatin states are associated with stable repression of the majority of genes on the Xi. Not all X-linked loci are affected in the same way, however, with some genes being silenced much faster than others or even entirely resisting repression (''escapees '') (Disteche and Berletch, 2015). The reason for this striking diversity in gene inactivation dynamics remains unclear. It is likely that the susceptibility of loci to Xist spreading plays a role in this process (Borensztein et al., 2017; Engreitz et al., 2013); however, differences in their chromatin status could also underpin transcriptional silencing dynamics.

✭)②•✐❝③ ❡t ❛•✳✱ ❈❡••✳ ✷✵✶✾✮ ❚❤❡ ♦✈❡r❛•• ❣♦❛• ♦❢ ♠② P❤❉ ✇❛s t♦ ❣❛✐♥ ❛ ❜❡tt❡r ✉♥❞❡rst❛♥❞✐♥❣ ♦❢ t❤❡ ♠❡❝❤❛♥✐s♠s ❛t ♣•❛② ❞✉r✐♥❣ ✐♥✐t✐❛t✐♦♥ ♦❢ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❞✉r✐♥❣ ❳ ❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥✳ ❲❤❡♥ ■ •♦✐♥❡❞ t❤❡ •❛❜ ✐♥ ✷✵✶✼✱ ❛ ♣r♦•❡❝t •❡❞ ❜② t✇♦ ♣♦st❞♦❝s ✭❏❛♥ )②•✐❝③ ❛♥❞ ❆✉ré•✐❡ ❇♦✉s❛r❞✮ ✉♥❞❡rt♦♦❦ t♦ ❣❛✐♥ s✉❝❤ ✐♥s✐❣❤ts ❜② ♣r♦✜•✐♥❣ t❤❡ ❞②♥❛♠✐❝s ♦❢ ❤✐st♦♥❡ ♠♦❞✐✜✲ ❝❛t✐♦♥

HDAC3-FLAG WT

Although some chromatin marks have previously been mapped on the X chromosome in female embryonic stem cells (ESCs) and differentiated cells, nothing is known about the dynamics of the XCI process, especially during early stages when gene silencing actually takes place (Marks et al., 2009; Pinter et al., 2012). Specifically, the order of chromatin changes and their possible role(s) in mediating transcriptional silencing remain largely unknown. Previous studies have relied on ESC differentiation for XCI initiation, which results in highly asynchronous Xist induction and elevated levels of heterogeneity in the population. These complications entirely mask primary chromatin events occurring in a small subset of cells embarking on the process of XCI. Recent advances in the identification of Xist binding proteins revealed that most histone-modifying activities are not directly recruited by Xist RNA but, rather, by its binding partners, like SPEN and HNRNPK (Chu et al., 2015; McHugh et al., 2015; Monfort et al., 2015; Pintacuda et al., 2017). A major question is whether the chromatin changes that occur early on in XCI are actually involved in gene silencing during XCI and, if so, how.

Here we investigate the earliest events accompanying transcriptional silencing of the X chromosome. Using a female ESC line in which one X chromosome can be specifically inactivated via an inducible Xist gene, we performed allele-specific native chromatin immunoprecipitation sequencing (ChIP-seq) as well as nascent transcript profiling at the initiation stages of XCI. Using 4-hr time resolution, we were able to reveal the precise order of chromosome-wide epigenetic events that are intricately coupled to gene repression during XCI. We find that loss of histone acetylation and, in particular H3K27ac, is one of the first events following Xist RNA accumulation during initiation of XCI. We also show that histone deacetylation, via HDAC3, is vital for efficient silencing of most genes on the Xist-coated X chromosome. Contrary to the prevailing hypothesis, HDAC3 is not acutely recruited to the X chromosome by Xist; rather, it is pre-loaded, mainly at putative enhancers. Upon Xist coating, pre-loaded HDAC3 likely mediates histone deacetylation and facilitates transcriptional silencing. We also uncover a surprisingly rapid accumulation of the PRC1-dependent H2AK119Ub mark on the X chromosome, particularly at intergenic regions lying in proximity to Xist RNA entry sites, which are pre-marked by Polycomb (PcG) marks. Accumulation of H3K27me3 appears slightly later and is delayed compared with gene silencing. Using Hdac3 and Xist mutant cells, we also show that spread of Polycomb marks into gene bodies can only occur when transcriptional repression begins. Our study reveals the chromatin choreography across the X chromosome during the early steps of XCI initiation and uncovers a role for histone deacetylation in the establishment of gene silencing.

RESULTS

Allele-Specific Native ChIP-Seq Monitors Chromatin Changes during XCI

A molecular roadmap of the earliest chromatin changes during the initiation of XCI has not yet been established. To reduce heterogeneity and obtain a highly resolved time series of chromatin events upon Xist RNA coating, we used the TX1072 ESC line (Figure 1A; Schulz et al., 2014). This hybrid (Mus musculus castaneus x C57BL/6) line harbors a doxycycline (DOX) inducible promoter upstream of the endogenous Xist at the C57BL/6 (B6) allele (Schulz et al., 2014; Wutz et al., 2002). By adding DOX, we synchronously induce Xist expression and can thus decouple the onset of XCI from differentiation-dependent chro-matin changes (Figure S1C). We performed allele-specific native ChIP-seq (nChIP-seq) for seven histone modifications across five time points at up to 4-hr resolution on biological duplicates (Figure 1A; Figures S1A andS1B). We focused on histone modifications associated with active promoters (H3K4me3, H3K9ac, H4ac, and H3K27ac) and active enhancers (H3K4me1 and H3K27ac) as well as repressive Polycomb-dependent marks (H3K27me3 and H2AK119Ub). All nChIP-seq datasets were sequenced to a depth of at least 40 million reads and showed a high signal-to-noise ratio (Figures S1B andS1E). The reads were split according to content of allele-specific SNPs (B6, mapping to Xi; Cast, mapping to active X); this allelic information was then analyzed in detail to define XCI-specific changes.

Histone Deacetylation Is One of the Earliest Events during Initiation of XCI Upon Xist coating, the Xi becomes rapidly depleted of active histone modifications. Although this global loss of euchromatin occurs in a similar time window as the changes in X-linked transcriptional activity (Chaumeil et al., 2002), the precise chronology and possible mechanistic relationship of these two events have remained unclear. To address this, we first analyzed nChIP-seq datasets for a panel of active histone modifications (H3K27ac, H3K4me1, H3K4me3, H3K9ac, and H4ac; Figure S1E). This revealed progressive depletion of B6-specific reads (originating from Xi) upon Xist induction, with different marks showing different kinetics of loss (Figure S1D). To analyze this in detail, we performed peak calling on all the reads (STAR Methods). For each peak with at least 50 allele-specific reads, we calculated its d-score (reads B6 /[reads Cast + reads B6 ]). This parameter of allelic skewing uses the active X chromosome as a powerful internal control, rendering the analysis less sensitive to variability in nChIP-seq quality and, thus, more stable between replicates.

We first set out to track the relative dynamics of active chromatin marks by comparing d-score dynamics of all peaks. We found that loss of H3K27ac and H4ac is among the earliest and most robust chromatin events on the X chromosome following Xist induction (Figure 1B). Furthermore, we found that not only did different histone modifications show distinct behaviors but, also, that specific regions of the X chromosome followed rather different dynamics of inactivation (Figure S1G). Thus, there is a temporal hierarchy of chromatin events both at the level of different histone marks and genomic loci.

To better understand in which context chromatin changes might affect gene silencing, we separately focused on promoters and putative enhancers. For each window, we plotted a d-score (normalized to t = 0 hr) as a function of time and fitted a sigmoidal curve (STAR Methods). We found that the maximal dynamic range of inactivation was from 0.5 to $0.2 and, thus, set a threshold for when the curve reached 50% of this dynamic range; i.e., d = 0.35 (STAR Methods). This IC35 (inactivation criterium 0.35) parameter indicates how quickly a given peak becomes significantly decreased or lost (Figure S1F). Pairwise comparisons of the IC35 at X-linked gene promoters revealed that H3K27ac becomes efficiently deacetylated prior to demethylation of H3K4me3 (Figure 1C). This trend holds true for $88% of measured promoters (Figure S1H), including a representative example of the Pdk3 promoter (Figure 1D). Similar analysis of the enhancer inactivation dynamics revealed that H3K27ac is efficiently depleted prior to loss of H3K4me1 (Figure 1C). This robust difference is observed in $95% of the cases; e.g., at a putative Pdk3 enhancer element (Figure 1E; Figure S1I). Thus, histone deacetylation is an early and efficient event during XCI.

Next, to extract information about relative timing rather than the efficiency threshold, we obtained the time when the curve reaches its maximum slope (effective dose 50%, ED 50 ; Figure S1F). ED 50 analysis revealed that both H3K27ac and H3K4me3 become depleted concomitantly (Figure S1J). Together with the IC35 data, this indicates that, even though de-methylation of promoters and their deacetylation are concurrent, the latter process is significantly more efficient. Furthermore, unlike promoters, enhancers showed significant differences in the ED 50 parameter between H3K27ac and H3K4me1, indicating that enhancer demethylation is not only less efficient but also significantly delayed (Figure S1J).

To assess the relative deacetylation dynamics of enhancers and promoters, we compared the IC35 parameter for H3K27ac at both types of genomic regulatory elements. Intriguingly, enhancer deacetylation was slightly more efficient than that of promoters (Figure S1K). After pairing active promoters to their putative enhancers (STAR Methods), $76% (37 of 49) of them showed more efficient deacetylation at enhancers than at

(legend continued on next page)

Cell 176, 182-197, January 10, 2019 185 promoters (Figure S1M). On the other hand, loss of H3K4me1 on the Xi, which is indicative of enhancer decommissioning, was found to be significantly less efficient compared with the loss of H3K4me3 at promoters (Figure S1L). Thus, inactivation (loss of H3K27ac) of enhancers proceeds at a slightly higher efficiency than of promoters, although stable decommissioning (loss of H3K4me) of promoters precedes that of enhancers. Intriguingly, upon H3K4me3 loss following Xist induction, we observe a slight increase in H3K4me1 levels around transcriptional start sites (TSSs), which probably reflects an intermediate of stepwise enzymatic H3K4me3 demethylation (Figures S1N andS1O).

In summary, our data show that efficient histone deacetylation at promoters and enhancers is one of the first events during XCI, indicating that it might contribute to initiation of transcriptional silencing. In comparison, demethylation of H3K4me3 and H3K4me1 is less efficient and delayed, respectively. Finally, deacetylation of promoters and enhancers seems to follow broadly comparable dynamics, whereas their decommissioning differs significantly.

Histone Deacetylation Is Tightly Linked to Transcriptional Silencing

To gain insight into the relationship between loss of active chromatin marks and transcriptional silencing of X-linked genes, we compared our nChIP-seq dataset with transient transcriptome sequencing (TT-seq; Schwalb et al., 2016). This allows a direct measure of newly synthesized RNA and, thus, enabled us to measure transcriptional activity as the X chromosome becomes inactivated. Comparison of the IC35 for TT-seq and individual histone modifications at associated promoters revealed that transcriptional silencing dynamics are most tightly linked to promoter deacetylation at H3K27 and, to a lesser extent, to loss of H4ac, H3K9ac, and H3K4me3 (Figures 2A and2B; Figure S2A). A similar pattern of gene silencing and promoter deacetylation dynamics further exemplifies this correlation (Figure 2C).

The striking variability in dynamics of promoter chromatin changes begged the question of whether these differences could be due to transcriptional status or genomic context; for example, proximity to Xist (the locus and/or Xist RNAs entry sites; Engreitz et al., 2013), gene density, or LINE (long interspersed nuclear elements) density. Indeed, the latter feature has been proposed previously to influence the efficiency of XCI (Chow et al., 2010; Loda et al., 2017; Figure S2B andS2C). We found that gene promoters that are rapidly H3K27-deacetylated preferentially reside in gene poor, LINE-dense regions in proximity to lamina-associated domains (LADs) and the Xist locus (Figure 2D).

Given that the histone deacetylation is the primary chromatin change observed upon Xist induction and that its kinetics are tightly linked to transcriptional silencing, we decided to investigate whether the loss of histone acetylation might play a functional role in mediating the initiation of gene silencing during XCI.

HDAC3 Mediates Efficient Transcriptional Silencing during XCI

To investigate whether this rapid histone deacetylation is mediated by histone deacetylases (HDACs), we first tested selective pharmacological inhibitors against all HDACs expressed in ESCs (Figure S3A). Importantly, after 24 hr of DOX induction, only HDAC3 catalytic inhibition resulted in a significant silencing defect compared with the control (DMSO) (Figure 3A). We further validated these findings using nascent RNA FISH and, unexpectedly, found that HDAC1 and 2 inhibition promoted more efficient Xist upregulation, possibly because of rtTA upregulation (Figure 3B). The finding that HDAC3 is involved in XCI is in line with previous reports that it might interact with SPEN, which is one of the key Xist-silencing factors (Chu et al., 2015; McHugh et al., 2015; Monfort et al., 2015). Also, Hdac3 knockdown was recently shown to result in defective silencing of another X-linked gene: Gpc4 (McHugh et al., 2015).

To better interrogate the specific functions of HDAC3 in XCI, we generated two independent Hdac3 À/À TX1072 deletion clones using CRISPR/Cas9 technology (STAR Methods; Figure 3C). We performed RNA sequencing (RNA-seq) on these, both prior to and 24 hr following DOX addition. Differential gene expression analysis revealed relatively mild changes apart from some meiosis-associated genes (Figures S3B-S3D). Consistent with our inhibitor treatment results, allelespecific analysis revealed that both Hdac3 À/À clones are unable to efficiently silence X-linked genes (Figure S3E). Although the majority of genes across the X chromosome were affected, $24% of genes (43 of 182) were not, showing, instead, almost normal silencing (Figure 3D; Figures S3Fa n d S3G). We further validated our findings in the context of EpiLC (epiblast like-cells) differentiation both with and without DOX treatment (Figures 3E-3G; Figure S3H). In our pyrosequencing experiments, we monitored the allelic bias in expression of genes affected (AtrX, G6pdX,a n dPgk1) in their silencing by the HDAC3 loss and three less affected genes (Ints6l, Rnf12, and Elf4)( Figure 3E).

Our results indicate that loss of HDAC3 leads to a delay in transcriptional silencing of most X-linked genes. We noted that, over time, XCI is not fully prevented because gene silencing progressively ensues, although more slowly than in control cells (Figure 3E), indicating that mechanisms parallel to HDAC3 must also contribute to XCI. To address this further, we tested whether other HDACs might compensate for HDAC3 loss. To this end, we treated Hdac3 À/À ESCs with selective inhibitors against all expressed HDACs (1,2,3,4,5,6, and 8). We found that allelic skewing (i.e., XCI) of AtrX, G6pdX, and Rnf12 were not affected in any of the treated cells (Figure 3H), arguing against the involvement of other HDACs in XCI. Thus, our data reveal that HDAC3 is the main histone deacetylase important for the initiation of silencing of most genes on the X chromosome. Because HDAC3 can mediate deacetylation of many substrates, we next investigated whether its effect on gene silencing is mediated by changes in chromatin status.

HDAC3 Promotes Silencing by Rapidly Deacetylating Histones

Although our experiments using inhibitors and knockout ESCs indicated the importance of HDAC3 catalytic activity in mediating efficient XCI, it remained unclear whether histone deacetylation dynamics were also affected by loss of HDAC3. To address this question, we first monitored the global distribution of histone modifications by IF/RNA FISH. We observed that the Xi silent compartment formed much less efficiently in Hdac3 À/À ESCs, with increased levels of H3K27ac and H4ac remaining within the Xist cloud (Figures 4A and4B; Figures S4A-S4D). To further explore the chromatin phenotype of Hdac3 À/À ESCs, we performed nChIP-seq for H3K27ac and H4ac at 0, 8, and 24 hr of DOX treatment. Differential peak analysis has revealed that less than 9% of peaks significantly change in Hdac3 À/À compared with WT cells at t = 0 hr (Figure S4E). Consistent with the IF/RNA FISH results, allele-specific analysis revealed delayed histone deacetylation in the Hdac3 À/À clones compared with the parental line (Figure 4C; Figures S4F andS4G). As with transcriptional silencing, the deacetylation defects affected most of the X chromosome but were not uniform, with some peaks being almost entirely resistant to HDAC3 loss ($29%, 38 of 133). Indeed, protracted loss of H3K27ac at promoters was linked to a severe defect in transcriptional silencing in mutant cells (Figures 4C and4D). Together, our data show that HDAC3 mediates efficient transcriptional silencing of the majority of genes through deacetylating histones. However, it remains unclear how HDAC3 is targeted to the X chromosome to help silencing.

HDAC3 Is Pre-bound on the X Chromosome, Likely to Promote Deacetylation during XCI It has been proposed that HDAC3 might be either de novo recruited to the Xi by Xist through interaction with the SPEN protein (McHugh et al., 2015) or, alternatively, that Xist-coating might lead to the activation of already pre-bound HDAC3. To test these two hypotheses, we measured sub-nuclear localization of HDAC3 after 24 hr of DOX treatment. IF analysis revealed no HDAC3 enrichment on the Xi and even a slight depletion (Fig-ure 5A). This surprising result prompted us to further explore the question of HDAC3 recruitment. To circumvent problems with antibody quality, we knocked in a 3xFLAG tag into the 3 0 region of the endogenous Hdac3 gene (Figure 5B). This novel ESC line (Hdac3 FLAG/FLAG ) expressed nearly normal levels of HDAC3 (Figure S5A) and robustly silenced the X chromosome, indicating that HDAC3 remained functional (Figure S5B). We then set out to identify potential common interactors of Xist and HDAC3 in DOX-treated cells. To this end, we performed anti-FLAG immunoprecipitation followed by quantitative label-free mass spectrometry analysis (Hdac3 FLAG/FLAG versus Hdac3 +/+ in five replicates) (Figure 5C). Among significant interactors, we identified nearly all components of the NCOR and SMRT complexes as well as their chaperone complex CCT (Figures 5D and5E; Guenther et al., 2002). Intriguingly, of all Xist binding partners (Chu et al., 2015), only RYBP (a PRC1 factor) showed a weak binding to HDAC3 (Figure 5C). Our results confirm the previously reported interactions between HDAC3 and NCOR and SMRT but argue against strong HDAC3 recruitment by Xist RNA binding partners like SPEN.

To directly measure HDAC3 recruitment to the Xi, we performed anti-FLAG ChIP-seq prior to and during XCI. We found robust binding of HDAC3 to multiple H3K27ac and H4ac peaks throughout the genome ($65% of HDAC3 peaks are enriched for H3K27ac [$65% on the X chromosome] and $61% for H4ac [$58% on the X chromosome]) (Figure 5F). The vast majority of HDAC3 peaks were intriguingly found to reside within putative enhancers rather than at gene promoters (Figure 5F; Figure S5C). Allele-specific analysis revealed a transient and modest enrichment after 4 hr of induction on the Xi (B6 reads) at the Xist locus and at some HDAC3 peaks (Figure 5G; Figures S5D andS5E). Together, this indicates that HDAC3 indirectly and unstably interacts with some of the Xist binding partners to allow for low-level and transient recruitment to the X chromosome. However, no significant correlation between HDAC3 de novo recruitment and timing of deacetylation was found (Figure S5F). On the other hand, the histone acetylation peaks that are lost early during XCI did show higher levels of HDAC3 prebinding (Figure S5G). In fact, HDAC3-bound regions show more efficient (low IC35) deacetylation of H4ac and a similar but statistically not significant trend for H3K27ac (Figure 5H; Figure S5H). Finally, we observed a mild but statistically insignificant (C) Immunoblot analysis of whole-cell lysates from TX1072 (Hdac3 +/+ ) and two Hdac3 À/À clones probed with antibodies specific for the N terminus of HDAC3 and Lamin B1. (D) Heatmap showing gene silencing after 0 and 24 hr of DOX treatment in TX1072 (Hdac3 +/+ ) and two Hdac3 À/À clones. Rows were ordered by their genomic position from centromere to telomere (the green triangle indicates the Xist location). (E) RNA-seq validation using pyrosequencing on ESCs (TX1072: Hdac3 +/+ and Hdac3 À/À ) differentiated to EpiLCs with DOX. Shown are averages (± SEM) from trend for HDAC3 pre-bound enhancers to surround rapidly silenced genes (Figure S5I). In summary, we show that HDAC3 does not appear to be robustly recruited de novo to the X chromosome via Xist RNA coating and does not strongly bind known partners of Xist. Rather, the majority of HDAC3 is pre-bound at putative enhancers, and, upon Xist induction, this results in efficient histone deacetylation, promoting transcriptional silencing.

PRC1-Dependent H2AK119Ub Accumulation Precedes H3K27me3 Deposition

Repressive chromatin modifications are also known to appear shortly after Xist RNA coating, in particular H2AK119Ub (PRC1-dependent) and H3K27me3 (PRC2-dependent). However, the early dynamics of this process, as well as the relative timing of H3K27me3 and H2AK119Ub deposition, have remained largely unexplored. To address this question, we have performed nChIP-seq for both PcG-dependent marks. These datasets showed a reproducible and stable pattern of enrichment on autosomes, both between time points and biological replicates (Figure S6A). In contrast, the X chromosome showed a bias in H2AK119Ub enrichment toward the B6 allele (Xi) after 4 hr of DOX induction and after 8 hr in the case of H3K27me3 (Figure S6B). First, we analyzed relative B6-read enrichment within 10-kb windows across the whole X chromosome normalized to t = 0 hr and observed rapid deposition of H2AK119Ub /+ ) and two Hdac3 À/À ESC clones induced with DOX for 24 hr. Cells were probed using anti-H3K27ac (red) antibodies and a Xist intronic probe (green). The white dotted line encircles the Xist domain. Because of technical variability, contrast settings were individually adjusted to reflect relative enrichment. Scale bar, 10 mm. (B) Quantification of (A) over at least 100 Xist clouds. Shown is the average signal for Xist and H3K27ac when profiles were aligned to the Xist cloud boundary (for more details, refer to Figure S4A andD). (C) Relationship between gene repression (RNA-seq) and promoter deacetylation (H3K27ac) at 24 hr in TX1072 (Hdac3 +/+ , blue) and Hdac3 À/À (red). On the scatterplot, the black dotted line represents fitted linear regression. r = 0.860. Density plots show the distribution of the normalized d-score for H3K27ac (horizontal) and RNA-seq (vertical); note that d-scores in mutant cells are shifted toward biallelic levels (0.5, dotted lines on density plots). Each dot represents the average normalized d-score from biological duplicates. (D) Comparison of promoter deacetylation dynamics and transcriptional silencing after 24 hr of DOX treatment in TX1072 (Hdac3 +/+ ) and Hdac3 À/À ESCs. Examples of genes sensitive (top) and more resistant (bottom) to HDAC3 loss are shown. For RNA-seq, shown is the average from biological duplicates (± SD). See also Figure S4.

Cell 176, 182-197, January 10, 2019 189 (legend continued on next page)

preceding that of H3K27me3 (Figure 6A). To quantify the dynamics at individual windows, we extracted the ED 50 parameter, as was done for active marks, but using B6-read accumulation (STAR Methods; Figure S1F). Pairwise comparison of ED 50 at all 10-kb windows across the X chromosome showed that H2AK119Ub accumulates most efficiently prior to H3K27me3 (Figure 6B), with very few exceptions ($1%) (Figure S6C).We also validated this finding using IF/RNA FISH during ESC differentiation toward EpiLCs (Figure S6D). Evidently, the Xi becomes enriched for H2AK119Ub prior to the PRC2-dependent H3K27me3 across the whole chromosome, although there may be local differences in the dynamics of this process. Indeed, certain regions preferentially accumulate Polycomb marks already at early time points (i.e., by 4 hr for H2AK119Ub or 8 hr for H3K27me3; Figure 6D). This early accumulation was found to be prominent around the Xist locus (green line) as well as the first regions with which it associates (''entry sites,'' blue lines) (Engreitz et al., 2013). There is also significant overlap between H2AK119Ub and H3K27me3 patterns of accumulation, and, interestingly, these primary deposition sites are typically marked by PcG prior to Xist upregulation (Figure 6C, Figure S6E). Thus, most H2AK119Ub accumulation occurs at sites pre-marked by PcG and rapidly coated by Xist. Subsequent H3K27me3 deposition closely follows this early deposition pattern. We next investigated the accumulation dynamics of both marks at specific types of genomic windows. ED 50 analysis revealed that H2AK119Ub accumulates first intergenically and is deposited only later at active gene bodies, promoters, and putative enhancers (Figure S6F). This pattern of accumulation is tightly followed by H3K27me3 deposition and spread. What is more, PcG-dependent mark accumulation seems to be closely linked to gene silencing kinetics (Figure 6E). This is exemplified by tracks for genes that are either silenced rapidly (Rnf12)o r slowly (Huwe1 and Alg13) or escape the process altogether (Kdm6a)( Figures 6F and6G). Direct comparison of gene silencing and promoter deacetylation dynamics with PcG mark accumulation at gene bodies revealed a significantly lower ED 50 parameter for H2AK119Ub (Figure S6G). This indicates that the H2AK119Ub deposition reaches its maximum rate earlier than gene silencing and promoter deacetylation. Next, we measured the time when the sigmoid's second derivative reaches its minimum or maximum. We use this parameter as a proxy for when the curve starts to increase or decrease (Figure S6I). This revealed that gene silencing, promoter deacetylation (H3K27ac loss), and H2AK119Ub accumulation at gene bodies are all initiated at the same time, whereas H3K27me3 is significantly delayed (Figure S6H). Thus, we see dynamic spreading of H2AK119Ub from intergenic regions to gene bodies coinciding with transcriptional silencing.

We also investigated genes that normally escape from XCI (constitutive) or escape more variably and in specific tissues (facultative escapees) (Disteche and Berletch, 2015). We examined a highly expressed constitutive escapee (Kdm6a; Figures 6F and6G) and found no PcG-dependent mark accumulation at its promoter or gene body in our dataset. In line with this, both constitutive and facultative escapees show no or lower accumulation of H2AK119Ub and H3K27me3 at their gene bodies (Figures S6J andS6K). This is consistent with significantly delayed H3K27ac loss at their promoters (Figure S6L). Thus, genes escaping transcriptional silencing do not undergo promoter deacetylation and do not accumulate PcG-associated marks.

All in all, our analysis reveals the hierarchy of PcG marks during XCI, with H2AK119Ub accumulating significantly prior to H3K27me3. This process is first initiated at large chromatin domains that are pre-marked by PcG (megabase scale) and that correspond to some of the first regions with which Xist RNA interacts (Engreitz et al., 2013). We demonstrate that PcG marks accumulate first intergenically and subsequently spread into regulatory elements and gene bodies. This spread into genes coincides with transcriptional silencing, raising the question of whether the two processes are functionally linked.

Transcriptional Silencing Is Necessary for PcG Spreading

To address whether spreading of these repressive histone modifications from intergenic regions into genes is a cause or consequence of transcriptional silencing, we decided to investigate PcG mark distribution in a Xist mutant lacking the A-repeat element (Xist:DA). Xist:DA RNA is able to coat the X chromosome and lead to PcG mark accumulation (based on IF) but cannot initiate gene silencing (Kohlmaier et al., 2004; Sakata et al., 2017; Schoeftner et al., 2006; Wutz et al., 2002). Xist:DA thus allows the relationship between repressive chromatin changes and transcriptional repression to be addressed. For this, we used previously published male ESCs harboring wild-type (TXY:Xist) or mutant Xist (TXY:XistDA) under a DOX-inducible promoter at the endogenous locus (Wutz et al., 2002; Figure 7A). These ESCs do not require allele-specific analysis, as all X-mapping reads will originate from the single X chromosome. After collecting DOX-treated and untreated differentiated cells (48 hr), we performed nChIP-seq for three histone modifications (H3K27ac, H3K27me3, and H2AK119Ub) in biological duplicates. Samples were normalized for ChIP quality prior to further analysis (STAR Methods).

First, we investigated the relationship between PcG-associated mark spread and transcriptional silencing in WT and Xist:DA cells. We observed that most regions of the X chromosome recruit nearly normal levels of H3K27me3 in the Xist:DA cells. However, clusters of active genes are a notable exception to this (Figure 7B; Figure S7A). The same regions are also found not to accumulate H2AK119Ub. Intriguingly, reduced recruitment of H2AK119Ub was observed throughout the chromosome in Xist:DA cells compared with WT Xist (Figure 7B). We next quantitated the deposition of both Polycomb marks in relation to regions with actively transcribed genes (Figure 7C). Indeed, (G) HDAC3 accumulation dynamics (shading is the interquartile range) on peaks at the X chromosome (left) and autosomes (right). (H) Comparison of peak deacetylation (H4ac) dynamics (IC35) and HDAC3 pre-binding. The p value is from a Wilcoxon rank-sum test. See also Figure S5 and Table S2. 7G). Thus, Xist:DA leads to PRC2 and PRC1 recruitment to regions without active transcription; however, both complexes seem impeded from spreading into domains of active genes. In line with this, we observed no H3K27me3 or H2AK119Ub enrichment within promoters and bodies of active genes (Figure 7D; Figures S7B-S7E).

Our finding that H3K27 deacetylation promotes gene silencing during XCI prompted us to investigate H3K27ac loss at gene regulatory elements upon coating by Xist:DA RNA (Figure 7E; Figures S7C and S7E). Importantly, we did not observe significant deacetylation of either promoters or enhancers in the Xist:DA mutant. This was in stark contrast to robust inactivation of such regulatory elements when using full-length Xist. Thus, promoter and enhancer deacetylation are tightly linked to transcriptional silencing and dependent on the Xist A-repeat. This is in line with our HDAC3 experiments indicating a functional role for histone deacetylation in initiating gene repression.

To further assess how PcG accumulation relates to histone deacetylation and gene silencing, we performed nChIP-seq in Hdac3 À/À cells. We mapped H2AK119Ub and H3K27me3 in two independent Hdac3 À/À clones at 0 and 24 hr of DOX treatment. Although HDAC3 loss results in inefficient transcriptional silencing, it should not have major direct effects on PcG marks. Indeed, we found that the pattern of both PcG marks at t = 0 hr was very similar along the X chromosome (Figure 7H). After 24 hr of DOX treatment, both marks accumulate globally along the X chromosome in a similar pattern to that observed in Hdac3 +/+ cells (Figure 7H), albeit with slightly lower efficiency. This result differs from the patterns observed in Xist A-repeat mutant cells because, in Hdac3 À/À cells, even gene-rich regions accumulate both PcG-associated marks. We then focused on what happens at genic regions. This revealed that genes that were not silenced in Hdac3 À/À cells (i.e., sensitive) accumulate neither H2AK119Ub nor H3K27me3 (Figure 7I; Figure S7F). On the other hand, genes efficiently repressed in Hdac3 À/À (resistant) robustly accumulated both marks. These relationships are exemplified by a AtrX (resistant gene) and Rnf12 (sensitive gene) (Figure S7G). Thus, Xist coating and intergenic PcG mark deposition are not sufficient for H2AK119Ub spread into active genes. Only the induction of transcriptional silencing enables H2AK119Ub spread to occur.

In conclusion, we uncoupled the general deposition of the Polycomb marks over the X chromosome from transcriptional silencing. By doing so, we demonstrate that neither PRC1 nor PRC2 dependent marks are able to spread into actively transcribed regions. In the A-repeat mutant, this may be a result of defective Xist spreading or the presence of transcription (Engreitz et al., 2013). On the other hand, our experiments in Hdac3 À/À cells revealed that reduced transcription and histone deacetylation are required to enable PcG spreading into actively transcribed regions.

DISCUSSION

X chromosome inactivation is a powerful model for the study of transcriptional repression and the formation of facultative heterochromatin. The link between chromatin changes and transcriptional silencing during XCI has remained rather elusive. In this study, we provide the detailed choreography of early transcriptional and chromatin changes during the initiation of XCI at high molecular and temporal resolution (Figure S7H). We also explore the chromatin regulatory pathways that help to achieve efficient gene repression. All in all, our findings point to multiple parallel epigenetic mechanisms being at play during the initiation of XCI to ensure rapid and robust transcriptional silencing.

Xist RNA as the Architect for Early Chromatin Changes along the X Chromosome Our results indicate that the dynamics and efficiency of chromatin alterations along the X chromosome are, in part, shaped by the spreading of Xist RNA itself. Indeed, rapid histone deacetylation is most efficient in regions proximal to the Xist locus, in line with previous allele-specific RNA-seq experiments (Borensztein et al., 2017; Marks et al., 2015).The PcG-dependent marks accumulate first at regions pre-marked by H3K27me3 and H2AK119Ub, which were previously identified as Xist entry sites (XESs), lying in 3D proximity to the Xist locus (Engreitz et al., 2013). This striking PcG mark enrichment and 3D proximity are reminiscent of the Polycomb-enriched regions specifically interacting together in Drosophila melanogaster ( Cheutin and Cavalli, 2014). Thus, it is an exciting possibility that the chromatin landscape of the X chromosome prior to its inactivation instructs the folding of the chromosome, resulting in a specific pattern of Xist spreading and subsequent dynamics in gene silencing.

Spreading of PcG Marks, but Not Their Initial

Recruitment, Requires Gene Silencing Our high-resolution XCI analysis also sheds light on the kinetics of PcG mark deposition and spreading along the X chromosome. The very rapid accumulation of PRC1-dependent H2AK119Ub (D and E) Average plots showing accumulation or depletion of H3K27me3 (D, left), H2AK119Ub (D, right), and H3K27ac (E) over all X-linked active genes (D), promoters (E), or enhancers (E) in Xist:DA-and Xist:WT-expressing cells. (F and G) Genome browser plots showing H3K27me3 and H2AK119Ub enrichments in regions with a high (F) or low (G) density of active genes. Signals from DOXtreated (red) and untreated (blue) samples were overlaid. Plotted are only expressed genes. (H) H3K27me3 (left) and H2AK119Ub (right) enrichment across the Xi at 0 hr (top) and their accumulation at 24 hr (bottom) in Hdac3 +/+ (TX1072, gray) and Hdac3 À/À cells. Lines indicate LOESS regression of 10-kb windows spanning the whole X chromosome. (I) Quantification of H3K27me3 (red) and H2AK119Ub (yellow) accumulation on gene bodies at 24 hr compared to 0 hr in Hdac3 À/À . Genes were separated based on their sensitivity to HDAC3 loss. The p values are from a Wilcoxon rank-sum test. (J) Correlation between the transcriptional silencing defect and H3K27me3 (red) and H2AK119Ub (yellow) accumulation defect in Hdac3 À/À compared with Hdac3 +/+ (TX1072). The black line represents linear regression fitting. r and p values from Pearson's correlation are shown. See also Figure S7.

that occurs prior to the deposition of H3K27me3 is in contrast with some previous reports suggesting direct PRC2 recruitment by the Xist RNA (Zhao et al., 2008) but is in line with recent reports where a non-canonical PRC1 complex (including PCGF3 and PCGF5) is first recruited, and this then recruits PRC2 (Almeida et al., 2017). Using Xist transgenes on autosomes, this was proposed to occur through HNRNPK bridging the B-repeat region of Xist with PRC1 (Pintacuda et al., 2017). This finding appears to be contradicted by the defect in H2AK119Ub deposition in the Xist A-repeat mutant. However, we propose that transcriptional silencing may be required to allow for secondary canonical PRC1 recruitment. Indeed, initial H2AK119Ub enrichment is thought to recruit the PRC2 complex via JARID2 or AEBP2, allowing H3K27me3 accumulation, which, in turn, would allow canonical PRC1 binding (da Rocha et al., 2014). Further studies of how transcription affects this feedback and of the timing of PRC1 and PRC2 recruitment are necessary to further validate the revised model.

Here we find that, during the first hours of XCI, PcG marks initially accumulate at intergenic regions and later spread into genes. In cells expressing the Xist A-repeat mutant, neither PRC1 nor PRC2-dependent marks are able to spread into genic regions because they are probably impeded by ongoing transcription (Kaneko et al., 2014). Alternatively, the Xist A-repeat deletion might lead to local defects in Xist spreading, as suggested by Engreitz et al. (2013). Furthermore, our detailed analysis of Hdac3 mutant cells revealed that H2AK119Ub spreading to genes requires their-at least partial-deacetylation and transcriptional inactivation. This indicates that PRC1 mark spreading is not the trigger for initiating gene repression. On the other hand, because both processes seem to occur around the same time, there might be a potential role of PRC1 in facilitating efficient XCI but not initiating it. Indeed, in an autosomal context, a mutant of Xist, which is unable to recruit PRC1, has slightly reduced silencing capacity (Almeida et al., 2017; Pintacuda et al., 2017).

Histone Deacetylation Has an Early Role in Gene

Silencing Events during XCI Here we identify H3K27 deacetylation as one of the earliest chromatin alterations during XCI, tightly linked to transcriptional silencing. This is in line with previous IF/FISH studies in differentiating ESCs and embryos (Chaumeil et al., 2002; Okamoto et al., 2004). Moreover, by using Hdac3 loss-of-function experiments, we demonstrate that histone deacetylation is not simply due to reduced transcription. Rather, it appears to be an active process that promotes gene silencing. We propose a model whereby histone deacetylation by the pre-bound HDAC3 promotes efficient silencing of most X-linked genes (Figure S7I). A previous study has shown that Hdac3 knockdown results in defective silencing of a single locus during XCI (McHugh et al., 2015). However, our study reveals that other Xist A-repeat-dependent but HDAC3-independent mechanisms are also at play during XCI. This is presumably to safeguard and mediate timely gene repression, especially at genes that need to become rapidly silenced; e.g., Rnf12. Although we have excluded the involvement of other histone deacetylases during XCI initiation, future studies will reveal whether these HDAC3-independent mechanisms act via other chromatin-modifying or -remodeling activities or whether they occur through entirely different pathways.

STAR+METHODS

Detailed methods are provided in the online version of this paper and include the following:

d KEY RESOURCES

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Edith Heard (edith.heard@curie.fr)

METHOD DETAILS

Doxycycline and inhibitor treatment TX1072 based ESC lines were plated at a density of 1.8mln/T75 flask. After 24hrs of culture in DMEM/15%FCS+LIF+2i cell the medium was supplemented with doxycycline (1ug/ml). Cells were then collected at regular intervals. For TXY based ESC lines we have plated them at a density of 1mln/T75. After 24hrs of culture in DMEM/15%FCS+LIF cells were washed twice with PBS and medium was changed to DMEM/10%FCS-LIF with or without doxycycline (1.5ug/ml). Cells were collected after 48hrs of differentiation. For HDAC inhibition cells were pre-treated for 1hr with: 10 mM BRD668; 10 mM RGFP966; 10 mM Tasquinimod; 5 mM Tubastatin A; 5 mM PCI-34051; 30nM LMK235.

Native ChIP-seq Cells were collected using Accutase (Thermo Fisher Scientific), washed twice in ice-cold PBS and counted. Typically, 3.5mln cells were used per immunoprecipitation (IP). A fraction of cells was always used for RNA/FISH verification of Xist induction. Cell pellet was resuspended in 90 mL (per 10 mln cells) of Lysis Buffer (50 mM Tris-HCl, pH7.5; 150mM NaCl; 0.1% sodium deoxycholate; 1% Triton X-100; 5mM CaCl 2 ; Protease Inhibitor Cocktail; 5 mM sodium butyrate). After lysing cells on ice for 10 min we added 62 mL (per 10 mln cells) of Lysis Buffer with MNase (500 mL buffer + 0.5 mL MNase). Chromatin was digested for exactly 10 min at 37 C and reaction was stopped by the addition of 20 mM EGTA. To remove undigested debris the lysates were centrifuged at 13000 rpm for 5 min at 4 C. Supernatant was transferred to a fresh tube, an equal volume of STOP Buffer (50 mM Tris-HCl, pH7.5; 150mM NaCl; 0.1% sodium deoxycholate; 1% Triton X-100; 30 mM EGTA; 30 mM EDTA; Protease Inhibitor Cocktail; 5 mM sodium butyrate) was added, samples were stored on ice. 5 mL of lysate was digested in 45 mL of ProtK Digestion Buffer (20 mM HEPES; 1 mM EDTA; 0.5% SDS) for 30 min at 56 C. 50 mLof AMPure XP beads were added to the digested lysate together with 60 mL of 20% PEG8000 1.25M NaCl. After mixing the samples were incubated for 15 min at RT. Beads were separated on a magnet and washed twice with 80% Ethanol for 30sec. DNA was eluted in 12 mL of Low-EDTA TE and DNA concentration was measured using Qubit DNA High-Sensitivity kit. These measurements were used to normalize lysate concentration between samples. DNA isolated in this step was used as the input sample. The volume of each undigested lysate was adjusted for equal concentration and to obtain 1mL per IP using a 1:1 mix of Lysis Buffer and STOP Buffer.

Anti-mouse Dynabeads (50ul/IP) and Protein-A Dynabeads (10ul/IP) were washed twice in Blocking Buffer (0.5% BSA; 0.5% Tween in PBS). Beads were then resuspended in Blocking buffer and coated with antibodies for 4hrs at 4 Following day beads were magnet-separated and washed quickly with ice-cold washing buffers. anti-H3K4me3 IP was washed 8 times with Low Salt Buffer (0.1% SDS; 1% Triton X-100; 2 mM EDTA; 20 mM Tris-HCl, pH 8.1; 150 mM NaCl; 0.1% sodium deoxycholate). All remaining IPs were washed 4-times with Low Salt Buffer, 2-times with High Salt Buffer (0.1% SDS; 1% Triton X-100; 2 mM EDTA; 20 mM Tris-HCl, pH 8.1; 360 mM NaCl; 0.1% sodium deoxycholate) and 2-times with LiCl buffer (0.25 M LiCl; 1% NP40;1.1% sodium deoxycholate; 1 mM EDTA; 10 mM Tris-HCl pH 8.1). Prior to elution all samples were rinsed once in TE. ChIP-DNA was eluted in ProtK-Digestion buffer for 15 min at 56 C. Beads were separated and the supernatant was further digested for another 2 hr at 56 C. DNA was isolated using AMPure XP beads as described for the input sample.

For each nChIP-seq, 0.5 mL of each sample was used for qPCR validation of enrichment at control regions. 0.5 mL of input samples were also used to verify the digestion efficiency using D1000 tapestation. Remaining DNA concentration was adjusted and used for library preparation using Ovation â Ultralow Library System V2 following suppliers protocol. Amplified libraries were size-selected for dinucleotide fraction (350-600bp fragments) using agarose gel-separation and MinElute Gel Extraction Kit (QIAGEN). Sample quality was inspected using D1000 tapestation. Samples were sequenced with HiSeq2500 using PE100 mode for nChIP-seq on TX1072 cell line and SE50 mode for nChIP-seq on TXY cell line.

EpiLC induction 2.5x10 5 of ESC were plated onto fibronectin-coated 6-well in N2B27 medium (50%DMEM/F12 and 50% Neurobasal Medium supplemented with: 2-Mercaptoethanol[0.1mM], L-Glutamine[20mM]; B27 supplement[1x], Ndiff Neuro-2 Medium Supplement[1x]) with bFGF (12 ng/ml) and ActivinA (20 ng/ml) and with or without doxycycline (1ug/mL). Medium was changed every day and cells were collected at appropriate intervals.

RNA FISH

ESC were dissociated using Accutase (Invitrogen) and adsorbed onto Poly-L-Lysine (Sigma) coated coverslips #1.5 (1mm) for 5 min. Cells were fixed with 3% paraformaldehyde in PBS for 10 min at room temperature and permeabilised for 5 min on ice in PBS containing 0.5%Triton X-100 and 2mM Vanadyl-ribonucleoside complex (New England Biolabs). Coverslips were preserved in 70% EtOH at À20 C. Prior to FISH, samples were dehydrated through an ethanol series (80%, 95%,100% twice) and air-dried quickly. For detecting Huwe1 and Atrx, a BAC spanning the respective genomic region (RP24-157H12, RP23-160I15) was labeled by nick translation (Roche) using spectrum green (Abbot). Per coverslip, 60ng probe was ethanol precipitated with Cot1 repeats, resuspended in formamide, denatured (10min 75 C) and competed for 45min at 37 C. For Xist detection or intron-spanning plasmid probe p510 was labeled by nick translation (Roche) using spectrum red (Abbot). Probe was ethanol precipitated and resuspended in formamide and denatured (10min 75oC). Both Xist and AtrX or Huwe1 probes were co-hybridized in FISH hybrization buffer (50% Formamide, 20% Dextran sulfate, 2x SSC, 1 mg/ml BSA, 10mM Vanadyl-ribonucleoside) over-night. Washes were carried out at 42 C three times 5min in 50% formamide in 2X SSC at pH = 7.2 and three times 5min in 2X SSC. 0.2mg/ml DAPI was used for counterstaining and mounting medium consisted of Vectashield (Vectorlabs). Images were acquired using an Inverted Confocal Spinning Disk Roper/Nikon.

IF with RNA FISH

Cells were grown on fibronectin coated coverslips #1.5 (1mm) and treated with doxycycline (1ug/mL) for 24hrs. Cells were fixed with 3% paraformaldehyde in PBS for 10 min at room temperature and permeabilised for 5 min on ice in PBS containing 0.5%Triton X-100 and 2mM Vanadyl-ribonucleoside complex (New England Biolabs). Samples were blocked for 15 min at room temperature with 1% BSA/PBS. Coverslips were incubated with primary antibodies diluted in blocking solution (anti-H4ac: 1/500; anti-H3K27ac:1/100; anti-H2AK119Ub: 1/200) in the presence of a Ribonuclease Inhibitor (0.8u/mL; Euromedex) for 45 min at room temperature. Coverslips were washed thrice with PBS for 5min and subsequently incubated with secondary antibody solution (goat anti-mouse or rabbit conjugated with Alexa fluorophores; 1/500; supplemented with Ribonuclease Inhibitor [0.8u/mL; Euromedex]) for 45 min at room temperature. Coverslips were washed three times with PBS for 5 min at room temperature. Cells were fixed again with 2% paraformaldehyde in PBS for 10 min at room temperature. After rinsing twice in 2xSSC coverslips were hybridized for Xist detection. Here we used a custom designed strand-specific probe that covers all exons with $75 bp long oligo nucleotides end-labeled with the Alexa 488 fluorophore (Roche). Probes was hybridized in FISH hybridization buffer (50% Formamide, 20% Dextran sulfate, 2x SSC, 1 mg/ml BSA, 10mM Vanadyl-ribonucleoside) over-night. Washes were carried out at 42 C three times 5min in 50% formamide in 2X SSC at pH = 7.2 and three times 5min in 2X SSC. 0.2mg/ml DAPI was used for counterstaining and mounting medium consisted of Vectashield (Vectorlabs). Images were acquired using an Inverted Confocal Spinning Disk Roper/Nikon. For signal quantification, signal intensity was measured along an axis across the Xist domain (z-plane with Xist maximum intensity) and part of the nucleus. Signal was not measured across the nucleoli. Profiles from 70-100 nuclei were centered at the point of Xist cloud boundary (maximum inflection point of Xist signal profile). Signal was normalized at per cell basis. In Figure 5B and Figure S5B shown are average profiles from all nucleoli with shading representing 25 and 75 quantiles.

RNA extraction, reverse transcription, pyrosequencing, qPCR RNA was extracted according to the manufacturer's recommendations using RNeasy Mini Kit (QIAGEN) with on-column DNase digestion (QIAGEN). For cDNA synthesis 1.1 mg RNA was reverse transcribed using Superscript III Reverse Transcriptase (Thermo Fisher Scientific). For allelic-skewing analysis the cDNA was PCR-amplified with biotinylated primers and sequenced using the Pyromark Q24 system (QIAGEN). To quantify ChIP DNA enrichments at control regions 2x SybRGreen Master Mix (Applied Biosystems) and a ViiA7 system (Applied biosystems) were used.

RNA-seq RNA was prepared as described above and quality of samples were verified by Tapestation. Only samples with RIN score above 9 were processed. 100 ng of RNA was used for library preparation using Ovation Mouse RNA-Seq System that uses mouse-specific InDA-C depletion of rRNA. Manufacturers recommendations were followed. Libraries were sequenced using HiSeq2500 at PE100 settings.

TT-seq

The TT-seq protocol was performed as previously described with modifications (Schwalb et al., 2016). Cells were induced with DOX as described above in two T75 flasks per sample. After 0, 4, 8, 12 and 24 hr, RNA was labeled by the addition of 0.5 mM 4-thiouridine (4sU, Carbosynth) for 5 min. Medium was quickly removed and cells were lysed with 10 mL of TRI reagent (Sigma-Aldrich). Lysates were stored at À80 C.

The spike-in mix, containing three 4sU-labeled and three unlabelled RNAs, was synthesized in vitro with MEGAscript T7 Transcription Kit (Thermo Fisher), where 1/10 UTP in the transcription reactions was replaced with 4sUTP (Jena BioScience) for the 4sU-labeled spike-ins. RNA lysates were thawed on ice and 24 ng of spike-in mix was added per 10 million cells. The RNA was then purified according to the manufacturer's protocol. 4sU-labeled RNA was isolated from 300 mg total RNA, for this two aliquots of 150 mg were sheared in Covaris MicroTubes with the Covaris S220 System (Covaris) for 10 s with settings 100 W and 1% duty cycle. These were then pooled and 2 mg of sonicated total RNA was taken for total RNA fraction. The RNA was biotinylated with EZ-Link HPDP-Biotin (Thermo Fisher) in two biotinylation reactions per sample (150 mg RNA in 10 mM Tris pH 7.5; 1 mM EDTA, 40% DMSO; and 200 mg/ml HPDP-biotin) that were incubated 1.5 h at 24 C. The biotinylated RNA was extracted with chloroform and precipitated with isopropanol. 4sU-labeled RNA was isolated by incubating the biotinylated RNA with paramagnetic streptavidin mMACS MicroBeads (Miltenyi) for 15 min at room temperature and binding to MACS columns. The columns were washed three times with wash buffer (100 mM Tris pH 7.5; 10 mM EDTA; 100 mM NaCl; and 0.1% Tween 20) at 65 C and three times with wash buffer at room temperature. The RNA was eluted from the columns with 100 mM DTT. The eluted 4sU-labeled RNA and reserved total RNA were purified with the miRNeasy Micro kit (QIAGEN) according to the manufacturer's protocol with on-column DNase I treatment. 150 ng and 500 ng of isolated newly synthesized RNA and total RNA fractions, respectively, were used to prepare NGS libraries with the TruSeq Stranded Total RNA Library Prep Kit (Illumina). The sample libraries were pooled and sequenced as a single run on the Nextseq550 (Illumina) using PE150 mode.

CRISPR/Cas9 knockout of Hdac3

Two gRNAs have been designed as previously described (Haeussler et al., 2016) and cloned into pX462 vector allowing for their expression under U6 promoter. Additionally, this vector leads to expression of Cas9. TX1072 ESCs were transfected using AmaxaTM 4D-NucleofectorTM system (Lonza) as per manufacturer's protocol. After a pulse of puromycin selection cells were plated at clonal density. Single clones were picked, expanded and screened for deletion by PCR. Final two knockout clones were further validated by sequencing both alleles. Hdac3 À/À #1 harbors a deletion of exon 4-7 on the Cast allele and exon 9 on the B6 allele. Hdac3 À/À #2 harbors a deletion of exon 4-7 on the B6 allele and exon 7 on the B6 allele. Both mutants showed negligible expression on the mRNA level as well as no detectable protein when using a N terminus specific antibody (Figure 4B).

CRISPR/Cas9 FLAG tagging of Hdac3

The Hdac3-Aid-3xFlag targeting construct was generated as follows: 500bp homology arms (flanking both sides of, but excluding the stop codon of Hdac3) were PCR amplified from mouse genomic DNA. 1-step Gibson cloning (NEB) was subsequently used to simultaneously surround the digested AID-3xFLAG insert (carrying a puromycin resistance gene under the control of the PGK promoter) in frame with the homology arms and clone the insert into the pBR322 vector. Synonymous mutations in the PAM/SEED target sequence (located on the 5 0 homology arm) were then introduced using the QuickChange II XL site-directed mutagenesis kit (Agilent) to prevent Cas9 mediated cleavage of the targeting vector upon transfection and of the 3xFLAG tagged allele(s) upon integration.

Single gRNAs has been designed as previously described (Haeussler et al., 2016) and cloned into pX462 vector allowing for its expression under U6 promoter.

Hdac3 targeting was performed using the 4-D nucleofector system from Lonza. 5 million cells were electroporated with 2.5 mg each of non-linearized targeting vector and gRNA/Cas9 encoding plasmids. The day after, puromycin was added at a concentration of 0.4ug/mL. Single clones were picked, expanded and screened for insertion by PCR. One clone showing homozygous insertion of the FLAG tagged cassette at the Hdac3 locus was validated by sanger sequencing and western blotting and kept for subsequent experiments (Figure S5).

HDAC3-FLAG IP-Mass Spectrometry 50 mln (per IP) ESCs (TX1072 -DOX; Hdac3 Flag/Flag +DOX 24h) were trypsinised and washed in PBS. Pellets were snap frozen and stored at À80 C. Defrosted pellets were resuspended in 10 mL of buffer A (10 mM HEPES pH 7.9; 5 mM MgCl 2 ; 10 mM KCl; 1 mM DTT; 0.1% NP-40, protease inhibitors) and incubated for 10 min at 4 C. Nuclei were pelleted at 2000 rpm at 4 C for 10 minutes. Nuclei were resuspended in 500mL of buffer C (20mM HEPES pH 7.9; 20% (v/v) glycerol; 150 mM KCl; 5 mM MgCl2; 2 mM EDTA; 2 mM EDTA; 1 mM DTT; protease inhibitors) and sonicated 3 times for 10 s (Bioruptor, medium setting). Lysates were cleared by centrifugation 12000 rpm at 4 C for 20 minutes. Lysate concentrations were equalised based on protein concentration. Anti-FLAG M2 Magnetic Beads (Sigma-Aldrich) were washed twice in buffer C and then added to each lysate (50mL per IP). Lysates were left rotating over-night at 4 C. Next day beads were washed 5 times in wash buffer (20 mM HEPES pH 7.9; 0.1% NP-40; 150mM KCl; 5mM MgCl2; 2mM EDTA; 2 mM EDTA; 1 mM DTT).

Proteins on magnetic beads were washed twice with 100 mL of 25mM NH4HCO3 and on-beads digestion was performed with 0.2 mg of trypsin/LysC (Promega) for 1 hour in 100mL of 25mM NH4HCO3. Samples were then loaded onto a custom-made C18 StageTips for desalting. Peptides were eluted using 40/60 MeCN/H2O + 0.1% formic acid and vacuum concentrated to dryness.

Online chromatography was performed with an RSLCnano system (Ultimate 3000, Thermo Scientific) coupled online to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific). Peptides were trapped on a C18 column (75 mm inner diameter 3 2 cm; nanoViper Acclaim PepMap 100, Thermo Scientific) with buffer A (2/98 MeCN/H2O in 0.1% formic acid) at a flow rate of 4.0mL/min over 4 min. Separation was performed on a 50cm x 75 mm C18 column (nanoViper Acclaim PepMap RSLC, 2 mm, 100A ˚, Thermo e5 Cell 176, 182-197.e1-e9, January 10, 2019 Scientific) regulated to a temperature of 55 C with a linear gradient of 5% to 25% buffer B (100% MeCN in 0.1% formic acid) at a flow rate of 300 nL/min over 100 min. Full-scan MS was acquired in the Orbitrap analyzer with a resolution set to 120,000 and ions from each full scan were HCD fragmented and analyzed in the linear ion trap.

Anti-FLAG ChIP-seq Cells were grown in 145mm plate format. Cells were initially fixed in 1.5 mM ethylene glycolbis [succinimidyl succinate] (EGS) for 30 min, next 2% formaldehyde was added and further incubated for 10 min. After rinsing twice in PBS, cells were scraped and pellets directly used for ChIP.

Fixed pellets were lysed in 6 mL Nuclear Lysis Buffer (0.5% Triton X-100, 0.1 M sucrose, 5 mM MgCl2, 1 mM EDTA, 10 mM Tris-HCl pH 8.0, 1x Protease Inhibitors) for 10min on ice and dounce-homogenized. Nuclei were pelleted and resuspended in 0.9 mL Lysis Buffer (1 mM EDTA, 0.5 mM EGTA, 10 mM Tris pH 8.0, 0.5% N-Lauroylsarcosine, 1x Protease Inhibitors). Chromatin was sheared using a BioruptorPlus (Diagenode) set to high for 30 cycles. Unsonicated chromatin was removed by centrifugation. Supernatant was diluted with 3.6 mL of Dilution Buffer (1.25% Triton, 0.125% sodium deoxycholate, 6 mM EDTA, 10 mM Tris-HCL pH8.0, 1x Protease Inhibitors) and antibody coated beads were added (per IP: 10 mL of anti-FLAG antibody). Samples were left rotating overnight at 4 C.

Following day beads were magnet-separated and washed 5-times with LiCl buffer (0.50 M LiCl; 1% NP40; 1.1% sodium deoxycholate; 1 mM EDTA; 10 mM Tris-HCl pH 8.1) and 5 times with High Salt Buffer (0.1% SDS; 1% Triton X-100; 2 mM EDTA; 20 mM Tris-HCl, pH 8.1; 1M NaCl; 0.1% sodium deoxycholate). Each wash was performed for 10 min on a rotating wheel at 4 C. Prior to elution all samples were rinsed once in TE. ChIP-DNA was eluted in ProtK-Digestion Buffer (20mM HEPES; 1mM EDTA; 0.5% SDS; 0.8mg/mL Proteinase K) for 15min at 56 C. Beads were separated and the supernatant was further digested for another 2hrs at 56 C and then decrosslinked for 4hrs at 68 C. DNA was isolated using AMPure XP beads. Libraries were prepared using Ovation Ultralow Library System V2 following suppliers protocol. Samples were sequenced with HiSeq2500 using PE100 mode.

QUANTIFICATION AND STATISTICAL ANALYSIS

Time course analysis on TX1072 cell line nChIP-seq Data processing Adapters and low quality bases (< Q20) have been removed with TrimGalore (v0.4.0; http://www.bioinformatics.babraham.ac.uk/ projects/trim_galore) and Cutadapt (1.8.2) (Martin, 2011). An 'N-masked '' genome has been generated with SNPSplit (0.3.2) (Krueger and Andrews, 2016) which is a version of the mouse reference genome mm10 where all the polymorphic sites for the hybrid strain Mus musculus CAST/EiJ and Mus musculus C57BL/6 are masked by ambiguity nucleobase 'N'. For all samples, reads were then mapped to the 'N-masked '' genome with Bowtie2 (2.2.5) with options [--end-to-end -N1 -q] (Langmead and Salzberg, 2012). Duplicates were discarded with Picard MarkDuplicates (1.65) with options [REMOVE_DUPLICATES = true] (https://broadinstitute.github. io/picard/) and reads mapped on blacklisted regions from Encode Consortium were discarded. SNPSplit (0.3.2) (Krueger and Andrews, 2016) was then used to generate allele-specific BAM files by separating the alignment into two distinct alleles (CAST and B6) based on SNPs information downloaded from Sanger. Bigwig files were created with bedtools genomeCoverageBed (2.25.0) (Quinlan and Hall, 2010), using a scale factor calculated on the total library (10.000.000/total reads) for both allele specific bigwigs, and loaded on UCSC genome browser.

Analysis of active histone marks (H3K27ac, H4ac, H3K9ac, H3K4me3, H3K4me1) Peak identification Peak calling was done with MACS2 (2.0.10) (Zhang et al., 2008) with options [--broad -B -f BAMPE --broad-cutoff 0.01], on total (non allele-specific) ChIP-seq signal with input as control. Then, peaks with a fold change inferior to 3 were filtered out. For each histone mark, consensus peaks were defined as follow. For each replicate, all peaks coordinates were merged using bedtools merge (2.25.0) (Quinlan and Hall, 2010). Then, common regions between merged peaks coordinates of each replicate were selected using bedtools intersectBed (2.25.0) (Quinlan and Hall, 2010).

Counts and normalization

Total and allelic reads overlapping consensus peaks were counted using featureCounts (1.5.1), with options [ -C -p -P] (Liao et al., 2014). Only peaks with more than 50 total allelic reads were selected. Next, ratios of allelic counts, called d-scores [reads B6 /(reads Cast + reads B6 )], were calculated.

Analysis of the dynamics

The dynamic evolution of d-score with time was analyzed for each peak with a d-score calculated on the 10 samples (5 times, 2 replicates). Moreover, only peaks that had a biallelic d-score at time 0 (comprised between 0.3 and 0.7) were selected. Then normalized d-scores were calculated by dividing each d-score by the corresponding initial d-score (time 0) and by 2, so that initial normalized d-scores start at 0.5. Initially we have fitted either exponential or sigmoidal curve to these values. We compared the quality of this fitting by taking into account the number of parameters used in the models (AIC and BIC criteria) and concluded that the sigmoidal model best reflects the observed dynamics (data not shown). Sigmoidal fitting of normalized d-scores in function of time has been done for each peak with a four-parameter log-logistic function from drc R package (Ritz et al., 2015). Sigmoidal fittings with low residuals (< 0.2; corresponding to more than 98% of the peaks) were selected, and three parameters were obtained from these fittings: IC35, which is the time where the normalized d-score reaches 0.35 on the sigmoidal fitting; ED 50 , which corresponds to the maximum slope of the sigmoidal fitting; and the minimum second derivative used as a proxy for when the curve starts to decrease. The minimum of the second derivative has been calculated using optimize function from R in the interval between 0 and the calculated ED 50 to avoid finding a local minimum not corresponding to the real minimum. IC/ED/minimum second derivative superior to 24h (outside the time range of the experiment) or not calculated were considered windows with a late loss of active histone mark, and replaced by 24h.

Moreover, for all active histone marks, IC25, IC30 and IC40 (thresholds of normalized d-score:0.25; 0.3; 0.4) were also tested and were able to replicate all our major findings (data not shown).

Features comparison between early and late silenced peaks

Early, intermediate and late silenced peaks located on TSS were defined with a 3 clusters k-means based on IC35 values. Early and late silenced peaks were compared for several features using Wilcoxon test: level of expression of associated genes (from TT-seqsee below), distance to Xist, distance to the closest TAD boundary (Dixon et al., 2012), distance to the closest LAD (Peric-Hupkes et al., 2010), LINE density in a window of 100kb around the peak (from RepeatMasker database) and gene density in a window of 100kb around the peak. Moreover, IC35 was compared for peaks that are inside or close to Xist entry sites (Engreitz et al., 2013)(< 100kb) and those that are distant.

For comparison of dynamics (Figure 6E) of deacetylation and accumulation of repressive marks, early, mid-early, mid-late and late silenced genes were defined with a 4 clusters k-means based on IC35 values. H3K4me1 accumulation at TSS Average plots of H3K4me1 signal were created using DeepTools (3.0.2) (Ramı ´rez et al., 2014). Matrix counts were created using DeepTools computeMatrix around active TSS (see above) on chrX and autosomes separately (with option [--binSize 50]), plots were then created using DeepTools PlotProfile.

Analysis of repressive marks (H3K27me3, H2AK119Ub) Windows definition

For repressive marks, global analysis was first done on fixed windows (10 kb) spanning the whole genome, then on different genomic subcategories: active gene bodies, active promoters, active enhancers and intergenic regions. Active genes were defined as genes with a transcript having its TSS (refFlat annotation) overlapping a consensus peak of H3K9ac and a consensus peak of H3K4me3. For genes having several active transcripts detected, the active gene was defined as starting at the minimum start of transcripts, and ending at the maximum end of transcripts. Then, the active gene bodies were defined as those active genes excluding the 2 first kb downstream of TSS. Active promoters were defined as ± 2kb windows around the TSS of active genes. Putative, active enhancers were defined as intersect between consensus H3K27ac peaks and consensus H3K4me1 peaks, located at a minimal distance of 1kb from a TSS. Intergenic regions were defined as 10 kb windows not overlapping a gene (active or inactive) and its promoter (2kb downstream) or an active enhancer. Enhancer coordinates were also used for the analysis of active histone mark (H3K4me1 and H3K27ac).

Counts and normalization

It should be noted that a d-score analysis, as used for active mark loss, involves the use of the active allele as an internal control, but in the case of H2AK119Ub and H3K27me3, this was hindered by very low enrichment for repressive marks across the Xa. For all defined windows, total and allelic reads overlapping those features were then counted using featureCounts (1.5.1), with options [-C -p -P] (Liao et al., 2014). Then, analysis was done based on normalized reads from B6 allele (allele of the inactive X chromosome). For each sample, a normalization factor was calculated with the trimmed mean of M-values method (TMM) from edgeR package (Robinson et al., 2010), based on B6 reads overlapping consensus peaks located on autosomes (identified and defined as consensus such as for active histone marks analysis). To correct for chromatin accessibility or mappability bias, 10kb windows with outliers counts in the input (counts superior or inferior to mean +-1.5 sd) were discarded from the analysis. Moreover, to represent B6 read accumulation compared to time 0, subtraction of normalized initial counts (time 0) was then applied to all other time points. Analysis of the dynamics Sigmoidal fitting of B6 read accumulation in function of time has also been done with the four-parameter log-logistic function from drc R package. Sigmoidal fittings with low residuals (< mean(residuals) + 1.5 sd(residuals)) were selected, representing more than 90% of the analyzed windows. The ED 50 and the maximum second derivative of the sigmoidal fitting were calculated for each window. Windows with ED 50 /maximum 2nd derivative superior to 24h, or not calculated, were considered as windows with a late accumulation of repressive marks, those were then replaced by 24h. For pairwise comparison between marks, only windows with ED 50 /maximum 2nd derivative inferior to 24h for both marks were selected. For comparison of ED 50 between different genomic subcategories (active promoters, gene bodies, enhancers, intergenic regions) for a same histone mark, no filter was done.

To test the stability of our results, main analysis was also performed with different thresholds of residuals (0.2; 0.15; 0.1; 0.05). Moreover, repressive marks dynamics were also analyzed using allelic ratio (d-scores), such as for analysis of active histone marks, using ED 50 , IC60 and IC65 as criteria. All those analysis revealed stable and reproducible results (data not shown).

TT-seq

Adapters (''TruSeq Universal Adapter '') and low quality bases (< Q20) have been removed with Cutadapt (v1.16) (Martin, 2011). For all samples, reads were then mapped to the 'N-masked' genome (masked for SNPs of the hybrid strain Mus musculus CAST/EiJ and Mus musculus C57BL/6) with STAR 2.3.0 (Dobin and Gingeras, 2015) with maximum 2 percent mismatches. Only unique alignments were retained and soft clipping was disabled [mapping parameters: '--outFilterMismatchNoverReadLmax 0.02 --outFilterMultimap ScoreRange 0 --alignEndsType EndToEnd']. Samtools (Li et al., 2009) was used to quality filter SAM files, where alignments with MAPQ smaller than 7 (-q 7) were skipped and only proper pairs (-f 2) were selected. SNPSplit (v0.3.2) (Krueger and Andrews, 2016) was used to generate allele-specific SAM files. Then, total and allelic reads per gene were counted with featureCounts (1.5.1) [options: -C -p -s 2 -T 8] (Liao et al., 2014) on longest gene coordinates (refFlat annotation). The next steps of analysis (from d-scores to sigmoidal fitting) were done such as for nChIP-seq analysis, except that d-scores were calculated for genes with minimum 10 allelic reads instead of 50. Total normalized expression used for features comparison of early and late silenced peaks (see upper) was calculated based on signal in introns and exons, normalized by the size of the library and the size of the entire gene.

Analysis of WT and Hdac3 À/À RNA-seq Reads were first mapped on rRNA with Tophat (2.1.0) (Trapnell et al., 2009), with options [-g 1 --no-coverage-search --library-type frsecondstrand]. Paired unmapped reads were then used to reconstruct fastq files with bedTools bamToFastq (2.25.0) (Quinlan and Hall, 2010). Those files were then mapped with Tophat (2.1.0) (Trapnell et al., 2009), with options [-p 8 -g 1 -x 1 -N 3 --read-edit-dist 3 --no-coverage-search --library-type fr-secondstrand], with refFlat annotation. Reads covering exons of each gene were then counted with featureCounts (1.5.1) with options [-C -p -s 1 -T 8] ( Liao et al., 2014). D-scores were then calculated for genes with minimum 10 allelic reads.

For differential gene expression analysis prior to DOX treatment, analysis was done on genes for which 2 among the 4 samples have a TPM superior to 1. Normalization was done using TMM (Robinson et al., 2010) method from edgeR and differential analysis using Voom function from Limma R package (Ritchie et al., 2015).

nChIP-seq

The processing from raw data (fastq) to clean mapped file was done such as for time course nChIP-seq. Then, for active histone marks, the reads counts of H3K27ac and H4ac nChIP-seq were done on consensus H3K27ac peaks and consensus H4ac peaks defined in time course analysis, using featureCounts (1.5.1), with options [-C -p -P] (Liao et al., 2014). D-score was calculated with a slight modification for one of the 2 mutant sample. Indeed, this sample showed a non biallelic signal on X chromosome in its input, due to the presence of some XO cells. To take this bias in account, a normalization factor was calculated as follow: normFactor = (reads B6 /(reads B6 + reads CAST )) / (1-(reads B6 /(reads B6 + reads CAST ))).

Then d-score for this mutant was adjusted from classic d-score as follow: d-score = d-score/(d-score + normFactor -normFactor * d-score). After this adjustment, only the peaks that were biallelic at time 0 in WT and mutant samples were selected (d-scores between 0.3 and 0.7) for analysis. For repressive marks, analysis was done such as for time course nChIP-seq, based on signal accumulation of 10 kb windows all along the X chromosome.

For differential peak signal analysis at t = 0hr, normalization was done using TMM (Robinson et al., 2010) method from edgeR and differential analysis using Voom function from Limma R package (Ritchie et al., 2015). Comparison of sensitive and resistant peaks (nChIP-seq on active histone marks) / genes (RNA-seq, ChIP-seq on repressive histone marks) Sensitive and resistant peaks/genes were defined as follow. For nChIP-seq on active histone marks and RNA-seq, only peaks/genes with d-scores considered as stable between replicates (diff replicate < median + 2sd of all differences between replicates) for both clones (WT, mutant) at each time point were selected. D-scores were normalized to time 0 (d-score / d-score t0), then only peaks/ genes considered as not escaping were selected (normalized d-score at t24 < 0.8). Difference of normalized d-score at 24h between WT and mutant was then calculated, and divided by the difference of normalized WT d-score between 0 and 24h for the WT sample. K-means with 3 clusters was done on this normalized difference, identifying 3 groups: sensitive, intermediate and resistant peaks/ genes. For nChIP-seq on repressive histone marks, differential enrichment between WT and mutant was defined as the difference between WT normalized BL6 enrichment relative to time 0 and Mutant normalized BL6 enrichment relative to time 0, divided by WT normalized BL6 enrichment relative to time 0.

Analysis of FLAG ChIP-seq on Hdac3 Flag/Flag ESC cell line

The processing from raw data (fastq) to d-score calculation was done such as for time course nChIP-seq, with slight modifications. The peak calling was done with MACS2 (2.0.10) (Zhang et al., 2008) with options [-B -f BAMPE], on total (non allele-specific) ChIP-seq signal against the FLAG ChIP-seq on TX1072 cell line as control. Consensus peaks were defined as peak regions present at least in 2 of the 4 samples, using bedtools multiIntersectBed (2.25.0) (Quinlan and Hall, 2010). Only peaks with more than 20 total allelic reads were selected.

Heatmaps and average plots of HDAC3 signal in comparison with H3K27ac and H4ac signal were created using DeepTools (3.0.2) (Ramı ´rez et al., 2014). Merged peaks of H3K27ac and H4ac at time 0 were split in 2 categories: active promoters for merged peaks overlapping a TSS and putative active enhancer for merged peaks at a distance of minimum 2 kb of a TSS. The merged peaks were ordered by normalized signal of HDAC3 peaks (signal/length of the peak) overlapping those peaks. For HDAC3, signal of the control (FLAG ChIP-seq on TX1072 cell line) was subtracted from the signal of FLAG ChIP-seq on Hdac3 Flag/Flag ESC cell line using DeepTools bamCompare with options [--ratio subtract --binSize 100] before to proceed to the next step. Then, matrix counts were created using DeepTools computeMatrix around TSS of active promoters (with option [--binSize 100]) or centers of putative enhancers, plots were then created using DeepTools PlotHeatmap.

For H3K27ac and HDAC3 peaks annotation comparison (Figure S5C), peaks were annotated as follow: peaks at a distance < 2000 bp of a TSS were considered in promoters, other peaks were considered as intragenic if overlapping a gene body, intergenic if not.

Category of peaks accumulating signal or not on Xi (Figures S5E andS5F) were created based on clustering with hclust function with parameter [method = 'Ward.D'], using Pearson correlation as distance. 11 clusters were identified using cutree function. Those clusters were grouped in 2 super groups according to the presence or not of an accumulation of signal on Xi at 4hr in each cluster.

Analysis of TXY:Xist and TXY:XistDA nChIP-seq Processing steps to clean bam files were similar to time course nChIP-seq analysis, except that mapping was not allele-specific but done on mm10 genome and that reads mapped with low quality (< q10) were removed with samtools (1.3) (Li et al., 2009). Such as for time course nChIP-seq analysis, ChIP-seq signal was analyzed differently depending on the histone mark: per peak for active mark (H3K27ac) and per window for repressive marks (H3K27me3, H2AK119Ub). Peak calling was done similarly to time course nChIPseq analysis of active histone marks. Here, consensus peaks were defined as common regions between peaks identified in minimum 2 among the 4 samples using bedtools multiIntersectBed (2.25.0) and bedtools merge (2.25.0).

The windows for repressive mark analysis were defined similarly as for nChIP-seq time course analysis for repressive marks, except that active TSS were defined here based on consensus peaks of H3K27ac overlapping TSS, and putative active enhancers as H3K27ac peaks located at minimum 1kb of TSS.

Reads overlapping defined peaks (for active marks) or windows (for repressive marks) were then counted with featureCounts (1.5.1) (Liao et al., 2014) with default options.

For global analysis on repressive marks, counts normalization was done such as for time course nChIP-seq analysis, based on counts falling in autosomal consensus peaks. For windows and peaks analysis, windows that had less than one read per 50kb for more than 2 among the 8 samples were removed for the analysis. Normalization factors were calculated based on windows located on autosomes (with selection of the 10000 most higher signal for intergenic window analysis), with TMM method using edgeR (Robinson et al., 2010).

Because of the high variability in proportion of cells involved in X chromosome inactivation quantified by the presence of Xist Cloud by FISH experiments (TXY:XistWT#1 46.64%, TXY:XistWT#2 59.44%, TXY:XistDA#1 50.61%, TXY:XistDA#2 48.24%), linear regression including the percentage of induction calculated by FISH was fitted for each window according the following model: $0+ clones + clones: induction, using Voom function from Limma R package (Ritchie et al., 2015). The slope of this regression represents then the logFC between noDox and Dox conditions if the induction of the cell population was complete (corrected logFC).

Metaplots were created using DeepTools (3.0.2) (Ramı ´rez et al., 2014). Bigwigs of log2(FC) between Dox and noDox samples were created with personalized scaling according to normalized factors calculated above using DeepTools bamCompare, bigwig of mean of log2(FC) between replicates was then created using DeepTools bigwigCompare (binSize: 100 bp for H3K27ac, 1000 bp for H3K27me3 and H2Ub), matrix counts was then created using DeepTools computeMatrix on active genes coordinates (see above) on chrX and autosomes separately and plots were then created using DeepTools PlotProfile.

Proteomics data analysis

For identification, the data were searched against the Mus musculus (UP000000589) UniProt database using Sequest HF through proteome discoverer (version 2.2). Enzyme specificity was set to trypsin and a maximum of two-missed cleavage sites were allowed. Oxidized methionine, N-terminal acetylation, and carbamidomethyl cysteine were set as variable modifications. Maximum allowed mass deviation was set to 10 ppm for monoisotopic precursor ions and 0.6 Da for MS/MS peaks.

The resulting files were further processed using myProMS v3.6 (Poullet et al., 2007). FDR calculation used Percolator and was set to 1% at the peptide level for the whole study. The label free quantification was performed by peptide Extracted Ion Chromatograms (XICs) computed with MassChroQ version 2.2.2 (Valot et al., 2011). For protein quantification, XICs from proteotypic peptides shared between compared conditions (TopN matching) with no missed cleavages were used. Median and scale normalization was applied on the total signal to correct the XICs for each biological replicate. To estimate the significance of the change in protein abundance, a linear model (adjusted on peptides and biological replicates) was performed and p-values were adjusted with a Benjamini-Hochberg FDR procedure with a control threshold set to 0.05.

DATA AND SOFTWARE AVAILABILITY

The accession number for the sequencing datasets reported in this paper is GEO: GSE116480. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD011344 The IC35 measures when the loss of a histone mark reaches an efficiency threshold and not the timing when this process occurs most rapidly. For example, if two marks show different efficiency of loss from the Xi (i.e., different IC35) the process might still be occurring at the same time. Shown is H3K27 deacetylation dynamics for two promoters (Fmr1:red, Pgk1:blue). Both promoters have the same ED 50 (i.e., time when sigmoid reaches its maximum slope), however show very different efficiency of deacetylation. The latter difference is captured by the IC35 parameter (i.e., time when sigmoid reaches the 0.35 threshold). (G) Heatmaps showing the d-scores of all peaks on the X chromosome. Windows were sorted from centromere (top) to telomere (bottom). (H-I) Plots showing the distribution of differential IC35 (H): H3K4me3-H3K27ac; (I): H3K4me1-H3K27ac) for all promoters (H) and putative enhancers (I). Each bar represents a single window, these were ordered accordingly to their differential IC35. (J) Violin plots comparing the dynamics of H3K27ac and H3K4 methylation loss at active promoters (left) and putative active enhancers (right). Plots compare the ED 50 parameter of H3K27ac and H3K4me peaks within the quantitative range of the experiment (IC35 < 24hrs). p value was calculated using paired Wilcoxon rank sum test.(K-L) Violin plots comparing the distribution of IC35 for H3K27ac (K) or H3K4me1/3 (L) at enhancers and promoters. p values calculated using Wilcoxon rank sum test. (M) Plots showing the distribution of differential IC35 for H3K27ac at promoterputative enhancer pairs. Windows within one TAD were paired based on proximity. Individual pairs were ordered accordingly to their differential IC35. (N) Average H3K4me1 distribution plots around the transcriptional start site of active genes (TSS ± 1kb). Plots were done using all mapped reads on TSS of active genes on X chromosome (top) and on autosomes (bottom). Average profiles show duplicates at 0hr (gray) and 12hrs (orange) of DOX treatment. (O) Boxplots showing the d-score distribution for H3K4me1 around TSS of active genes (±200bp) after 0 (gray) and 12hrs (orange) of DOX treatment, on X chromosome (top) and on autosomes (bottom). p values calculated using Wilcoxon rank sum test. showing the distribution of d-scores at H3K27ac (left) and H4ac (right) peaks on the X chromosome in Hdac3 +/+ and Hdac3 À/À . Initially, peaks are on average biallelic (0h) and become skewed toward the Cast allele after 24h of DOX treatment. Shown is an overlay of biological duplicates including two independent Hdac3 À/À clones. (G) Heatmaps showing the d-score evolution of all H3K27ac and H4ac peaks in Hdac3 +/+ and Hdac3 À/À . X-linked peaks were sorted based on their genomic position from the centromere to telomere.

B6 -Xi Cast -Xa

H2AK119Ub H3K27me3

Hdac3 -/-0h

Hdac3 +/+ 0h Hdac3 -/-24h Hdac3 +/+ 24h Hdac3 -/-0h Hdac3 +/+ 0h Hdac3 -/-24h Hdac3 +/+ 24h
Atrx -HDAC3 sensitive gene Step 0

Step 1

Step 2

Step 3

Step 4 |❡s✉•ts

H

❙P❊◆ ❢✉♥❝t✐♦♥ ❛♥❞ ♠❡❝❤❛♥✐s♠s ✐♥ ❳❈■ ❈❤❛♣t❡r ✻ ❙P❊◆ ❢✉♥❝t✐♦♥ ❛♥❞ ♠❡❝❤❛♥✐s♠s ✐♥ ❳❈■ ✭❉♦ss✐♥ ❡t ❛•✳✱ ◆❛t✉r❡✳ ✷✵✷✵✮ ✻✳✶ |❛t✐♦♥❛•❡ ❢♦r ✐♥✈❡st✐❣❛t✐♥❣ ❙P❊◆ ❛♥❞ ❳❈■ ❆•t❤♦✉❣❤ ❳❈■ ✐s ❛ss♦❝✐❛t❡❞ ✇✐t❤ ❛ ♣•❡t❤♦r❛ ♦❢ ❝❤r♦♠❛t✐♥ ❝❤❛♥❣❡s ✭s❡❡ s❡❝t✐♦♥ ✶✳✽ ❛♥❞ ❝❤❛♣t❡r ✺✮✱ ♥♦ ❝❤r♦♠❛t✐♥ ♠♦❞✐❢②✐♥❣ ❡♥③②♠❡s ✇❡r❡ ❢♦✉♥❞ t♦ ❜❡ r❡❝r✉✐t❡❞ ❞✐r❡❝t•② ❜② ❳✐st |◆❆ ❬▼✐♥❛•✐❣✐ ❡t ❛•✳✱ ✷✵✶✺❀ ❈❤✉ ❡t ❛•✳✱ ✷✵✶✺❀ ▼❝❍✉❣❤ ❡t ❛•✳✱ ✷✵✶✺(✳ ■♥tr✐❣✉✐♥❣•②✱ ❳✐st ✐♥st❡❛❞ ✐♥t❡r❛❝ts ✇✐t❤ ❛ ✈❛st ❛rr❛② ♦❢ ♣r♦t❡✐♥s✱ ✇❤✐❝❤ ✇❡r❡ ❤✐t❤❡rt♦ ♥❡✈❡r ❛ss♦❝✐❛t❡❞ ✇✐t❤ ❳❈■ ❛♥❞ ✇❤♦s❡ ❢✉♥❝t✐♦♥s ❛r❡ ♣♦♦r•② ❝❤❛r❛❝t❡r✐③❡❞ ✭s❡❡ s❡❝t✐♦♥ ✶✳✶✸✮✳ ❯♥❞❡rst❛♥❞✐♥❣ ❤♦✇ t❤❡s❡ ❳✐st✲❜✐♥❞✐♥❣ ♣r♦t❡✐♥s ❣♦✈❡r♥ s✉❜s❡q✉❡♥t ❝❤❛♥❣❡s ✐♥ tr❛♥s❝r✐♣t✐♦♥✱ ❝❤r♦♠❛t✐♥✱ ❛♥❞ s♣❛t✐❛• ♦r❣❛♥✐③❛t✐♦♥ ♦❢ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡ ✐s ♥♦✇ ♦❢ ♣❛r❛♠♦✉♥t ✐♠♣♦rt❛♥❝❡✳ ❲❤❡♥ ■ •♦✐♥❡❞ ❊❞✐t❤✬s •❛❜ ✐♥ ✷✵✶✼✱ t❤❡ •❛❜ ♦❢ ◆❡✐• ❇r♦❝❦❞♦r✛ ❤❛❞ ❡①t❡♥s✐✈❡•② ❝❤❛r❛❝✲ t❡r✐③❡❞ ❤♦✇ ♦♥❡ ♦❢ t❤❡s❡ ♣r♦t❡✐♥s✱ ❤♥|◆P❑✱ ❝♦♥tr✐❜✉t❡s t♦ t❤❡ ♣r♦❝❡ss ♦❢ ❳❈■ ✭❢r♦♠ ✐ts ❞✐r❡❝t ❛ss♦❝✐❛t✐♦♥ ✇✐t❤ t❤❡ ❇✲r❡♣❡❛t ♦❢ ❳✐st t♦ ✐ts r❡q✉✐r❡♠❡♥t ❢♦r P♦•②❝♦♠❜ r❡❝r✉✐t♠❡♥t✱ s❡❡ s✉❜s❡❝t✐♦♥ ✶✳✶✸✳✶✮✳ ❍♦✇❡✈❡r✱ s✉❝❤ ❝❤❛r❛❝t❡r✐③❛t✐♦♥ ❤❛❞ ♥♦t ❜❡❡♥ ❞♦♥❡ ❢♦r ❙P❊◆✳ ❚❤❡ ♦r✐❣✐♥❛••② ♣r♦♣♦s❡❞ ♠♦❞❡• ❢♦r t❤❡ r♦•❡ ♦❢ ❙P❊◆ ✐♥ ❳ ❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥ s✉❣❣❡st❡❞ t❤❛t ❳✐st r❡❝r✉✐ts ❙P❊◆ ✇❤✐❝❤ ✐♥ t✉r♥ r❡❝r✉✐ts ◆❈♦|✴❙▼|❚ ❛♥❞ ❍❉❆❈✸ t♦ s✐•❡♥❝❡ tr❛♥s❝r✐♣t✐♦♥ ❬▼❝❍✉❣❤ ❡t ❛•✳✱ ✷✵✶✺(✳ ■♠♣♦rt❛♥t•② ✐♥ t❤✐s st✉❞②✱ t❤❡r❡ ✇❡r❡ ♥♦ ❞✐r❡❝t ❡✈✐❞❡♥❝❡ t❤❛t ❙P❊◆ ✐s ✐♥❞❡❡❞ r❡❝r✉✐t❡❞ ❜② ❳✐st t♦ t❤❡ ❳✐ ❞✉r✐♥❣ ❳❈■✳ ❋✉rt❤❡r♠♦r❡✱ ✐ts ♠❡❝❤❛♥✐s♠ ♦❢ ❛❝t✐♦♥ ✭t❤r♦✉❣❤ ❍❉❆❈✸✮ ♦♥•② r❡•✐❡❞ ♦♥ t❤❡ ♦❜s❡r✈❛t✐♦♥ t❤❛t s✐|◆❆✲ ♠❡❞✐❛t❡❞✱ ✐♥❞✐✈✐❞✉❛• ❦♥♦❝❦❞♦✇♥s ♦❢ ❍❞❛❝✸ ♦r ❙♣❡♥ s❤♦✇ s✐♠✐•❛r ❞❡❢❡❝ts ✐♥ s✐•❡♥❝✐♥❣ ♦❢ ♦♥❡ ❳✲•✐♥❦❡❞ ❣❡♥❡✱ •♣❝✹ ❬▼❝❍✉❣❤ ❡t ❛•✳✱ ✷✵✶✺(✱ ✇✐t❤ ♥♦ ♣❤②s✐❝❛• ❡✈✐❞❡♥❝❡ ❢♦r ❙P❊◆✲ ❞❡♣❡♥❞❡♥t r❡❝r✉✐t♠❡♥t ♦❢ ❍❉❆❈✸ ❞✉r✐♥❣ ❳❈■✳ ✶✵✺ |❡s✉•ts ❙P❊◆ ❢✉♥❝t✐♦♥ ❛♥❞ ♠❡❝❤❛♥✐s♠s ✐♥ ❳❈■ ❖✉r ♦❜s❡r✈❛t✐♦♥ t❤❛t ❍❉❆❈✸ ✐s ♥♦t r❡❝r✉✐t❡❞ ❞✉r✐♥❣ ❳❈■ ❜✉t ✐s ✐♥st❡❛❞ ♣r❡✲❜♦✉♥❞ t♦ ❛❝t✐✈❡ ❡♥❤❛♥❝❡rs ✭s❡❡ s✉❜s❡❝t✐♦♥ ✺✳✸✳✷ ❛♥❞ ❬➏②•✐❝③ ❡t ❛•✳✱ ✷✵✶✾(✮ s✉❣❣❡sts t❤❛t t❤✐s ♠♦❞❡• ♥❡❡❞s t♦ ❜❡ r❡✈✐s❡❞✳ ❋✉rt❤❡r♠♦r❡✱ ♦✉r ❛♥❛•②s✐s ♦❢ ❍❞❛❝✸ ❑❖ ❝❡••s✱ ✇❤✐❝❤ s❤♦✇ ❞❡•❛②❡❞ ❜✉t ♥♦t ❢✉••② ❞❡✜❝✐❡♥t ❳✲•✐♥❦❡❞ ❣❡♥❡ s✐•❡♥❝✐♥❣ ✭s❡❡ s✉❜s❡❝t✐♦♥ ✺✳✷✳✸ ❛♥❞ ❬➏②•✐❝③ ❡t ❛•✳✱ ✷✵✶✾(✮✱ ❞❡♠♦♥str❛t❡s t❤❛t ❍❉❆❈✸ ✐s ♥♦t t❤❡ ♦♥•② ❢❛❝t♦r ❛t ♣•❛② ✐♥ ✐♥✐t✐❛t✐♥❣ ❳❈■✳ ❍♦✇ ❛❜♦✉t ❙P❊◆❄ ■s ❙P❊◆ ❛•s♦ ♦♥•② ♣❛rt✐❛••② ✐♠♣♦rt❛♥t ❢♦r ❳❈■✱ ♦r ✐s ✐t r❡q✉✐r❡❞ t♦ ❛ •❛r❣❡r ❡①t❡♥t t❤❛♥ ❍❉❆❈✸❄ ■s ❙P❊◆ ❛❝t✐✈❡•② r❡❝r✉✐t❡❞ t♦ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡❄ ■❢ s♦✱ ✇✐t❤ ✇❤✐❝❤ ❞②♥❛♠✐❝s ❛♥❞ ✇❤❡r❡ ❞♦❡s ✐t ♣r❡❢❡r❡♥t✐❛••② ❜✐♥❞❄ ❲❤✐❝❤ ♣r♦t❡✐♥ ♣❛rt♥❡rs ❞♦❡s ✐t ❡♥❣❛❣❡ ✇✐t❤❄ ■s ❙P❊◆ ❛•s♦ ✐♠♣♦rt❛♥t ❢♦r ♠❛✐♥t❡♥❛♥❝❡ ♦❢ ❳❈■✱ ♥❛♠❡•② t♦ ❦❡❡♣ t❤❡ ✐♥❛❝t✐✈❡ ❳ ❝❤r♦♠♦s♦♠❡ s✐•❡♥❝❡❞ ❛♥❞ ♣r♦♣❡r•② str✉❝t✉r❡❞ ✐♥ s♦♠❛t✐❝ ❝❡••s❄ ❉❡s♣✐t❡ ✜✈❡ st✉❞✐❡s ✉♥✐✈❡rs❛••② ♣♦✐♥t✐♥❣ ♦✉t t♦ ❙P❊◆ ❛s ❛♥ ✐♠♣♦rt❛♥t ♣•❛②❡r ❞✉r✐♥❣ ❳❈■ ❬▼❝❍✉❣❤ ❡t ❛•✳✱ ✷✵✶✺❀ ❈❤✉ ❡t ❛•✳✱ ✷✵✶✺❀ ▼✐♥❛•✐❣✐ ❡t ❛•✳✱ ✷✵✶✺❀ ▼♦♥❢♦rt ❡t ❛•✳✱ ✷✵✶✺❀ ▼♦✐♥❞r♦t ❡t ❛•✳✱ ✷✵✶✺(✱ t❤❡s❡ ❢✉♥❞❛♠❡♥t❛• q✉❡st✐♦♥s r❡♠❛✐♥❡❞ ✉♥❛♥s✇❡r❡❞✳ ■ ❤❡♥❝❡ ❞❡❝✐❞❡❞ t❤❛t ■ ✇♦✉•❞ ❞❡❞✐❝❛t❡ t❤❡ ❝♦r❡ ♦❢ ♠② P❤❉ ✇♦r❦ t♦ ♣r♦♣❡r•② ❝❤❛r❛❝t❡r✐③❡ t❤❡ ❢✉♥❝t✐♦♥ ❛♥❞ ❡ss❡♥t✐❛•✐t② ♦❢ ❙P❊◆ ✐♥ t❤❡ ♣r♦❝❡ss ♦❢ ❳❈■✱ ❛♥❞ ❛tt❡♠♣t t♦ ❞✐ss❡❝t ✐ts ♠❡❝❤❛♥✐s♠ ♦❢ ❛❝t✐♦♥✳ ■♥ t❤❡ ❢♦••♦✇✐♥❣ s❡❝t✐♦♥s ♦❢ t❤✐s ❝❤❛♣t❡r✱ ■ ✐♥t❡♥❞ t♦ r❡♣♦rt t❤❡ s❝✐❡♥t✐✜❝ ♠❡t❤♦❞♦•♦❣② ■ ❢♦••♦✇❡❞ ✕ ♦✉t•✐♥✐♥❣ ✐t ❝❤r♦♥♦•♦❣✐❝❛••②✱ ❛s ✐t ✇❡♥t ❜② t❤r♦✉❣❤♦✉t ♠② P❤❉ ✕ t♦ ✉♥❝♦✈❡r ❞✐✛❡r❡♥t ❝•✉❡s ❛❜♦✉t ❙P❊◆✬s ❢✉♥❝t✐♦♥ ✭•♦❛• ✶✱ s❡❡ s❡❝t✐♦♥ ✻✳✷✮ ❛♥❞ ♠❡❝❤❛♥✐s♠ ♦❢ ❛❝t✐♦♥ ✭•♦❛• ✷✱ s❡❡ s❡❝t✐♦♥ ✻✳✸✮ ✐♥ ❳❈■ ✭❋✐❣✉r❡ ✻✳✶✮✳
❋✐❣✉r❡ ✻✳✶ ✕ ❋•♦✇❝❤❛rt ❞❡♣✐❝t✐♥❣ t❤❡ q✉❡st✐♦♥s ❛❞❞r❡ss❡❞ ❛♥❞ ♠❡t❤♦❞s ❡♠♣•♦②❡❞ ❢♦r ❡❛❝❤ ❣♦❛• ♦❢ t❤❡ ❙P❊◆ ♣r♦ •❡❝t✳ ✶✵✻ |❡s✉•ts ❙P❊◆ ❢✉♥❝t✐♦♥ ❛♥❞ ♠❡❝❤❛♥✐s♠s ✐♥ ❳❈■ ❚❤❡ r❡s✉•ts ■ ♦❜t❛✐♥❡❞ ✇✐•• ❜❡ ♣r❡s❡♥t❡❞ ✈❡r② s✉♠♠❛r✐•② ✐♥ t❤❡ ❢♦••♦✇✐♥❣ s❡❝t✐♦♥s✱ ❛s t❤❡② ✇❡r❡ ♣✉❜•✐s❤❡❞ ❡❛r•② ✷✵✷✵ ✐♥ ◆❛t✉r❡✱ ❛♥❞ ❛r❡ t♦ ❜❡ ❢♦✉♥❞ ✐♥ t❤❡ ♠❛✐♥ t❡①t✴✜❣✉r❡s ❛s ✇❡•• ❛s t❤❡ ❡①t❡♥❞❡❞ ❞❛t❛ ✜❣✉r❡s ♦❢ t❤❡ ❛rt✐❝•❡ ❛♣♣❡♥❞❡❞ ❛t t❤❡ ❡♥❞ ♦❢ t❤✐s ❝❤❛♣t❡r ✭s❡❝t✐♦♥ ✻✳✹ ❛♥❞ ❬❉♦ss✐♥ ❡t ❛•✳✱ ✷✵✷✵(✮✳ ✻✳✷ •♦❛• ✶✿ ❆❞❞r❡ss✐♥❣ ❙P❊◆ ❢✉♥❝t✐♦♥ ❛♥❞ ✇✐♥❞♦✇ ♦❢ ♦♣♣♦rt✉♥✐t② ❞✉r✐♥❣ ❳❈■ ❚❤❡ ✜rst •✐♥❡ ♦❢ ❡①♣❡r✐♠❡♥ts ■ s❡t ✉♣ ✇❡r❡ ❛✐♠❡❞ t♦ ❣❛✐♥ ✐♥s✐❣❤ts ✐♥t♦ ✭❋✐❣✉r❡ ✻✳✶✮✿ ✭✐✮ ❚❤❡ ❡①t❡♥t t♦ ✇❤✐❝❤ ❙P❊◆ r❡❣✉•❛t❡s ❣❡♥❡ s✐•❡♥❝✐♥❣ ❝❤r♦♠♦s♦♠❡ ✇✐❞❡ ❞✉r✐♥❣ ✐♥✐t✐✲ ❛t✐♦♥ ♦❢ ❳❈■✳ ✭✐✐✮ ❲❤❡t❤❡r ❙P❊◆ ♣•❛②s ❛ r♦•❡ ❢♦r ❦❡❡♣✐♥❣ ❣❡♥❡s s✐•❡♥❝❡❞ ✭❛♥❞ t❤❡ ❳✐ str✉❝t✉r❡❞✮ ❞✉r✐♥❣ ♠❛✐♥t❡♥❛♥❝❡ ♦❢ ❳❈■ ✐♥ s♦♠❛t✐❝ ❝❡••s✳ ✭✐✐✐✮ ❲❤❡t❤❡r ❙P❊◆ ✐s ❛❝t✐✈❡•② r❡❝r✉✐t❡❞ t♦ t❤❡ ❳✐ t❤r♦✉❣❤♦✉t t❤❡ ❳❈■ ♣r♦❝❡ss✳ ✻✳✷✳✶ ❉✐ss❡❝t✐♥❣ ❙P❊◆ ❢✉♥❝t✐♦♥ ✐♥ ✐♥✐t✐❛t✐♥❣ ❳❈■ ✐♥ ✈✐tr♦ ❚♦ ❛❞❞r❡ss ♣♦✐♥ts ✭✐✮ ❛♥❞ ✭✐✐✮✱ ❛ ❙P❊◆ •♦ss ♦❢ ❢✉♥❝t✐♦♥ ❛♣♣r♦❛❝❤ ❤❛❞ t♦ ❜❡ ❡♠♣•♦②❡❞✳ ❚❤❡ ♦❜s❡r✈❛t✐♦♥ t❤❛t ❙♣❡♥ ❑❖ ♠♦✉s❡ ❡♠❜r②♦s ❞✐❡ ♦♥•② ❛t ❊✶✸✳✺ ❬(❛❜❡ ❡t ❛•✳✱ ✷✵✵✼(✱ •♦♥❣ ❛❢t❡r t❤❡ ❜•❛st♦❝②st st❛❣❡✱ s✉❣❣❡sts t❤❛t ❙P❊◆ ✐s ♥♦t ❡ss❡♥t✐❛• ✐♥ ❡♠❜r②♦♥✐❝ st❡♠ ❝❡••s✳ ❍❡♥❝❡✱ ❛ ❝•❛ss✐❝❛• ❝♦♥st✐t✉t✐✈❡ ❦♥♦❝❦✲♦✉t ♦❢ ❙♣❡♥ ❝♦✉•❞ ❤❛✈❡ ❜❡❡♥ ❝❛rr✐❡❞ ♦✉t ✐♥ ♦✉r ❚❳✶✵✼✷ ♠❊❙❈ s②st❡♠ t♦ ✐♥t❡rr♦❣❛t❡ ✐ts ❢✉♥❝t✐♦♥❛• ✐♠♣♦rt❛♥❝❡ ❞✉r✐♥❣ ✐♥✐t✐❛t✐♦♥ ♦❢ ❳❈■✳ ❍♦✇❡✈❡r✱ s✉❝❤ ❝♦♥st✐t✉t✐✈❡ ❑❖ ❛♣♣r♦❛❝❤ ✇♦✉•❞ ❤❛✈❡ ❜❡❡♥ ✐♥❝♦♠♣❛t✐❜•❡ ✇✐t❤ ♠② ❛✐♠ t♦ ❛❞❞r❡ss ✇❤❡t❤❡r ❙P❊◆ ❛•s♦ r❡❣✉•❛t❡s ♠❛✐♥t❡♥❛♥❝❡ ♦❢ ❳❈■ ✐♥ ❞✐✛❡r❡♥t✐❛t❡❞ ❝❡••s✳ ■♥✲ ❞❡❡❞✱ s✉❝❤ ❛✐♠ r❡q✉✐r❡s t❤❛t ♠❊❙❈s ❛r❡ ✜rst ❞✐✛❡r❡♥t✐❛t❡❞ ✐♥ t❤❡ ♣r❡s❡♥❝❡ ♦❢ ❙P❊◆✱ ♦♥•② ❛❢t❡r ✇❤✐❝❤ ❙P❊◆ ❝♦✉•❞ ❜❡ r❡♠♦✈❡❞ ❛♥❞ t❤❡ ❳❈■ st❛t✉s ✐♥t❡rr♦❣❛t❡❞✳ ❋✉rt❤❡r♠♦r❡✱ ♥♦ ♣r❡✈✐♦✉s r❡♣♦rts ❛••♦✇❡❞ ✉s t♦ ✐♥❢❡r ✇❤❡t❤❡r ❙P❊◆✲❞❡✜❝✐❡♥t ♠❊❙❈s ❝♦✉•❞ ✉♥❞❡r❣♦ ❞✐✛❡r✲ ❡♥t✐❛t✐♦♥ ♣r♦♣❡r•②✳ ❍❡♥❝❡✱ ❛ ❝♦♥❞✐t✐♦♥❛• ❙P❊◆ •♦ss ♦❢ ❢✉♥❝t✐♦♥ ❛♣♣r♦❛❝❤ ✇❛s ♣r❡❢❡rr❡❞✳ ■ ♠❛❞❡ ✉s❡ ♦❢ t❤❡ ❛✉①✐♥✲✐♥❞✉❝✐❜•❡ ❞❡❣r♦♥ str❛t❡❣② ❬◆✐s❤✐♠✉r❛ ❡t ❛•✳✱ ✷✵✵✾(✱ ✇❤✐❝❤ ❛••♦✇s ❛❝✉t❡ ❛♥❞ r❛♣✐❞ ❞❡♣•❡t✐♦♥ ♦❢ ❛♥② ♣r♦t❡✐♥ ♦❢ ✐♥t❡r❡st✱ ✉♣♦♥ tr❡❛t♠❡♥t ✇✐t❤ t❤❡ ♣•❛♥t ❤♦r♠♦♥❡ ❛✉①✐♥ ✭❋✐❣✉r❡ ✻✳✷✮✳ ❚❤✐s ❛♣♣r♦❛❝❤ r❡•✐❡s ♦♥ t❤❡ ❤❡t❡r❡♦•♦❣♦✉s ❡①♣r❡ss✐♦♥ ♦❢ ❚■|✶✱ ❛ r✐❝❡✲❞❡r✐✈❡❞ ❋✲❜♦① ♣r♦t❡✐♥ ❬•r❛② ❡t ❛•✳✱ ✷✵✵✶❀ ◆✐s❤✐♠✉r❛ ❡t ❛•✳✱ ✷✵✵✾(✳ ❊①♣r❡ss❡❞ ✐♥ ♠❛♠♠❛•✐❛♥ ❝❡••s✱ ❚■|✶ ❛ss♦❝✐❛t❡s ✇✐t❤ ❡♥❞♦❣❡♥♦✉s ❝♦♠♣♦♥❡♥ts ♦❢ t❤❡ ❙❈❋ ❝♦♠♣•❡①✱ r❡s♣♦♥s✐❜•❡ ❢♦r ♣♦•②✉❜✐q✉✐t②•❛t✐♦♥ ❛♥❞ s✉❜s❡q✉❡♥t ♣r♦t❡❛s♦♠❛• ❞❡❣r❛❞❛t✐♦♥ ♦❢ ♣r♦t❡✐♥ t❛r❣❡ts ❬❈❛r❞♦③♦ ❛♥❞ P❛❣❛♥♦✱ ✷✵✵✹(✳ |❡♠❛r❦❛❜•②✱ t❤r♦✉❣❤ ✐ts ❚■|✶ ♠♦✐❡t②✱ ❛♥❞ ♦♥•② ✐♥ ✶✵✼ |❡s✉•ts ❙P❊◆ ❢✉♥❝t✐♦♥ ❛♥❞ ♠❡❝❤❛♥✐s♠s ✐♥ ❳❈■ t❤❡ ♣r❡s❡♥❝❡ ❛✉①✐♥✱ t❤❡ ❙❈❋ ❚■|✶ ❝♦♠♣•❡① ❤❛s t❤❡ ❛❜✐•✐t② t♦ ✐♥t❡r❛❝t ✇✐t❤ ❛♥❞ ♣r♦♠♦t❡ t❤❡ ❞❡❣r❛❞❛t✐♦♥ ♦❢ ❛♥② ♣r♦t❡✐♥ ❝❛rr②✐♥❣ ❛♥ ❛✉①✐♥✲✐♥❞✉❝✐❜•❡ ❞ ❡❣r♦♥ ✭❆■❉✮ ♠♦t✐❢ ✭❋✐❣✉r❡ ✻✳✷✮ ❬❉❤❛r♠❛s✐r✐ ❡t ❛•✳✱ ✷✵✵✺❀ •r❛② ❡t ❛•✳✱ ✷✵✵✶❀ ❚❛♥ ❡t ❛•✳✱ ✷✵✵✼(✳
ub ub ub E2 RBX1 SPEN AID SPEN AID ❋✐❣✉r❡ ✻✳✷ ✕ ❙❝❤❡♠❡ ❞❡♣✐❝t✐♥❣ t❤❡ ❛✉①✐♥ ✐♥❞✉❝✐❜•❡ ❞❡❣r♦♥ s②st❡♠ ❛♣♣•✐❡❞ t♦ ❙P❊◆✳ ❆✉①✐♥ •❡❛❞s t♦ ❚■|✶✲❞❡♣❡♥❞❡♥t ♣♦•②✉❜✐q✉✐t②•❛t✐♦♥ ❛♥❞ ♣r♦t❡❛s♦♠❛• ❞❡❣r❛❞❛t✐♦♥ ♦❢ ❙P❊◆ t❛❣❣❡❞ ✇✐t❤ t❤❡ ❛✉①✐♥✲✐♥❞✉❝✐❜•❡ ❞❡❣r♦♥ ✭❆■❉✮✳ ❆✉①✐♥ r❡♠♦✈❛• •❡❛❞s t♦ ♣r♦t❡✐♥ r❡❝♦✈❡r②✳ ❚❤✐s s②st❡♠ ❤❛s r♦✉t✐♥❡•② ❜❡❡♥ ✉s❡❞ ❛❝r♦ss s❡✈❡r❛• ♠♦❞❡• ♦r❣❛♥✐s♠s✱ ❛♥❞ ❛••♦✇s t♦ ❛❞❞r❡ss ♣r♦t❡✐♥ ❢✉♥❝t✐♦♥ ❞✐r❡❝t•②✱ ✇✐t❤ ❤✐❣❤❡st s♣❡❝✐✜❝✐t②✱ ✇✐t❤♦✉t ♣❡rt✉r❜✐♥❣ ♥❡✐t❤❡r t❤❡ ❣❡♥❡ ♥♦r t❤❡ tr❛♥s❝r✐♣t ❡♥❝♦❞✐♥❣ s✉❝❤ ♣r♦t❡✐♥✳ ❋✉rt❤❡r♠♦r❡✱ t❤❡ r❛♣✐❞✐t② ✇✐t❤ ✇❤✐❝❤ t❛r❣❡t ♣r♦t❡✐♥s ❛r❡ ❞❡♣•❡t❡❞ ✐♥ t❤✐s s②st❡♠ ♠✐♥✐♠✐③❡s t❤❡ ❛♣♣❡❛r❛♥❝❡ ♦❢ ❝♦♠♣❡♥s❛t♦r② ❡✛❡❝ts t❤❛t ✇♦✉•❞ ♦t❤❡r✇✐s❡ ❝♦♠♣•✐❝❛t❡ ❞❛t❛ ✐♥t❡r♣r❡t❛t✐♦♥✳ ✻✳✷✳✶✳✶ •❡♥❡r❛t✐♦♥ ♦❢ ❙P❊◆ ❛✉①✐♥✲✐♥❞✉❝✐❜•❡ ❞❡❣r♦♥ ♠❊❙❈s ❚♦ ✐♠♣•❡♠❡♥t t❤✐s ❛♣♣r♦❛❝❤ ✐♥ ♦✉r ❚❳✶✵✼✷ ♠❊❙❈ s②st❡♠ ✭s❡❡ s✉❜s❡❝t✐♦♥ ✺✳✶✳✶✮✱ ■ ✉s❡❞ ❈|■❙P|✴❈❛s✾ t♦ t❛r❣❡t ❛ ❝♦♥st✐t✉t✐✈❡•② ❡①♣r❡ss❡❞ ❖s❚✐r✶ ❣❡♥❡ ❛t t❤❡ ❚✐❣r❡ •♦❝✉s ❛♥❞✱ ✉s✐♥❣ t❤❡ s❛♠❡ ♠❡t❤♦❞♦•♦❣② ❛s ■ ❞✐❞ ❢♦r ❍❞❛❝✸ ✭s❡❡ s✉❜s❡❝t✐♦♥ ✺✳✸✳✶ ❛♥❞ ❋✐❣✉r❡ ✺✳✷❆✮✱ ■ ♣❡r❢♦r♠❡❞ ❛ ❤♦♠♦③②❣♦✉s ❦♥♦❝❦✲✐♥ ♦❢ t❤❡ ❆■❉ ❞❡❣r♦♥ ¶ ❛t t❤❡ ❈✲t❡r♠✐♥✉s ♦❢ ❙♣❡♥ ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❊①t❡r♥❛• ❉❛t❛ ❋✐❣✳ ✶❛✮✳ ■ ❛•s♦ ✐♥❝•✉❞❡❞ ❛ ❍❛•♦❚❛❣ ❬▲♦s ❡t ❛•✳✱ ✷✵✵✽(✱ ❛❞❞✐t✐♦♥❛••② t♦ t❤❡ ❆■❉✱ ❛••♦✇✐♥❣ ❢♦r ✈✐s✉❛•✐③❛t✐♦♥ ♦❢ ❙P❊◆ ❜♦t❤ ❜② ✇❡st❡r♥ ❜•♦t ❛♥❞ ♠✐❝r♦s❝♦♣②✱ ✉s✐♥❣ ❍❛•♦❚❛❣ ❛♥t✐❜♦❞✐❡s ❛♥❞ ✢✉♦r❡s❝❡♥t✲•✐❣❛♥❞s r❡s♣❡❝t✐✈❡•②✳ ❚❤✐s ✇♦r❦ ✇❛s ❞♦♥❡ ✉♥❞❡r t❤❡ ♠❡♥t♦rs❤✐♣ ♦❢ ■♥ês P✐♥❤❡✐r♦✱ ❛ ♣♦st❞♦❝ ✇❤♦ ✐♥✐t✐❛••② tr❛✐♥❡❞ ♠❡ ✉♣♦♥ ♠② ❛rr✐✈❛• ✐♥ ❊❞✐t❤✬s •❛❜✳ ❲✐t❤ t❤✐s s②st❡♠✱ ❙P❊◆ ❝♦✉•❞ ❜❡ ❡✣❝✐❡♥t•② ❞❡♣•❡t❡❞ ✇✐t❤✐♥ ✶ ❤♦✉r ♦❢ ❛✉①✐♥ tr❡❛t♠❡♥t ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✶❜ ❛♥❞ ❊①t❡r♥❛• ❉❛t❛ ❋✐❣✳ ✶❜✮✳ ❍❡♥❝❡✱ t❤✐s ❝❡••✉•❛r s②st❡♠ ❛••♦✇s ✉s t♦ ❝♦♥tr♦••❛❜•② ✐♥❞✉❝❡ ❳❈■ ✇❤✐•❡ s✐♠✉•t❛♥❡♦✉s•② ❞❡♣•❡t✐♥❣ ❙P❊◆ ✭❋✐❣✉r❡ ✻✳✸✮✳ ¶ ❚❤r♦✉❣❤♦✉t ♠② P❤❉✱ ■ ❣❡♥❡r❛t❡❞ s❡✈❡r❛• ❝♦♥str✉❝ts t♦ t❛r❣❡t ❆■❉✲❢✉s✐♦♥ ❝❛ss❡tt❡s ❛t t❤❡ ◆✲ ♦r ❈✲t❡r♠✐♥✐ ♦❢ ❡♥❞♦❣❡♥♦✉s ❣❡♥❡s✴♣r♦t❡✐♥s✳ ❚❤❡s❡ ❝♦♥str✉❝ts ✇❡r❡ ✉s❡❞ ✐♥ t❤❡ ❝♦♥t❡①t ♦❢ ❛ ❝♦••❛❜♦r❛t✐♦♥ ✇✐t❤ t❤❡ ❇♦✉r❝✬❤✐s •❛❜✱ t♦ ✐♥✈❡st✐❣❛t❡ t❤❡ r♦•❡ ♦❢ t❤❡ ♠ ✻ ❆ |◆❆ ♠❡t❤②•❛t✐♦♥ ♣❛t❤✇❛② ✐♥ ❝♦♥tr♦••✐♥❣ t❤❡ ❡①♣r❡ss✐♦♥ ♦❢ tr❛♥s♣♦s❛❜•❡ ❡•❡♠❡♥ts✱ r❡s✉•t✐♥❣ ✐♥ ❛ ♣✉❜•✐❝❛t✐♦♥ ✐♥ ◆❛t✉r❡ ❬❈❤❡•♠✐❝❦✐ ❡t ❛•✳✱ ✷✵✷✶(✱ ✇❤✐❝❤ ✐s ❛♣♣❡♥❞❡❞ ❛t s❡❝t✐♦♥ ❆✳✶✳ ✶✵✽ |❡s✉•ts ❙P❊◆ ❢✉♥❝t✐♦♥ ❛♥❞ ♠❡❝❤❛♥✐s♠s ✐♥ ❳❈■

SPEN-degron mESCs

X chromosome inactivation

Xist

❋✐❣✉r❡ ✻✳✸ ✕ ❙❝❤❡♠❡ ❞❡♣✐❝t✐♥❣ t❤❡ str❛t❡❣② t♦ ✐♥✈❡st✐❣❛t❡ ❙P❊◆✬s ✐♠♣♦rt❛♥❝❡ ❞✉r✐♥❣ ✐♥✐t✐❛t✐♦♥ ♦❢ ❳❈■ ✐♥ ✈✐tr♦✳ ❙P❊◆✲❞❡❣r♦♥ ❚❳✶✵✼✷ ♠❊❙❈s ❛••♦✇ ❝♦♥❞✐t✐♦♥❛• ❞♦①✲tr✐❣❣❡r❡❞ ❳❈■ ❝♦♠❜✐♥❡❞ ✇✐t❤ ❛✉①✐♥✲♠❡❞✐❛t❡❞ ❙P❊◆ ❞❡♣•❡t✐♦♥✳ ✻✳✷✳✶✳✷ ❙P❊◆ ✐s ❡ss❡♥t✐❛• ❢♦r ✐♥✐t✐❛t✐♥❣ ❳❈■ ✐♥ ✈✐tr♦ ❯s✐♥❣ ❛••❡•❡ s♣❡❝✐✜❝ |◆❆✲s❡q✱ ♣②r♦s❡q✉❡♥❝✐♥❣ ❛♥❞ |◆❆ ❋■❙❍✱ ■ r❡✈❡❛•❡❞ t❤❛t ❛❝✉t❡ ❞❡♣•❡t✐♦♥ ♦❢ ❙P❊◆ ✐♥ ♠❊❙❈s •❡❛❞s t♦ ❞r❛st✐❝ ❞❡❢❡❝ts ✐♥ ❳✲•✐♥❦❡❞ ❣❡♥❡ s✐•❡♥❝✐♥❣✱ ✇✐t❤♦✉t ❛✛❡❝t✐♥❣ ❳✐st |◆❆ •♦❝❛•✐③❛t✐♦♥ ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✶❛✲❢ ❛♥❞ ❊①t❡r♥❛• ❉❛t❛ ❋✐❣✳ ✶❛✲❡✱ ❛♥❞ ❝❤❛♣t❡r ✽ ❢♦r ❢✉rt❤❡r ❞✐s❝✉ss✐♦♥✮✳ ✻✳✷✳✷ ❉✐ss❡❝t✐♥❣ ❙P❊◆ ❢✉♥❝t✐♦♥ ✐♥ ✐♥✐t✐❛t✐♥❣ ✐♠♣r✐♥t❡❞ ❳❈■ ✐♥ ✈✐✈♦ ❚♦ ❡①t❡♥❞ t❤✐s ♦❜s❡r✈❛t✐♦♥ ✐♥ ✈✐✈♦✱ ■ ❛❞r❡ss❡❞ t❤❡ st❛t✉s ♦❢ ❳❈■ ✐♥ ❙♣❡♥ ❑❖ ♠♦✉s❡ ❡♠❜r②♦s✱ ❝♦••❛❜♦r❛t✐♥❣ ✇✐t❤ ❏❛♥ )②•✐❝③✱ ❛♥ ❡♠❜r②♦•♦❣✐st ❛♥❞ ♣♦st❞♦❝ ❢r♦♠ ❊❞✐t❤✬s •❛❜✿ ❆s ♣r❡✈✐♦✉s•② ♠❡♥t✐♦♥❡❞ ✐♥ s❡❝t✐♦♥ ✶✳✸✱ t✇♦ ✇❛✈❡s ♦❢ ❳❈■ ✭✐♠♣r✐♥t❡❞ ❛♥❞ r❛♥❞♦♠✮ ♦❝❝✉r ❞✉r✐♥❣ ♠♦✉s❡ ❡♠❜r②♦♥✐❝ ❞❡✈❡•♦♣♠❡♥t ✭❋✐❣✉r❡ ✶✳✷✮✳ ▼♦♥✐t♦r✐♥❣ r❛♥❞♦♠ ❳❈■ ✭✇❤✐❝❤ ✐♥✐t✐❛t❡s ❛t t❤❡ •❛t❡ ❜•❛st♦❝②st st❛❣❡✮ ✉s✐♥❣ ❛♥ ❛••❡•❡ s♣❡❝✐✜❝ r❡❛❞♦✉t✱ ❝❤r♦♠♦s♦♠❡✲ ✇✐❞❡✱ ✐s ❝❤❛••❡♥❣✐♥❣✳ ■♥❞❡❡❞✱ ✐t r❡q✉✐r❡s t❤❛t t❤❡ ❡①tr❛❡♠❜r②♦♥✐❝ ❝❡••s ❛r❡ r❡♠♦✈❡❞ ✭s✐♥❝❡ t❤❡② ❤❛✈❡ ✉♥❞❡r❣♦♥❡ ✐♠♣r✐♥t❡❞ ❳❈■✮✱ ❛♥❞ t❤❡♥ ✐✈♦•✈❡s t❤❡ ✉s❡ ♦❢ s✐♥❣•❡✲❝❡•• |◆❆✲s❡q✱ ❛s ❤❛•❢ ♦❢ t❤❡ ❡♠❜r②♦♥✐❝ ❝❡••s ✇✐•• ✐♥❛❝t✐✈❛t❡ ♣❛t❡r♥❛• ❳✲•✐♥❦❡❞ ❛••❡•❡s✱ ❛♥❞ t❤❡ ♦t❤❡r ❤❛•❢ ✇✐•• ✐♥❛❝t✐✈❛t❡ ♠❛t❡r♥❛• ❳✲•✐♥❦❡❞ ❛••❡•❡s✳ ■♥st❡❛❞✱ ♠♦♥✐t♦r✐♥❣ ✐♠♣r✐♥t❡❞ ❳❈■ ✭✇❤✐❝❤ ❤❛s ❜❡❡♥ ❡st❛❜•✐s❤❡❞ ✐♥ ❛•• ❝❡••s ❜② t❤❡ ❡❛r•② ❜•❛st♦❝②st st❛❣❡✮ ✐s ❝♦♠♣❛t✐❜•❡ ✇✐t❤ t♦t❛•✲❡♠❜r②♦ ❛••❡•❡ s♣❡❝✐✜❝ |◆❆s❡q✱ s✐♥❝❡ ❛•• t❤❡ ❝❡••s ❤❛✈❡ ✐♥❛❝t✐✈❛t❡❞ str✐❝t•② t❤❡ ♣❛t❡r♥❛• ❳✲•✐♥❦❡❞ ❛••❡•❡s✳ ✻✳✷✳✷✳✶ ▼♦✉s❡ ❝r♦ss✐♥❣ str❛t❡❣② ❍❡♥❝❡✱ ❢♦r ♣r❛❝t✐❝❛• r❡❛s♦♥s✱ ✇❡ ❛❞❞r❡ss❡❞ t❤❡ ✐♠♣♦rt❛♥❝❡ ♦❢ ❙P❊◆ ♦♥•② ❞✉r✐♥❣ ✐♠✲ ♣r✐♥t❡❞ ❳❈■✳ ❚♦ ❞♦ s♦✱ ✇❡ ✉s❡❞ ❛ ♣✉❜•✐s❤❡❞ ❝♦♥❞✐t✐♦♥❛• ✭✐✳❡✳ ✢♦①❡❞✮ ❙♣❡♥ ❑❖ ❛••❡•❡ ❬(❛❜❡ ✶✵✾ |❡s✉•ts ❙P❊◆ ❢✉♥❝t✐♦♥ ❛♥❞ ♠❡❝❤❛♥✐s♠s ✐♥ ❳❈■ ❡t ❛•✳✱ ✷✵✵✼(✱ ❛♥❞ ❣❡♥❡r❛t❡❞ ❋ ✶ ❤②❜r✐❞ ❙♣❡♥ ❑❖✴❲❚ ♠❛•❡s ❝❛rr②✐♥❣ ❛ ❈❛st✴❊✐❏ ✲❞❡r✐✈❡❞ ❳ ❝❤r♦♠♦s♦♠❡ ✭❋✐❣✉r❡ ✻✳✹❆✮✳ ❚❤❡s❡ ♠❛•❡s ✇❡r❡ ❝r♦ss❡❞ ✇✐t❤ ❈✺✼❇▲✴✻❏ ❙♣❡♥ ✢♦①✴✢♦① ❢❡✲ ♠❛•❡s ✭❋✐❣✉r❡ ✻✳✹❆✮✱ ❝❛rr②✐♥❣ ❛ |♦s❛✷✻✿)♣✸✲❈r❡ ❛••❡•❡ ❬❉❡ ❱r✐❡s ❡t ❛•✳✱ ✷✵✵✵(✱ ❛••♦✇✐♥❣ ❢♦r ♠❛t❡r♥❛• ❙♣❡♥ ❞❡•❡t✐♦♥ ✐♥ ♦♦❝②t❡s ✭❋✐❣✉r❡ ✻✳✹❇✮✳ ❋♦••♦✇✐♥❣ t❤✐s ❝r♦ss✱ ❜♦t❤ ♠❛t❡r♥❛• ✭✐✳❡✳ ✐♥❤❡r✐t✐♥❣ ♦♥•② ❛ ❢✉♥❝t✐♦♥❛• ❙♣❡♥ ❛••❡•❡ ♦❢ ♣❛t❡r♥❛• ♦r✐❣✐♥✮ ❛♥❞ ♠❛t❡r♥❛•✲③②❣♦t✐❝ ✭✐✳❡✳ ✐♥❤❡r✐t✐♥❣ ♥♦♥✲❢✉♥❝t✐♦♥❛• ❙♣❡♥ ❛••❡•❡s ❢r♦♠ ❜♦t❤ ♣❛r❡♥ts✮ ❙♣❡♥ ❑❖ ❢❡♠❛•❡ ❡♠❜r②♦s ✇❡r❡ ♦❜t❛✐♥❡❞ ✭❋✐❣✉r❡ ✻✳✹❈✮✳ ❇② t❤❡ ❡❛r•② ❜•❛st♦❝②st st❛❣❡ ✭❊✸✳✺✮✱ ♦♥•② t❤❡ ♣❛t❡r♥❛• ❳ ❝❤r♦♠♦s♦♠❡ ✕ ♦❢ ❈❛st✴❊✐❏ ♦r✐❣✐♥ ✕ s❤♦✉•❞ ❜❡ ✐♥❛❝t✐✈❛t❡❞ ✭❋✐❣✉r❡ ✻✳✹❉✮✳ ❳✲•✐♥❦❡❞ tr❛♥s❝r✐♣t✐♦♥ ✐♥ t❤❡s❡ ❢❡♠❛•❡ ❡♠❜r②♦s ✇❛s ♣r♦✜•❡❞ ✉s✐♥❣ ❛••❡•❡✲s♣❡❝✐✜❝ s✐♥❣•❡ ❡♠❜r②♦ |◆❆s❡q ✭❋✐❣✉r❡ ✻✳✹❉✮✳ cross B6 Cast/ B6 ♂ Spen KO/WT ♀ Spen flox/flox R26:Zp3-Cre X Cast Y B6 X B6 X B6 x "female" sperm gametes Spen KO Spen WT 50% 50% X Cast oocyte 100% Spen KO X B6 ♀ zygotes Spen KO/WT maternal KO Spen KO/KO maternal & zygotic KO Xm B6 Xp Cast single embryo RNAseq imprinted XCI? ♀ early blastocysts Xm B6 Xp Cast E3.5 A D B C ❋✐❣✉r❡ ✻✳✹ ✕ ▼♦✉s❡ ❝r♦ss✐♥❣ str❛t❡❣② t♦ ✐♥✈❡st✐❣❛t❡ ❙P❊◆✬s ✐♠♣♦rt❛♥❝❡ ❞✉r✐♥❣ ✐♠✲ ♣r✐♥t❡❞ ❳❈■ ✐♥ ✈✐✈♦✳ ■♠♣r✐♥t❡❞ ❳❈■ ✐s ❛ss❛②❡❞ ✉s✐♥❣ s✐♥❣•❡ ❡♠❜r②♦ |◆❆s❡q ✐♥ ❙♣❡♥ ♠❛t❡r♥❛• ❛♥❞ ♠❛t❡r♥❛•✲③②❣♦t✐❝ ❑❖ ❡❛r•② ❜•❛st♦❝②sts✳ ✻✳✷✳✷✳✷ ❙P❊◆ ✐s ❡ss❡♥t✐❛• ❢♦r ✐♠♣r✐♥t❡❞ ❳❈■ ✐♥ ✈✐✈♦ ❚❤✐s ❡①♣❡r✐♠❡♥t r❡✈❡❛•❡❞ t❤❛t s✐♠✐•❛r•② t♦ ✇❤❛t ✐s ♦❜s❡r✈❡❞ ✐♥ ❙P❊◆✲❞❡❣r♦♥ ♠❊❙❈s ♦r ❳✐st ❑❖ ❡♠❜r②♦s ❬❇♦r❡♥s③t❡✐♥ ❡t ❛•✳✱ ✷✵✶✼❜(✱ ❙♣❡♥ ❑❖ ❢❡♠❛•❡ ❡♠❜r②♦s ❛r❡ ❛•♠♦st ❢✉••② ❞❡✜❝✐❡♥t ❢♦r ❳✲•✐♥❦❡❞ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❞✉r✐♥❣ ✐♠♣r✐♥t❡❞ ❳❈■✱ ❞❡s♣✐t❡ s✉st❛✐♥❡❞ ♣❛t❡r♥❛• ❳✐st ❡①♣r❡ss✐♦♥ ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✶❣✱ ❤ ❛♥❞ ❊①t❡r♥❛• ❉❛t❛ ❋✐❣✳ ✶❢✲❤✱ ❛♥❞ s❡❝t✐♦♥ ✽✳✹ ❢♦r ❢✉rt❤❡r ❞✐s❝✉ss✐♦♥✮✳ ✻✳✷✳✸ ❉✐ss❡❝t✐♥❣ ❙P❊◆ ❢✉♥❝t✐♦♥ ❞✉r✐♥❣ ♠❛✐♥t❡♥❛♥❝❡ ♦❢ ❳❈■ ✐♥ s♦♠❛t✐❝ ❝❡••s ❍❛✈✐♥❣ ❡st❛❜•✐s❤❡❞ t❤❛t ❙P❊◆ ✐s ❡ss❡♥t✐❛• ❢♦r ✐♥✐t✐❛t✐♥❣ ❳✐st ✲❞❡♣❡♥❞❡♥t ❣❡♥❡ s✐•❡♥❝✐♥❣ ❞✉r✐♥❣ ❳❈■✱ ✐t ❜❡❝❛♠❡ r❡•❡✈❛♥t t♦ ✐♥✈❡st✐❣❛t❡ ✇❤❡t❤❡r ❙P❊◆ ✐s ❛•s♦ r❡q✉✐r❡❞ t♦ ♠❛✐♥t❛✐♥ ✶✶✵ |❡s✉•ts ❙P❊◆ ❢✉♥❝t✐♦♥ ❛♥❞ ♠❡❝❤❛♥✐s♠s ✐♥ ❳❈■ ❣❡♥❡s s✐•❡♥❝❡❞ ❛❢t❡r ❳❈■ ❤❛s ❜❡❡♥ ❡st❛❜•✐s❤❡❞ ✐♥ ❞✐✛❡r❡♥t✐❛t❡❞ ❝❡••s✳ ❍❡♥❝❡✱ ■ ❞✐✛❡r❡♥t✐❛t❡❞ ❙P❊◆✲❞❡❣r♦♥ ♠❊❙❈s ✐♥t♦ ♥❡✉r❛• ♣r♦❣❡♥✐t♦r ❝❡••s ✭◆P❈s✮✱ ✐s♦•❛t❡❞ ✐♥❞❡♥❞❡♥t ◆P❈ ❝•♦♥❡s ❛♥❞ ♣❡r❢♦r♠❡❞ ❛••❡•❡✲s♣❡❝✐✜❝ |◆❆✲s❡q ❢♦••♦✇✐♥❣ ❡✐t❤❡r ✷✹ ♦r ✹✽ ❤♦✉rs ♦❢ ❙P❊◆ ❞❡♣•❡t✐♦♥
❋✐❣✉r❡ ✻✳✺ ✕ ❙❝❤❡♠❡ ❞❡♣✐❝t✐♥❣ t❤❡ str❛t❡❣② t♦ ✐♥✈❡st✐❣❛t❡ ❙P❊◆✬s ✐♠♣♦rt❛♥❝❡ ❞✉r✐♥❣ ♠❛✐♥t❡♥❛♥❝❡ ♦❢ ❳❈■ ✐♥ ◆P❈s✳ ❋♦••♦✇✐♥❣ ❞✐✛❡r❡♥t✐❛t✐♦♥ ♦❢ ❙P❊◆✲❞❡❣r♦♥ ♠❊❙❈s ✐♥t♦ st❛❜•❡ ◆P❈ ❝•♦♥❡s✱ t❤❡ ✐♠♣♦rt❛♥❝❡ ♦❢ ❙P❊◆ ✐♥ ♠❛✐♥t❛✐♥✐♥❣ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❛♥❞ str✉❝t✉r❡ ♦❢ t❤❡ ❳✐ ✐s ❛ss❛②❡❞ ✉s✐♥❣ |◆❆s❡q ❛♥❞ ❍✐✲❈ r❡s♣❡❝t✐✈❡•②✱ ❢♦••♦✇✐♥❣ ✷✹ ♦r ✹✽ ❤♦✉rs ♦❢ ❙P❊◆ ❞❡♣•❡t✐♦♥✳ ✻✳✷✳✸✳✶ ❙P❊◆ ✐s ❞✐s♣❡♥s❛❜•❡ ❢♦r ❦❡❡♣✐♥❣ ❣❡♥❡s s✐•❡♥❝❡❞✱ ❜✉t ❜✉✛❡rs tr❛♥✲ s❝r✐♣t✐♦♥ ♦❢ ❡s❝❛♣❡❡s ▲♦ss ♦❢ ❙P❊◆ ❞✐❞ ♥♦t •❡❛❞ t♦ r❡❛❝t✐✈❛t✐♦♥ ♦❢ ❢✉••② s✐•❡♥❝❡❞ ❣❡♥❡s ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✷❝✱ ❞✮✱ ❞❡♠♦♥str❛t✐♥❣ t❤❛t ♠❛✐♥t❡♥❛♥❝❡ ♦❢ ❣❡♥❡ s✐•❡♥❝✐♥❣ ♦♥ t❤❡ ❳✐ ✐s ❡♥s✉r❡❞ ✐♥❞❡♣❡♥❞❡♥t•② ♦❢ ❙P❊◆✳ ❍♦✇❡✈❡r✱ tr❛♥s❝r✐♣t✐♦♥ ♦❢ ❡s❝❛♣✐♥❣ ❣❡♥❡s ✇❛s ♠♦❞❡r❛t❡•②✱ ❜✉t s✐❣♥✐✜❝❛♥t•②✱ ✉♣r❡❣✉•❛t❡❞ ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✷❡✱ ❢✮✱ s✉❣❣❡st✐♥❣ t❤❛t ❙P❊◆ ❜✉✛❡rs ♦✈❡r❡①♣r❡ss✐♦♥ ♦❢ ❡s❝❛♣❡❡s ✐♥ ❢❡♠❛•❡ s♦♠❛t✐❝ ❝❡••s ✭❞✐s❝✉ss❡❞ ✐♥ s❡❝t✐♦♥ ✽✳✺✮✳ ✻✳✷✳✸✳✷ ❚❤❡ ♣❡❝✉•✐❛r ♦r❣❛♥✐③❛t✐♦♥ ♦❢ t❤❡ ❳✐ ✐s ♠❛✐♥t❛✐♥❡❞ ✐♥❞❡♣❡♥❞❡♥t•② ♦❢ ❙P❊◆ •✐✈❡♥ t❤❛t ❳✐st ♣•❛②s ❛ ❝❡♥tr❛• r♦•❡ ✐♥ r❡♦r❣❛♥✐③✐♥❣ t❤❡ ✸❉✲str✉❝t✉r❡ ♦❢ t❤❡ ❳✐ ✐♥ s♦♠❛t✐❝ ❝❡••s ✭s❡❡ s❡❝t✐♦♥ ✶✳✶✵✮✱ ■ ❢✉rt❤❡r ❡①❛♠✐♥❡❞ ✇❤❡t❤❡r ❙P❊◆ ✕ ❛s ❛ ❳✐st✲❜✐♥❞✐♥❣ ♣r♦t❡✐♥ ✕ ✐s ❛•s♦ ✐♥✈♦•✈❡❞ ✐♥ s✉❝❤ str✉❝t✉r❛• ❡✛❡❝ts✳ ❚♦ ❞♦ s♦✱ ■ ❞❡♣•❡t❡❞ ❙P❊◆ ❢♦r ✹✽ ❤♦✉rs ✐♥ ◆P❈s ❛♥❞✱ ✐♥ ❝♦••❛❜♦r❛t✐♦♥ ✇✐t❤ ❏♦❜ ❉❡❦❦❡r✬s •❛❜✱ ♣❡r❢♦r♠❡❞ ❛••❡•❡✲s♣❡❝✐✜❝ ❍✐✲❈ ✭❋✐❣✉r❡ ✻✳✺✮✳ ▲♦ss ♦❢ ❙P❊◆ ❤❛❞ ♥♦ ❡✛❡❝t ♦♥ t❤❡ ❳✐ ❝♦♥❢♦r♠❛t✐♦♥✱ ♥❡✐t❤❡r ❛t t❤❡ •❡✈❡• ♦❢ ♠❡❣❛❞♦✲ ♠❛✐♥s ♥♦r ❛t t❤❡ •❡✈❡• ♦❢ ❡s❝❛♣❡❡ ❚❆❉✲•✐❦❡ str✉❝t✉r❡s ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❊①t❡r♥❛• ❉❛t❛ ❋✐❣✳ ✷❡✲❣✮✱ ❤✐♥t✐♥❣ t❤❛t ❳✐st |◆❆ str✉❝t✉r❡s t❤❡ ❳✐ ✐♥❞❡♣❡♥❞❡♥t•② ♦❢ ❙P❊◆✳ ✶✶✶ |❡s✉•ts ❙P❊◆ ❢✉♥❝t✐♦♥ ❛♥❞ ♠❡❝❤❛♥✐s♠s ✐♥ ❳❈■ ✻✳✷✳✹ ■♠❛❣✐♥❣ ♦❢ ❙P❊◆ ❛♥❞ ❳✐st |◆❆ ❞②♥❛♠✐❝s ❞✉r✐♥❣ ❳❈■ ❚❤❡ ✐♠♣♦rt❛♥❝❡ ♦❢ ❙P❊◆ ❞✉r✐♥❣ ✐♥✐t✐❛t✐♦♥ ♦❢ ❳❈■ ♣r♦♠♣t❡❞ ♠❡ t♦ ❡①♣•♦r❡ ✐ts ❝❡••✉•❛r •♦❝❛•✐③❛t✐♦♥ r❡•❛t✐✈❡ t♦ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡✱ t❤r♦✉❣❤♦✉t t❤❡ ♣r♦❝❡ss ♦❢ ❳❈■✳ ■♠♣♦rt❛♥t•②✱ ■ ❛✐♠❡❞ t♦ ❛❞❞r❡ss ✇❤❡t❤❡r ❙P❊◆ ✐s ❛❝t✐✈❡•② r❡❝r✉✐t❡❞ ❜② ❳✐st✱ ❛♥❞ ✐❢ s♦✱ ✇✐t❤ ✇❤✐❝❤ ❞②♥❛♠✐❝s✳ ✻✳✷✳✹✳✶ ❋✐①❡❞✲❝❡•• ✐♠❛❣✐♥❣ ❋✐rst✱ ■ ♠♦♥✐t♦r❡❞ ❙P❊◆ •♦❝❛•✐③❛t✐♦♥ t❤r♦✉❣❤♦✉t ❛ t✐♠❡❝♦✉rs❡ ♦❢ ❳✐st ✐♥❞✉❝t✐♦♥✱ ✐♥ ✜①❡❞ ❝❡••s✳ ❚♦ ❞♦ s♦✱ ❞♦①✲✐♥❞✉❝❡❞ ❙P❊◆✲❍❛•♦❚❛❣ ❞❡❣r♦♥ ♠❊❙❈s ✇❡r❡ •❛❜❡•❡❞ ✐♥ ❝✉•t✉r❡ ✇✐t❤ ❛ ✢✉♦r❡s❝❡♥t ❍❛•♦✲•✐❣❛♥❞ ❬•r✐♠♠ ❡t ❛•✳✱ ✷✵✶✺(✱ ✜①❡❞✱ ❛♥❞ ♣r♦❝❡ss❡❞ ❢♦r |◆❆ ❋■❙❍ ❛❣❛✐♥st ❳✐st✳ |❡♠❛r❦❛❜•②✱ ❛s ❡❛r•② ❛s ✷ ❤♦✉rs ❢♦••♦✇✐♥❣ ❳✐st ❡①♣r❡ss✐♦♥✱ ❙P❊◆ str♦♥❣•② ❛❝❝✉♠✉•❛t❡❞ ✐♥t♦ ❞♦♠❛✐♥s ❝♦rr❡s♣♦♥❞✐♥❣ t♦ ❳✐st |◆❆ ❝•♦✉❞s ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✷❛✮✳ ❚❤✐s ❛❝❝✉♠✉•❛t✐♦♥ ✇❛s s✉st❛✐♥❡❞ ❛♥❞ ❛♠♣•✐✜❡❞ t❤r♦✉❣❤♦✉t •❛t❡r t✐♠❡ ♣♦✐♥ts❀ ❛♥❞ ❙P❊◆ ❛•s♦ ❛❝❝✉♠✉•❛t❡❞ ❛r♦✉♥❞ t❤❡ ❳✐ ✐♥ ❞✐✛❡r❡♥t✐❛t❡❞ ◆P❈s ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✷❛✱ ❜✮✳ ❲❤✐•❡ t❤✐s ✜rst s❡t ♦❢ r❡s✉•ts s✉❣❣❡st❡❞ t❤❛t ❙P❊◆ ✐s r❡❝r✉✐t❡❞ t♦ t❤❡ ❳✐ ❢❛✐r•② ❡❛r•② ✭✷ ❤♦✉rs✮ ✉♣♦♥ ❳✐st ❛❝❝✉♠✉•❛t✐♦♥✱ t❤❡ •♦✇ t❡♠♣♦r❛• r❡s♦•✉t✐♦♥ ❛❝❤✐❡✈❡❞ ✇✐t❤✐♥ t❤❡ ✜①❡❞✲ ❝❡•• ✐♠❛❣✐♥❣ t✐♠❡❝♦✉rs❡ ♣r❡✈❡♥t❡❞ ♠❡ ❢r♦♠ ♠♦♥✐t♦r✐♥❣ ✇❤❡t❤❡r ❙P❊◆ ✐s ✐♥ ❢❛❝t r❡❝r✉✐t❡❞ ✐♠♠❡❞✐❛t❡•② ❢♦••♦✇✐♥❣ ❳✐st ❡①♣r❡ss✐♦♥✳ ❚♦ ❝✐r❝✉♠✈❡♥t t❤✐s •✐♠✐t❛t✐♦♥✱ ■ ❤❛❞ t♦ tr❛❝❦ ❙P❊◆ ❛♥❞ ❳✐st ♠♦•❡❝✉•❡s ✐♥ •✐✈❡ ❝❡••s✱ ❛••♦✇✐♥❣ ♠❡ t♦ r❡❝♦r❞ ❳✐st✴❙P❊◆ ❞②♥❛♠✐❝s ✇✐t❤ ❤✐❣❤❡r t❡♠♣♦r❛• r❡s♦•✉t✐♦♥✱ ✐♥ ✐♥❞✐✈✐❞✉❛• ❝❡••s✳ ✻✳✷✳✹✳✷ ▲✐✈❡✲❝❡•• ✐♠❛❣✐♥❣ ❚♦ ❞✐r❡❝t•② ❢♦••♦✇ ❙P❊◆✱ ■ ♣❡r❢♦r♠❡❞ ❛ ❤♦♠♦③②❣♦✉s ❦♥♦❝❦✲✐♥ ♦❢ •❋P ❛t ✐ts ❈✲ t❡r♠✐♥✉s✳ ❚♦ ✈✐s✉❛•✐③❡ ❳✐st |◆❆ ✐♥ •✐✈❡ ❝❡••s✱ ❏✉•✐❛ |♦❡♥s❝❤✱ ❛ r❡s❡❛r❝❤❡r ❢r♦♠ t❤❡ •❛❜✱ ✐♥s❡rt❡❞ ❛♥ ❛rr❛② ♦❢ ❇❣• st❡♠ •♦♦♣s ✭❇❣•❙▲✱ ❬❈❤❡♥ ❡t ❛•✳✱ ✷✵✵✾(✮ ✇✐t❤✐♥ ❳✐st ❡①♦♥ ✼ ✭♦♥ t❤❡ ❞♦①✲✐♥❞✉❝✐❜•❡ ❛••❡•❡✮✱ ✐♥ ❛ ❜❛❝❦❣r♦✉♥❞ ✇❤❡r❡ ❛ ❇❣••✲♠❈❤❡rr② ❢✉s✐♦♥ ♣r♦t❡✐♥ ✐s ❝♦♥st✐t✉t✐✈❡•② ❡①♣r❡ss❡❞ ✭❋✐❣✉r❡ ✻✳✻❆✮✳ ❯♣♦♥ ❳✐st ❡①♣r❡ss✐♦♥✱ ❇❣••✲♠❈❤❡rr② ❜✐♥❞s t❤❡ ❇❣•❙▲✱ ❛••♦✇✐♥❣ ❞✐r❡❝t✱ •✐✈❡ ❝❡•• ✈✐s✉❛•✐③❛t✐♦♥ ♦❢ ❳✐st |◆❆ ♠♦•❡❝✉•❡s ✭❋✐❣✉r❡ ✻✳✻❇✮✳ ✶✶✷ |❡s✉•ts ❙P❊◆ ❢✉♥❝t✐♦♥ ❛♥❞ ♠❡❝❤❛♥✐s♠s ✐♥ ❳❈■

A-repeat

Xist

2um ❋✐❣✉r❡ ✻✳✻ ✕ ▲✐✈❡ ❝❡•• ✐♠❛❣✐♥❣ ♦❢ ❙P❊◆ ❛♥❞ ❳✐st |◆❆ ✐♥ ♠❊❙❈s✳ ✭❆✮ ❇❣•❙▲ ✐♥✲ s❡rt✐♦♥s ✇✐t❤✐♥ ❳✐st ❡①♦♥ ✼ ❛••♦✇ ❳✐st |◆❆ ✈✐s✉❛•✐③❛t✐♦♥ ✐♥ •✐✈❡ ❝❡••s t❤r♦✉❣❤ ❜✐♥❞✐♥❣ ♦❢ ❛ ❇❣••✲♠❈❤❡rr② ✢✉♦r❡s❝❡♥t ❢✉s✐♦♥ ♣r♦t❡✐♥✳ ❚❤❡ ❇❣••✲♠❈❤❡rr② ✐s ❝♦♥st✐t✉t✐✈❡•② ❡①♣r❡ss❡❞ ❢r♦♠ t❤❡ ❚✐❣r❡ •♦❝✉s✱ ✇❤✐•❡ ❙P❊◆ ✐s ❡♥❞♦❣❡♥♦✉s•② t❛❣❣❡❞ ✇✐t❤ ❛ •❋P✳ ✭❇✮ ▲✐✈❡✲❝❡•• ♠✐❝r♦s❝♦♣② s♥❛♣✲ s❤♦ts ♦❢ ❛ r❡♣r❡s❡♥t❛t✐✈❡ ❝❡•• s❤♦✇✐♥❣ ❳✐st ✭r❡❞✮ ❛♥❞ ❙P❊◆ ✭❣r❡❡♥✮ ❞②♥❛♠✐❝s ❡✈❡r② ✶✵ ♠✐♥✉t❡s ❢♦••♦✇✐♥❣ ❞♦①②❝②❝•✐♥❡✲♠❡❞✐❛t❡❞ ✐♥❞✉❝t✐♦♥ ♦❢ ❳✐st |◆❆✳ ❯s✐♥❣ t❤✐s ❝❡•• •✐♥❡✱ ❏✉•✐❛ ❛♥❞ ■ ♣❡r❢♦r♠❡❞ t✐♠❡✲•❛♣s❡ ♠✐❝r♦s❝♦♣②✱ ✐♠❛❣✐♥❣ ❝❡••s ❡✈❡r② ✶✵ ♠✐♥✉t❡s✱ ❢♦r ✹ ❤♦✉rs ❢♦••♦✇✐♥❣ ❞♦①②❝②❝•✐♥❡ ✐♥❞✉❝t✐♦♥✳ ❲❡ ❛❝q✉✐r❡❞ ❞♦③❡♥s ♦❢ ❣♦♦❞ q✉❛•✐t② ♠♦✈✐❡s✱ ❢r♦♠ ✇❤✐❝❤ ♥✉❝•❡✐ ❛♥❞ ❳✐st ❝•♦✉❞s ❝♦✉•❞ ❜❡ s❡❣♠❡♥t❡❞ ❢♦r ❡❛❝❤ t✐♠❡❢r❛♠❡✱ ❡✐t❤❡r ♠❛♥✉❛••②✱ ♦r ✇✐t❤ ♠❛❝❤✐♥❡ •❡❛r♥✐♥❣ ✉s✐♥❣ ■•❛st✐❦ ❬❇❡r❣ ❡t ❛•✳✱ ✷✵✶✾(✳ ❋♦r ❡❛❝❤ ♥✉❝•❡✉s ❛♥❞ ✇✐t❤✐♥ ❡❛❝❤ s❡❣♠❡♥t❡❞ ❝•♦✉❞ s✉r❢❛❝❡✱ ♣✐①❡• ✐♥t❡♥s✐t✐❡s ❢♦r ❜♦t❤ t❤❡ ❙P❊◆ ✭❣r❡❡♥✮ ❛♥❞ ❳✐st ✭r❡❞✮ ❝❤❛♥♥❡•s ✇❡r❡ r❡tr✐❡✈❡❞ ❛♥❞ tr❛❝❦❡❞ t❤r♦✉❣❤ t✐♠❡ ✭s❡❡ s❡❝t✐♦♥ ✸✳✶✺ ❢♦r ❢✉rt❤❡r ❞❡t❛✐•s✮✳ ❙tr✐❦✐♥❣•②✱ ❙P❊◆ ❛❝❝✉♠✉•❛t✐♦♥ ✇❛s ❞❡t❡❝t❡❞ ❛s s♦♦♥ ❛s ✭✇✐t❤✐♥ ±✶✵ ♠✐♥✉t❡s✮ ❳✐st ❞♦♠❛✐♥s ❛♣♣❡❛r❡❞ ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❊①t❡r♥❛• ❉❛t❛ ❋✐❣✳ ✷❛✲❞✮✳ ❆•t♦❣❡t❤❡r✱ t❤❡s❡ r❡s✉•ts ❞❡♠♦♥str❛t❡ t❤❛t ❙P❊◆ ✐s r❡❝r✉✐t❡❞ t♦ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡ t♦ ✐♥✐t✐❛t❡ ❣❡♥❡ s✐•❡♥❝✐♥❣ ✐♠✲ ♠❡❞✐❛t❡•② ✉♣♦♥ ❳✐st |◆❆ ✉♣r❡❣✉•❛t✐♦♥✳ ■ts s✉st❛✐♥❡❞ ❛❝❝✉♠✉•❛t✐♦♥ ❛r♦✉♥❞ t❤❡ ❳✐ ✐♥ ◆P❈s ✐s ❝♦♥s✐st❡♥t ✇✐t❤ ✐ts r♦•❡ ✐♥ ❜✉✛❡r✐♥❣ tr❛♥s❝r✐♣t✐♦♥ ♦❢ ❡s❝❛♣✐♥❣ ❣❡♥❡s✳ ✶✶✸ |❡s✉•ts ❙P❊◆ ❢✉♥❝t✐♦♥ ❛♥❞ ♠❡❝❤❛♥✐s♠s ✐♥ ❳❈■ ✻✳✸ •♦❛• ✷✿ •❛✐♥✐♥❣ ♠❡❝❤❛♥✐st✐❝ ✐♥s✐❣❤ts ✐♥t♦ ❙P❊◆ ❢✉♥❝t✐♦♥ ❞✉r✐♥❣ ❳❈■ ❍❛✈✐♥❣ ❡st❛❜•✐s❤❡❞ t❤❛t ❙P❊◆ ✐s ❛♥ ❡ss❡♥t✐❛• ♣•❛②❡r ❢♦r ❝❤r♦♠♦s♦♠❡✲✇✐❞❡ ❣❡♥❡ s✐✲ •❡♥❝✐♥❣ ❞✉r✐♥❣ ✐♥✐t✐❛t✐♦♥ ✭❜✉t ♥♦t ♠❛✐♥t❡♥❛♥❝❡✮ ♦❢ ❳❈■✱ ■ ♥❡①t s♦✉❣❤t t♦ ❣❛✐♥ ❢✉rt❤❡r ♠❡❝❤❛♥✐st✐❝ ✐♥s✐❣❤t ✐♥t♦ ❤♦✇ ❙P❊◆ ❝❛rr✐❡s ♦✉t s✉❝❤ ❢✉♥❝t✐♦♥✳ ❚❤❡ s❡❝♦♥❞ •✐♥❡ ♦❢ ❡①♣❡r✐♠❡♥ts ■ s❡t ✉♣ ✇❡r❡ ❛✐♠❡❞ t♦ ✐❞❡♥t✐❢② ✭❋✐❣✉r❡ ✻✳✶✮✿ ✭✐✈✮ ❲❤❛t ❛r❡ t❤❡ ❣❡♥♦♠✐❝ t❛r❣❡ts ✕ ✐❢ ❛♥② ✕ ♦❢ ❙P❊◆ ❞✉r✐♥❣ ✐♥✐t✐❛t✐♦♥ ♦❢ ❳❈■✳ ✭✈✮ ❲❤✐❝❤ ❞♦♠❛✐♥s ♦❢ ❙P❊◆ ♠❡❞✐❛t❡ ✐ts ❢✉♥❝t✐♦♥✱ ❛♥❞ ✇❤✐❝❤ ♣r♦t❡✐♥ ♣❛rt♥❡rs ❞♦ t❤❡② ✐♥t❡r❛❝t ✇✐t❤✳ ✻✳✸✳✶ Pr♦✜•✐♥❣ ❙P❊◆ ❝❤r♦♠❛t✐♥ ❜✐♥❞✐♥❣ ❞✉r✐♥❣ ❳❈■ ❚❤❡ r❡♠❛r❦❛❜•❡ ❛❝❝✉♠✉•❛t✐♦♥ ♦❢ ❙P❊◆ t♦ t❤❡ ✐♥❛❝t✐✈❛t✐♥❣ ❳ ❝❤r♦♠♦s♦♠❡ ✉♣♦♥ ❳✐st ✉♣r❡❣✉•❛t✐♦♥ ♦❜s❡r✈❡❞ ❜② ♠✐❝r♦s❝♦♣② s✉❣❣❡st❡❞ t❤❛t ✐❞❡♥t✐❢②✐♥❣ t❤❡ ❣❡♥♦♠✐❝ r❡❣✐♦♥s ❙P❊◆ t❛r❣❡ts t❤r♦✉❣❤♦✉t ❳❈■ ❝♦✉•❞ ❤❡•♣ ✉♥r❛✈❡• ❤♦✇ ✐t ❝♦♥tr✐❜✉t❡s t♦ ❣❡♥❡ s✐•❡♥❝✐♥❣✳ ❈•❛ss✐❝❛••②✱ ♦♥❡ ✇♦✉•❞ ♣❡r❢♦r♠ ❈❤■P✲s❡q ❛❣❛✐♥st ❙P❊◆ t♦ ✐❞❡♥t✐❢② ✐ts ❣❡♥♦♠✐❝ t❛r❣❡ts✳ ❍♦✇❡✈❡r✱ ✇❤❡t❤❡r ❙P❊◆ ❣❡♥✉✐♥❡•② ❜✐♥❞s ❉◆❆✴❝❤r♦♠❛t✐♥ ❞✐r❡❝t•② ✐s ✉♥❦♥♦✇♥✱ ❛♥❞ ❛•• ♠② ❛tt❡♠♣ts t♦ ✐♠♠✉♥♦♣r❡❝✐♣✐t❛t❡ ✐t✱ ❡✐t❤❡r ✉♥❞❡r ♥❛t✐✈❡ ♦r ❝r♦ss✲•✐♥❦❡❞ ❝♦♥❞✐t✐♦♥s✱ ❛♥❞ ✉s✐♥❣ ❛ ✇✐❞❡ ❛rr❛② ♦❢ ❛✣♥✐t② ❡♣✐t♦♣❡s ✭❍❆✱ •❋P✱ ❋•❛❣✱ ❍❛•♦❚❛❣✮ ✇❡r❡ ♥♦t s✉❝❝❡ss❢✉•✳ ❋✉rt❤❡r♠♦r❡✱ ✐♥ ❛ ❝•❛ss✐❝❛• ❈❤■P ♣r♦t♦❝♦•✱ ❝❤r♦♠❛t✐♥ ❢r❛❣♠❡♥t❛t✐♦♥ ✭t②♣✐❝❛••② ❛❝❤✐❡✈❡❞ ✉s✐♥❣ s♦♥✐❝❛t✐♦♥✮ ✇♦✉•❞ ♠❛•♦r•② ❞✐sr✉♣t t❤❡ ❳✐st |◆❆ ❞♦♠❛✐♥✱ ❛♥❞ ♣♦t❡♥t✐❛••② •❡❛❞ t♦ ❛ ♠❛•♦r •♦ss ✐♥ t❤❡ ❙P❊◆ ❢r❛❝t✐♦♥ ❡♥r✐❝❤❡❞ ♦♥ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡✳ ✻✳✸✳✶✳✶ ❆❞❛♣t✐♥❣ ❈❯❚✫|❯◆ ❢♦r ❙P❊◆ ❛♥❞ ❳❈■ ❆t t❤❛t t✐♠❡ ♦❢ ♠② P❤❉✱ ❛ ♥♦✈❡• ❝❤r♦♠❛t✐♥ ♣r♦✜•✐♥❣ str❛t❡❣②✱ ✇❤✐❝❤ ✐♥✈♦•✈❡❞ ♥❡✐t❤❡r ✐♠♠✉♥♦♣r❡❝✐♣✐t❛t✐♦♥ ♥♦r ❝❤r♦♠❛t✐♥ ❢r❛❣♠❡♥t❛t✐♦♥✱ ✇❛s ❞❡✈❡•♦♣❡❞ ❜② t❤❡ •❛❜ ♦❢ ❙t❡✈❡ ❍❡♥✐❦♦✛ ❬❙❦❡♥❡ ❛♥❞ ❍❡♥✐❦♦✛✱ ✷✵✶✼(✳ ❚❤✐s ♠❡t❤♦❞✱ ❝❛••❡❞ ❈❯❚✫|❯◆ ✭❝•❡❛✈❛❣❡ ✉♥❞❡r t❛r❣❡ts ❛♥❞ r❡•❡❛s❡ ✉s✐♥❣ ♥✉❝•❡❛s❡✮✱ ✐s ♣❡r❢♦r♠❡❞ ✐♥ s✐t✉✱ ♦♥ ✐♥t❛❝t ❝❡••s ✭❋✐❣✉r❡ ✻✳✼❆✮✿ ❋♦••♦✇✐♥❣ ❣❡♥t•❡ ♠❡♠❜r❛♥❡ ♣❡r♠❡❛❜✐•✐③❛t✐♦♥✱ ❝❡••s ❛r❡ ✐♥❝✉❜❛t❡❞ ✇✐t❤ ❛♥ ❛♥t✐❜♦❞② ❛❣❛✐♥st ❛♥② ❝❤r♦♠❛t✐♥✲❛ss♦❝✐❛t❡❞ ❢❛❝t♦r ♦❢ ✐♥t❡r❡st✳ ❚❤❡♥✱ ❛ ♣❆✲▼◆❛s❡ ❢✉s✐♦♥ ♣r♦t❡✐♥ ✐s t❡t❤❡r❡❞ t♦ t❤❡ ❛♥t✐❜♦❞②✴❝❤r♦♠❛t✐♥✲❛ss♦❝✐❛t❡❞ ❢❛❝t♦r ❝♦♠♣•❡① ✭t❤r♦✉❣❤ ✐ts ♣r♦t❡✐♥ ❆ ♠♦✐❡t②✮✳ ❯♣♦♥ ❛❝t✐✈❛t✐♦♥ ✇✐t❤ ❞✐✈❛•❡♥t ❝❛t✐♦♥s ✭❡✳❣✳ ❈❛ ✷✰ ✮✱ t❤❡ ▼◆❛s❡ ♠♦✐❡t② ❝•❡❛✈❡s ❉◆❆ ❛r♦✉♥❞ t❤❡ tr❛♥s❝r✐♣t✐♦♥ ❢❛❝t♦r ❜✐♥❞✐♥❣ s✐t❡✱ r❡s✉•t✐♥❣ ✐♥ t❤❡ ✧r❡•❡❛s❡✧ ♦❢ t❤✐s s♣❡❝✐✜❝ ✶✶✹ |❡s✉•ts ❙P❊◆ ❢✉♥❝t✐♦♥ ❛♥❞ ♠❡❝❤❛♥✐s♠s ✐♥ ❳❈■ ❝❤r♦♠❛t✐♥ ❢r❛❣♠❡♥t✱ ✇❤✐❝❤ ❛••❡❣❡❞•② ❞✐✛✉s❡s ♦✉t ♦❢ t❤❡ ♥✉❝•❡✉s✳ ❚❤❡s❡ r❡•❡❛s❡❞ ❢r❛❣♠❡♥ts ❛r❡ t❤❡♥ r❡tr✐❡✈❡❞ ❢r♦♠ t❤❡ s✉♣❡r♥❛t❛♥t✱ ❛♥❞ s✉❜s❡q✉❡♥t ❉◆❆ s❡q✉❡♥❝✐♥❣ ❞✐r❡❝t•② r❡✈❡❛•s t❤❡ ❣❡♥♦♠✐❝
❋✐❣✉r❡ ✻✳✼ ✕ ❈r♦ss✲•✐♥❦❡❞ ❈❯❚✫|❯◆ s✉❝❝❡ss❢✉••② ♠❛♣s tr❛♥s❝r✐♣t✐♦♥ ❢❛❝t♦r ❜✐♥❞✐♥❣ s✐t❡s✳ ✭❆✮ ❙❝❤❡♠❡ ❞❡♣✐❝t✐♥❣ t❤❡ ❈❯❚✫|❯◆ ♠❡t❤♦❞♦•♦❣②✳ ■♠❛❣❡ ❞✐r❡❝t•② r❡♣r♦❞✉❝❡❞ ❢r♦♠ ❬❙❦❡♥❡ ❛♥❞ ❍❡♥✐❦♦✛✱ ✷✵✶✼(✳ ✭❇✮ ▼✐❝r♦s❝♦♣② ✐♠❛❣❡ ♦❢ ❞♦①✲✐♥❞✉❝❡❞ ❙P❊◆✲•❋P ❝❡••s ♣r♦❝❡ss❡❞ ❢♦r ♦♣t✐♠✐③❡❞ ❝r♦ss✲•✐♥❦❡❞ ❈❯❚✫|❯◆ s❤♦✇s t❤❛t ❙P❊◆ ❛❝❝✉♠✉•❛t✐♦♥ ♦♥ t❤❡ ❳✲❝❤r♦♠♦s♦♠❡ ✐s ♣r❡s❡r✈❡❞ ❢♦r ✹✽ ❤♦✉rs ✉♥❞❡r s✉❝❤ ❝♦♥❞✐t✐♦♥s✳ ✭❈✮ ❯❈❙❈ ❣❡♥♦♠❡ ❜r♦✇s❡r tr❛❝❦s s❤♦✇ t❤❛t ❝r♦ss✲•✐♥❦❡❞ ❈❯❚✫|❯◆ ❢❛✐t❤❢✉••② ♠❛♣s ❈❚❈❋ ❜✐♥❞✐♥❣ s✐t❡s ✐♥ ♠❊❙❈s✳ ❆•• tr❛❝❦s ❛r❡ s❝❛•❡❞ ✐❞❡♥t✐❝❛••②✱ ❛♥❞ ❛ ❝•✉st❡r ♦❢ ❍♦① ❣❡♥❡s ✐s s❤♦✇♥ ❛s ❛♥ ❡①❛♠♣•❡✳ P❡r❢♦r♠❡❞ ✐♥ s✐t✉✱ t❤✐s ♠❡t❤♦❞ ✇♦✉•❞ ✐♥ ♣r✐♥❝✐♣•❡ ❜❡ ♠♦r❡ s✉✐t❛❜•❡ t❤❛♥ ❈❤■P t♦ ♣r❡s❡r✈❡ t❤❡ ❳✐st ✴❙P❊◆ ♥✉❝•❡❛r ❞♦♠❛✐♥ ✐♥t❛❝t✳ ❍♦✇❡✈❡r✱ ❞♦♠❛✐♥s ♦❢ ❳✐st |◆❆ ❛r❡ r❡❛❞✐•② ❞❡st❛❜✐•✐③❡❞ ❢♦••♦✇✐♥❣ ♣❡r♠❡❛❜✐•✐③❛t✐♦♥✱ ✇✐t❤ ♠❛•♦r |◆❆ ❞❡❣r❛❞❛t✐♦♥ ♦❝❝✉rr✐♥❣✳ •✐✈❡♥ t❤❛t ❙P❊◆✬s ❝❤r♦♠❛t✐♥ ❡♥r✐❝❤♠❡♥t ❞❡♣❡♥❞s ♦♥ ❳✐st |◆❆✱ ■ ♦♣t❡❞ t♦ ♣❡r❢♦r♠ ❈❯❚✫|❯◆ ✉♥❞❡r ❝r♦ss✲•✐♥❦❡❞ ❝♦♥❞✐t✐♦♥s✱ ✇❤✐❝❤ ✇♦✉•❞ ❡♥s✉r❡ st❛❜✐•✐③❛t✐♦♥ ♦❢ ❙P❊◆ ❞♦♠❛✐♥s✳ ✶✶✺ |❡s✉•ts ❙P❊◆ ❢✉♥❝t✐♦♥ ❛♥❞ ♠❡❝❤❛♥✐s♠s ✐♥ ❳❈■ ❲✐t❤ ♦♣t✐♠✐③❛t✐♦♥✱ ■ ✐❞❡♥t✐✜❡❞ ❝r♦ss✲•✐♥❦✐♥❣ ❛♥❞ ♣❡r♠❡❛❜✐•✐③❛t✐♦♥ ❝♦♥❞✐t✐♦♥s t❤❛t ♣r❡✲ s❡r✈❡❞ ❙P❊◆ ❛❝❝✉♠✉•❛t✐♦♥ t❤r♦✉❣❤♦✉t t❤❡ ❈❯❚✫|❯◆ ♣r♦❝❡❞✉r❡✱ ❢♦r ✉♣ t♦ ✹✽ ❤♦✉rs ❢♦••♦✇✐♥❣ t✐♠❡ ♦❢ ✜①❛t✐♦♥ ✭❋✐❣✉r❡ ✻✳✼❇✮✳ ❯♥❞❡r t❤❡s❡ ❝♦♥❞✐t✐♦♥s✱ ■ ✜rst ♣❡r❢♦r♠❡❞ ❈❯❚✫|❯◆ ❛❣❛✐♥st t❤❡ tr❛♥s❝r✐♣t✐♦♥ ❢❛❝t♦r ❈❚❈❋✱ ✇❤♦s❡ ❣❡♥♦♠✐❝ ❜✐♥❞✐♥❣ s✐t❡s ✐♥ ♠❊❙❈s ❤❛✈❡ ❜❡❡♥ ❝❤❛r❛❝t❡r✐③❡❞ ❡①t❡♥s✐✈❡•②✳ ■♠✲ ♣♦rt❛♥t•②✱ ■ ♠❛❞❡ ✉s❡ ♦❢ ❛ ♣✉❜•✐s❤❡❞ ❈❚❈❋✲•❋P ❛✉①✐♥✲❞❡❣r♦♥ ♠❊❙❈ •✐♥❡ ❬◆♦r❛ ❡t ❛•✳✱ ✷✵✶✼(✱ ♣❡r❢♦r♠✐♥❣ ❈❯❚✫|❯◆ ✇✐t❤ ❛ •❋P ❛♥t✐❜♦❞② ✭❋✐❣✉r❡ ✻✳✼❈✮✱ ❛s ■ ✇♦✉•❞ t❤❡♥ ♣❡r❢♦r♠ ❈❯❚✫|❯◆ ♦♥ ❛ ❙P❊◆✲•❋P ♠❊❙❈ •✐♥❡ ✇✐t❤ t❤❛t s❛♠❡ ❛♥t✐❜♦❞②✳ |❡♠❛r❦❛❜•②✱ ❈❚❈❋ ❜✐♥❞✐♥❣ ♣r♦✜•❡s •♦♦❦❡❞ ✈❡r② s✐♠✐•❛r ❜❡t✇❡❡♥ ♠② ❝r♦ss✲•✐♥❦❡❞ ❈❯❚✫|❯◆ ❞❛t❛s❡t ❛♥❞ ❛ ♣r❡✈✐♦✉s•② ♣✉❜•✐s❤❡❞ ❈❚❈❋ ❈❤■P✲s❡q ✐♥ ♠❊❙❈s ❬❍❛♥s❡♥ ❡t ❛•✳✱ ✷✵✶✼( ✭❋✐❣✉r❡ ✻✳✼❈✮✳ ❋✉rt❤❡r♠♦r❡✱ t❤❡ ♣r♦✜•❡s ♦❜t❛✐♥❡❞ ✉♣♦♥ ❛✉①✐♥✲♠❡❞✐❛t❡❞ ❈❚❈❋ ❞❡❣r❛❞❛t✐♦♥ ❞❡♠♦♥str❛t❡❞ t❤❛t t❤❡ s✐❣♥❛• ♦❜s❡r✈❡❞ ✐s s♣❡❝✐✜❝ t♦ •❋P✲t❛❣❣❡❞ ❈❚❈❋✱ ❛♥❞ t❤❛t t❤❡ •❋P ❛♥t✐❜♦❞② ❝❛♥ ❜❡ ✉s❡❞ s✉❝❝❡ss❢✉••② ❢♦r ❝r♦ss✲•✐♥❦❡❞ ❈❯❚✫|❯◆✳ ✻✳✸✳✶✳✷ ❙P❊◆ ❈❯❚✫|❯◆ s❤❡❞s •✐❣❤t ♦♥ ✐ts ♠❡❝❤❛♥✐s♠ ♦❢ ❛❝t✐♦♥ ❞✉r✐♥❣ ❳❈■ ■ ❤❡♥❝❡ ♣❡r❢♦r♠❡❞ ❝r♦ss✲•✐♥❦❡❞ ❈❯❚✫|❯◆ ❛❣❛✐♥st ❙P❊◆✱ ❞✉r✐♥❣ ❛ t✐♠❡✲❝♦✉rs❡ ♦❢ ❳✐st ✐♥❞✉❝t✐♦♥ ✭✵❤✱ ✹❤✱ ✽❤ ❛♥❞ ✷✹❤ ❞♦① ❛s ✇❡•• ❛s ✽❤ ❞♦①✰❛✉①✐♥ ❛s ❛ ♥❡❣❛t✐✈❡ ❝♦♥tr♦•✮ ✐♥ ✷ ❜✐♦•♦❣✐❝❛• r❡♣•✐❝❛t❡s✳ ❙❡✈❡r❛• r❡♠❛r❦❛❜•❡ ♦❜s❡r✈❛t✐♦♥s ❡♠❡r❣❡❞ ❢r♦♠ t❤✐s ❡①♣❡r✐♠❡♥t✿ ❋✐rst✱ ✐♥ t❤❡ ❛❜s❡♥❝❡ ♦❢ ❳✐st ✉♣r❡❣✉•❛t✐♦♥✱ ❙P❊◆ s❤♦✇s ✈❡r② ❢❡✇ ❜✐♥❞✐♥❣ s✐t❡s ❛❝r♦ss t❤❡ ❣❡♥♦♠❡ ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❊①t❡r♥❛• ❉❛t❛ ❋✐❣✳ ✷❛✮✱ s✉❣❣❡st✐♥❣ t❤❛t ❙P❊◆ ❞♦❡s ♥♦t r❡❛❞✐•② ❛ss♦❝✐❛t❡ ✇✐t❤ ❝❤r♦♠❛t✐♥ ✐♥ ♠❊❙❈s✳ ❚❤✐s ✐s ❝♦♥s✐st❡♥t ✇✐t❤ ♦✉r ♦❜s❡r✈❛t✐♦♥ t❤❛t ✈❡r② ❢❡✇ ❣❡♥❡s ❛r❡ ❞✐✛❡r❡♥t✐❛••② ❡①♣r❡ss❡❞ ❢♦••♦✇✐♥❣ ❙P❊◆ ❞❡♣•❡t✐♦♥ ✐♥ ❙P❊◆✲❞❡❣r♦♥ ♠❊❙❈s✳ ❯♣♦♥ ❳✐st |◆❆ ❝♦❛t✐♥❣ ✭❛s ❡❛r•② ❛s ✹ ❤♦✉rs ❢♦••♦✇✐♥❣ ❞♦①✲♠❡❞✐❛t❡❞ ✐♥❞✉❝t✐♦♥✮✱ s❡✈✲ ❡r❛• ❤✉♥❞r❡❞s ♦❢ ❙P❊◆ ❜✐♥❞✐♥❣ s✐t❡s ❛♣♣❡❛r s♣❡❝✐✜❝❛••② ♦♥ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡ ✉♥❞❡r❣♦✲ ✐♥❣ ✐♥❛❝t✐✈❛t✐♦♥ ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✹❛ ❛♥❞ ❊①t❡r♥❛• ❉❛t❛ ❋✐❣✳ ✹❛✮✱ ❝♦♥✜r♠✐♥❣ t❤❛t ❙P❊◆ ❛ss♦❝✐❛t✐♦♥ ✇✐t❤ ❳✲•✐♥❦❡❞ ❝❤r♦♠❛t✐♥ ✐s ❞❡♣❡♥❞❡♥t ♦♥ ❳✐st |◆❆✳ ❋✉rt❤❡r♠♦r❡✱ t❤❡ ❡♥r✐❝❤♠❡♥t ♦❢ ❙P❊◆✱ ❡❛r•② ❛❢t❡r ❳✐st ❝♦❛t✐♥❣✱ ✐s ❤✐❣❤❡st ✇✐t❤✐♥ ❳✐st ❡♥tr② s✐t❡s ✭s❡❡ s❡❝t✐♦♥ ✶✳✺✮✱ ❤✐♥t✐♥❣ t❤❛t ❙P❊◆ ❡♥r✐❝❤♠❡♥t ♦♥ ❳✲•✐♥❦❡❞ ❝❤r♦♠❛t✐♥ ❢♦••♦✇s t❤❡ s♣❛t✐❛• ❞②♥❛♠✐❝s ♦❢ ❳✐st ❝♦❛t✐♥❣ ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✹❡✮✳ ❆ str♦♥❣ ❡♥r✐❝❤♠❡♥t ♦❢ ❙P❊◆ ✐s ♦❜s❡r✈❡❞ ❛❝r♦ss ❛ •❛r❣❡ ❢r❛❝t✐♦♥ ♦❢ t❤❡ ❳✐st ❣❡♥❡ ❜♦❞②✱ ♠♦st•② ❛t t❤❡ ✺✬ ❤❛•❢ ♦❢ t❤❡ tr❛♥s❝r✐♣t✐♦♥ ✉♥✐t ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✹❜✮✳ •✐✈❡♥ t❤❛t t❤❡ ❆✲r❡♣❡❛t ♦❢ ❳✐st ✕ •♦❝❛t❡❞ ❛t t❤❡ ✈❡r② ❜❡❣✐♥♥✐♥❣ ♦❢ t❤❡ tr❛♥s❝r✐♣t ✕ ❞✐r❡❝t•② ❜✐♥❞s ❙P❊◆ ✐♥ ✈✐tr♦ ❬▼♦♥❢♦rt ❡t ❛•✳✱ ✷✵✶✺(✱ ■ ✐♥t❡r♣r❡t t❤✐s ❞✐✛✉s❡ ❈❯❚✫|❯◆ ♣r♦✜•❡ ❛s t❤❡ ▼◆❛s❡ ❢♦♦t♣r✐♥t r❡s✉•t✐♥❣ ❢r♦♠ ❙P❊◆ ❜✐♥❞✐♥❣ t♦ ♥❛s❝❡♥t ❳✐st |◆❆ tr❛♥s❝r✐♣ts ✶✶✻ |❡s✉•ts ❙P❊◆ ❢✉♥❝t✐♦♥ ❛♥❞ ♠❡❝❤❛♥✐s♠s ✐♥ ❳❈■ ✭❋✐❣✉r❡ ✻✳✽❇✮✳ ❙✉❝❤ ❝♦♥❝•✉s✐♦♥ ✐s ❝♦♥s✐st❡♥t ✇✐t❤ t❤❡ ♠✐❝r♦s❝♦♣②✲❜❛s❡❞ ♦❜s❡r✈❛t✐♦♥ t❤❛t ❙P❊◆ ✐s r❡❝r✉✐t❡❞ t♦ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡ ✐♠♠❡❞✐❛t❡•② ✉♣♦♥ ❳✐st ✉♣r❡❣✉•❛t✐♦♥ ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❊①t❡r♥❛• ❉❛t❛ ❋✐❣✳ ✷❛✲❞✮✳ ❆♣❛rt ❢r♦♠ ❳✐st✱ ♥♦ ♦t❤❡r ❳✲•✐♥❦❡❞ •♦❝✐ s❤♦✇ s✉❝❤ ♣❡❝✉•✐❛r ❞✐✛✉s❡ ❜✐♥❞✐♥❣ ♣❛tt❡r♥✳ ■♥st❡❛❞✱ ❙P❊◆ s❤♦✇s ❢♦❝❛• ❛♥❞ ❞✐s❝r❡t❡ ❡♥r✐❝❤♠❡♥t✱ s♣❡❝✐✜❝❛••② t❛r❣❡t❡❞ t♦ ❣❡♥❡ ♣r♦♠♦t✲ ❡rs ❛♥❞ ❡♥❤❛♥❝❡rs ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✹❝✱ ❞✱ ❊①t❡r♥❛• ❉❛t❛ ❋✐❣✳ ✹❝✱ ❛♥❞ ❊①t❡r♥❛• ❉❛t❛ ❋✐❣✳ ✺❛✲❣✮✳ ❙tr✐❦✐♥❣•②✱ ❙P❊◆ s♣❡❝✐✜❝❛••② t❛r❣❡ts ♣r♦♠♦t❡rs ♦❢ ✐♥✐t✐❛••② ❛❝t✐✈❡ ❣❡♥❡s ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✹❢ ❛♥❞ ❊①t❡r♥❛• ❉❛t❛ ❋✐❣✳ ✹❞✮✳ ❈♦♥s✐st❡♥t•②✱ ❳✲•✐♥❦❡❞ ❣❡♥❡s ✇❤✐❝❤ ❛r❡ ♠♦st ❞❡♣❡♥❞❡♥t ♦♥ ❙P❊◆ ❢♦r s✐•❡♥❝✐♥❣ ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✶❡✮ ✕ ❡①♣❡❝t❡❞•② s❤♦✇✐♥❣ ❤✐❣❤✲ ❡st •❡✈❡•s ♦❢ ❙P❊◆ r❡❝r✉✐t♠❡♥t ❛t t❤❡✐r ♣r♦♠♦t❡rs ✕ ❛r❡ ✐♥ ❢❛❝t ♠♦r❡ ❤✐❣❤•② ❡①♣r❡ss❡❞ ✐♥✐t✐❛••② t❤❛♥ ❣❡♥❡s ✇❤✐❝❤ ❛r❡ •❡ss ❞❡♣❡♥❞❡♥t ♦♥ ❙P❊◆ ❢♦r t❤❡✐r s✐•❡♥❝✐♥❣ ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✹❣✱ ❛♥❞ ❊①t❡r♥❛• ❉❛t❛ ❋✐❣✳ ✹❡✮✳ ❚❛❦❡♥ t♦❣❡t❤❡r✱ t❤❡s❡ ♦❜s❡r✈❛t✐♦♥s str♦♥❣•② s✉❣❣❡st t❤❛t ❛❝t✐✈❡ tr❛♥s❝r✐♣t✐♦♥ ♣r♦✈✐❞❡s ❛ ❢❛✈♦r❛❜•❡ ❝♦♥t❡①t ❢♦r ❙P❊◆ r❡❝r✉✐t♠❡♥t t♦ ❝❤r♦♠❛t✐♥ ✭❋✐❣✉r❡ ✻✳✽❈✮✱ ✇❤✐❝❤ ✐s ❢✉rt❤❡r ❞✐s❝✉ss❡❞ ✐♥ s✉❜s❡❝t✐♦♥ ✾✳✶✳✷ ❛♥❞ s✉❜s✉❜✲ s❡❝t✐♦♥ ✾✳✷✳✷✳✸✳ ❆♥♦t❤❡r ✐♠♣♦rt❛♥t ♦❜s❡r✈❛t✐♦♥ ✐s t❤❛t t❤❡ •❡✈❡•s ♦❢ ❡❛r•② ❙P❊◆ r❡❝r✉✐t♠❡♥t ❛t ❳✲ •✐♥❦❡❞ ♣r♦♠♦t❡rs ✭✇✐t❤✐♥ ✹ ❤♦✉rs ♦❢ ❳✐st ❝♦❛t✐♥❣✮ ❛r❡ ❤✐❣❤❡r ❢♦r ❣❡♥❡s ✇❤✐❝❤ ❛r❡ ❡✣❝✐❡♥t•② ✭✐✳❡✳ ❡❛r•②✮ s✐•❡♥❝❡❞ t❤❛♥ t❤❡② ❛r❡ ❢♦r ❣❡♥❡s ✇❤✐❝❤ ❛r❡ •❡ss ❡✣❝✐❡♥t•② ✭✐✳❡✳ •❛t❡✮ s✐•❡♥❝❡❞ ❞✉r✐♥❣ ❳❈■ ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✹❤✮✳ ❚❤❡ s❛♠❡ tr❡♥❞ ✐s ♦❜s❡r✈❡❞ ❜❡t✇❡❡♥ ❙P❊◆ r❡❝r✉✐t♠❡♥t ❛t ❳✲•✐♥❦❡❞ ❡♥❤❛♥❝❡rs ❛♥❞ t❤❡ ❡✣❝✐❡♥❝② ✇✐t❤ ✇❤✐❝❤ t❤❡s❡ ❡♥❤❛♥❝❡rs ❛r❡ ❞❡❛❝❡t②•❛t❡❞ ❞✉r✐♥❣ ❳❈■ ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✹✐✮✳ ❋✐♥❛••②✱ ❙P❊◆ ❜✐♥❞✐♥❣ ❛t ♣r♦♠♦t❡rs ✐s •♦✇❡st ✕ ♥❛② ❛❜s❡♥t ✕ ❛t ❣❡♥❡s t❤❛t ✉♥❞❡r❣♦ ♥♦ s✐❣♥✐✜❝❛♥t s✐•❡♥❝✐♥❣ ✕ ✐❢ ♥♦t ❝♦♠♣•❡t❡ ❡s❝❛♣❡ ✕ ✐♥ ♦✉r ❳✐st✲✐♥❞✉❝✐❜•❡ s②st❡♠ ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❊①t❡r♥❛• ❉❛t❛ ❋✐❣✳ ✹❢✱ ❣✮✳ ❚❛❦❡♥ t♦❣❡t❤❡r✱ t❤❡s❡ t❤r❡❡ ♦❜s❡r✈❛t✐♦♥s str♦♥❣•② ✐♥❞✐❝❛t❡ t❤❛t ❙P❊◆ r❡❝r✉✐t♠❡♥t t♦ ♣r♦♠♦t❡rs ❛♥❞ ❡♥❤❛♥❝❡rs ✐s ❞✐r❡❝t•② r❡s♣♦♥s✐❜•❡ ❢♦r tr❛♥s❝r✐♣t✐♦♥❛• s✐•❡♥❝✐♥❣ ❞✉r✐♥❣ ❳❈■ ✭❋✐❣✉r❡ ✻✳✽❈ ❛♥❞ ❉✮✳ |❡♠❛r❦❛❜•②✱ ❙P❊◆ ❜✐♥❞✐♥❣ t♦ ❳✲•✐♥❦❡❞ ❝❤r♦♠❛t✐♥ ❞❡❝r❡❛s❡s ❞r❛st✐❝❛••② ❛❢t❡r ✷✹ ❤♦✉rs ♦❢ ❳✐st ❡①♣r❡ss✐♦♥ ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✹❛ ❛♥❞ ❊①t❡♥❞❡❞ ❉❛t❛ ❋✐❣✳ ✹❝✮✱ ❞❡s♣✐t❡ ✈❡r② str♦♥❣ ❳✐st✴❙P❊◆ ❝•♦✉❞s ❜❡✐♥❣ ♦❜s❡r✈❡❞ ❜② ♠✐❝r♦s❝♦♣② ❛t t❤❛t t✐♠❡♣♦✐♥t✳ ❚♦ ❜❡tt❡r ✉♥❞❡rst❛♥❞ t❤❡ ❜✐♦•♦❣✐❝❛• s✐❣♥✐✜❝❛t✐♦♥ ♦❢ t❤✐s ❞r♦♣ ✐♥ ❙P❊◆ ❜✐♥❞✐♥❣✱ ❳✲ •✐♥❦❡❞ ♣r♦♠♦t❡rs ✇❡r❡ ❝•✉st❡r❡❞ ❛❝❝♦r❞✐♥❣ t♦ t❤❡✐r ❞②♥❛♠✐❝s ♦❢ ❙P❊◆ ❜✐♥❞✐♥❣✴✧r❡•❡❛s❡✧ ✇✐t❤✐♥ t❤❡ ✷✹❤ t✐♠❡❝♦✉rs❡ ♦❢ ❳✐st ❝♦❛t✐♥❣ ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❊①t❡♥❞❡❞ ❉❛t❛ ❋✐❣✳ ✹✐✮✱ ❛♥❞ t❤❡ s✐•❡♥❝✐♥❣ ❞②♥❛♠✐❝s ♦❢ t❤❡✐r ❝♦rr❡s♣♦♥❞✐♥❣ ❣❡♥❡s ✇❛s ❝♦♠♣❛r❡❞ ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❊①t❡♥❞❡❞ ❉❛t❛ ❋✐❣✳ ✹•✮✿ ✶✶✼ |❡s✉•ts ❙P❊◆ ❢✉♥❝t✐♦♥ ❛♥❞ ♠❡❝❤❛♥✐s♠s ✐♥ ❳❈■ ❚❤✐s ❛♥❛•②s✐s ❞✐st✐♥❣✉✐s❤❡❞ t❤r❡❡ ❣r♦✉♣s ♦❢ ♣r♦♠♦t❡rs✿ ■♥ t❤❡ ✜rst ❣r♦✉♣✱ ❙P❊◆ ❜✐♥❞✲ ✐♥❣ ✇❛s ♠❛①✐♠❛• ✇✐t❤✐♥ ✹❤ ♦❢ ❳✐st ❝♦❛t✐♥❣ ❜✉t ❞❡❝r❡❛s❡❞ t❤❡r❡❛❢t❡r✱ ✇✐t❤ ♠❛•♦r •♦ss ♦❢ ❙P❊◆ ❜✐♥❞✐♥❣ ♦❜s❡r✈❡❞ ❜② ✷✹ ❤♦✉rs ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✹•✮✳ ■♥ t❤❡ s❡❝♦♥❞ ❣r♦✉♣ ❤♦✇✲ ❡✈❡r✱ ❙P❊◆ ❜✐♥❞✐♥❣ ✇❛s ♠❛①✐♠❛• ♦♥•② ❛❢t❡r ✽❤ ♦❢ ❳✐st ❝♦❛t✐♥❣✱ ❛♥❞ •♦ss ♦❢ ❙P❊◆ ❜✐♥❞✐♥❣ ✇❛s ♦♥•② ♠✐•❞•② ♦❜s❡r✈❡❞ ❜② ✷✹ ❤♦✉rs ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✹•✮✳ ❋✐♥❛••②✱ t❤❡ t❤✐r❞ ❣r♦✉♣✱ ✇❤✐❝❤ ❝♦♠♣r✐s❡s s✐❣♥✐✜❝❛♥t•② ❢❡✇❡r ♣r♦♠♦t❡rs t❤❛♥ t❤❡ t✇♦ ♣r❡✈✐♦✉s ♦♥❡s✱ ✇❛s ❝❤❛r❛❝t❡r✲ ✐③❡❞ ❜② s✐❣♥✐✜❝❛♥t•② r❡❞✉❝❡❞ •❡✈❡•s ♦❢ ❙P❊◆ ❜✐♥❞✐♥❣ ✭❛♥❞ •♦ss t❤❡r❡♦❢✮ t❤r♦✉❣❤♦✉t t❤❡ ❳✐st ❝♦❛t✐♥❣ t✐♠❡❝♦✉rs❡ ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✹•✮✳
❋✐❣✉r❡ ✻✳✽ ✕ ▼♦❞❡• ❢♦r ❙P❊◆ ❝❤r♦♠❛t✐♥ r❡❝r✉✐t♠❡♥t ❞✉r✐♥❣ ❳❈■✱ ❞❡r✐✈❡❞ ❢r♦♠ ❈❯❚✫|❯◆ ❛♥❛•②s✐s✳ ✭❆✮ ❳❈■ ✐♥✐t✐❛t❡s ✇✐t❤ t❤❡ ♠♦♥♦❛••❡•✐❝ ✉♣r❡❣✉•❛t✐♦♥ ♦❢ ❳✐st✳ ✭❇✮❳✐st r❡❝r✉✐ts ❙P❊◆ ✐♠♠❡❞✐❛t❡•② ❛❢t❡r ✐ts ❆✲r❡♣❡❛t ❤❛s ❜❡❡♥ tr❛♥s❝r✐❜❡❞✱ •❡❛❞✐♥❣ t♦ ❈❯❚✫|❯◆ s✐❣♥❛• ❛❝r♦ss t❤❡ ❡♥t✐r❡ ❳✐st tr❛♥s❝r✐♣t✐♦♥ ✉♥✐t t❤r♦✉❣❤♦✉t ❳❈■✳ ✭❈✮ ❖♥❝❡ ❛ss♦❝✐❛t❡❞ ✇✐t❤ ❳✐st✱ ❙P❊◆ ✐s ❣✉✐❞❡❞ t♦ ❡♥❤❛♥❝❡rs ❛♥❞ ♣r♦♠♦t❡rs ♦❢ ❛❝t✐✈❡ ❣❡♥❡s s♣❡❝✐✜❝❛••②✱ ✭❉✮ ✇❤❡r❡ ✐t s✐•❡♥❝❡s tr❛♥s❝r✐♣t✐♦♥✳ •❡♥❡ s✐•❡♥❝✐♥❣ ✭✐✳❡✳ •♦ss ♦❢ tr❛♥s❝r✐❜✐♥❣ |◆❆P■■✮ r❡s✉•ts ✐♥ ✭❊✮ ❞✐ss♦❝✐❛t✐♦♥ ♦❢ ❙P❊◆ ❢r♦♠ ❝❤r♦♠❛t✐♥ ❲❤❡♥ ❝♦♠♣❛r✐♥❣ t❤❡ ❡✣❝✐❡♥❝② ♦❢ ❣❡♥❡ s✐•❡♥❝✐♥❣ ✭❜② ✷✹❤ ♦❢ ❳✐st ❝♦❛t✐♥❣✮ ❜❡t✇❡❡♥ ❡❛❝❤ ♦❢ t❤❡ t❤r❡❡ ♣r♦♠♦t❡r✴❣❡♥❡ ❣r♦✉♣s ❛❢♦r❡♠❡♥t✐♦♥❡❞✱ t❤❡ ❣r♦✉♣ ✇❤✐❝❤ •♦st ❙P❊◆ ❜✐♥❞✐♥❣ ♠♦st ❡✣❝✐❡♥t•② s❤♦✇❡❞ str♦♥❣❡st ❣❡♥❡ s✐•❡♥❝✐♥❣ ❜② ✷✹❤ ❝♦♠♣❛r❡❞ t♦ t❤❡ t✇♦ ♦t❤❡r ❣r♦✉♣s t❤❛t s❤♦✇❡❞ ♣r♦tr❛❝t❡❞ ❙P❊◆ ❜✐♥❞✐♥❣ ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✹❦✮✳ ❚❤✐s s✉❣❣❡sts t❤❛t ✶✶✽ |❡s✉•ts ❙P❊◆ ❢✉♥❝t✐♦♥ ❛♥❞ ♠❡❝❤❛♥✐s♠s ✐♥ ❳❈■ ✉♣♦♥ ❣❡♥❡ s✐•❡♥❝✐♥❣✱ ❙P❊◆ ❜✐♥❞✐♥❣ t♦ ❝❤r♦♠❛t✐♥ ✐s ♥♦ •♦♥❣❡r ♣♦ss✐❜•❡ ✭❋✐❣✉r❡ ✻✳✽❉ ❛♥❞ ❊✮✱ ❢✉rt❤❡r s✉♣♣♦rt✐♥❣ t❤❡ ✐❞❡❛ t❤❛t ❛❝t✐✈❡ tr❛♥s❝r✐♣t✐♦♥ ✐s r❡q✉✐r❡❞ ❢♦r ❙P❊◆ t❛r❣❡t✐♥❣✳ ❆•t♦❣❡t❤❡r✱ t❤✐s ❈❯❚✫|❯◆ ❛♥❛•②s✐s ❢❛✈♦rs ❛ ♠♦❞❡• ✇❤❡r❡✐♥ ❳✐st r❡❝r✉✐ts ❙P❊◆ t♦ ❣❡♥❡ ♣r♦♠♦t❡rs ❛♥❞ ❡♥❤❛♥❝❡rs✱ ✇❤✐❝❤ s❡r✈❡ ❛s t❤❡ s✉❜str❛t❡s ❢♦r ❙P❊◆✬s s✐•❡♥❝✐♥❣ ❛❝t✐♦♥✳ ❙P❊◆ ❛ss♦❝✐❛t❡s ✇✐t❤ ❝❤r♦♠❛t✐♥ ✐♥ ❛ tr❛♥s❝r✐♣t✐♦♥✲❞❡♣❡♥❞❡♥t ♠❛♥♥❡r✱ ❛s ✐t ♦♥•② t❛r❣❡ts ❛❝t✐✈❡ ♣r♦♠♦t❡rs ❛♥❞ ❡♥❤❛♥❝❡rs✱ ❛♥❞ ❞✐s❡♥❣❛❣❡s ❢r♦♠ ❝❤r♦♠❛t✐♥ ✉♣♦♥ ❣❡♥❡ s✐•❡♥❝✐♥❣ ✭❋✐❣✉r❡ ✻✳✽✮✳ ✻✳✸✳✷ ■❞❡♥t✐❢②✐♥❣ t❤❡ ❞♦♠❛✐♥s ♦❢ ❙P❊◆ r❡q✉✐r❡❞ ❢♦r ✐ts ❢✉♥❝t✐♦♥ ❞✉r✐♥❣ ❳❈■ ❆•t❤♦✉❣❤ ❛ •♦t ♦❢ ✐♥s✐❣❤t ✐♥t♦ ❤♦✇ ❙P❊◆ ✇♦r❦s t♦ s✐•❡♥❝❡ ❣❡♥❡s ✇❛s ❣❛✐♥❡❞ ❢r♦♠ ❈❯❚✫|❯◆ ❛♥❛•②s✐s✱ t❤❡ ♣r♦t❡✐♥ ❢❛❝t♦rs ❙P❊◆ ❝♦✉•❞ ❡♥❣❛❣❡ ✇✐t❤ t♦ ♠❡❞✐❛t❡ s✉❝❤ s✐•❡♥❝✲ ✐♥❣ r❡♠❛✐♥❡❞ ✉♥❦♥♦✇♥✳ ❖r✐❣✐♥❛••②✱ ■ ❤❛❞ ♣•❛♥♥❡❞ t♦ ♣❡r❢♦r♠ ❙P❊◆ ✐♠♠✉♥♦♣r❡❝✐♣✐t❛t✐♦♥ ❢♦••♦✇❡❞ ❜② ♠❛ss s♣❡❝tr♦♠❡tr② t♦ ✉♥r❛✈❡• t❤❡ ♣r♦t❡✐♥ ♣❛rt♥❡rs ♦❢ ❙P❊◆✳ ❯♥❢♦rt✉♥❛t❡•②✱ ❛♥❞ ❛s ♠❡♥t✐♦♥❡❞ ✐♥ s✉❜s❡❝t✐♦♥ ✻✳✸✳✶✱ ❛•• ♠② ❛tt❡♠♣ts ❛t ✐♠♠✉♥♦♣r❡❝✐♣✐t❛t✐♥❣ ❙P❊◆ ✭❝❛rr②✐♥❣ ❞✐✛❡r❡♥t ❛✣♥✐t② t❛❣s✮ ✇❡r❡ ✉♥s✉❝❝❡ss❢✉•✳ ■♠♣♦rt❛♥t•②✱ ♣r♦t❡✐♥ ❡①tr❛❝t✐♦♥ ✉♥❞❡r ♥❛t✐✈❡ ❝♦♥❞✐t✐♦♥s ❝♦♥s✐st❡♥t•② r❡s✉•t❡❞ ✐♥ s✐❣♥✐✜❝❛♥t ❢✉••✲•❡♥❣t❤ ❙P❊◆ ❞❡❣r❛❞❛t✐♦♥ ✭❞❛t❛ ♥♦t s❤♦✇♥✮✱ ❢✉rt❤❡r ❝♦♠♣•✐❝❛t✐♥❣ ♠② ❛tt❡♠♣ts ❛t ❝❤❛r❛❝t❡r✐③✐♥❣ ✐ts ♣r♦t❡✐♥ ✐♥t❡r❛❝t♦♠❡✳ ✻✳✸✳✷✳✶ ❙P❊◆ ❢✉♥❝t✐♦♥❛• ❝♦♠♣•❡♠❡♥t❛t✐♦♥ ♠❡t❤♦❞♦•♦❣② ❍❡♥❝❡✱ ■ ♦♣t❡❞ t♦ ✜rst ❞❡t❡r♠✐♥❡ ✇❤✐❝❤ r❡❣✐♦♥s ♦❢ t❤❡ ❙P❊◆ ♣r♦t❡✐♥ ❛r❡ r❡q✉✐r❡❞ ❢♦r ✐ts ❣❡♥❡ s✐•❡♥❝✐♥❣ ❢✉♥❝t✐♦♥ ❞✉r✐♥❣ ❳❈■✱ ❛♥❞ ♦♥•② t❤❡♥ ❝❤❛r❛❝t❡r✐③❡ t❤❡ ♣r♦t❡✐♥ ✐♥t❡r❛❝t♦♠❡ ♦❢ t❤❡s❡ s♣❡❝✐✜❝ r❡❣✐♦♥s✳ ■ ❤❡♥❝❡ ❞❡✈❡•♦♣❡❞ ❛ str❛t❡❣② t♦ ❡①♣r❡ss ❛ ✈❛r✐❡t② ♦❢ tr✉♥❝❛t❡❞ ❙P❊◆ ♣r♦t❡✐♥s ✭❋✐❣✉r❡ ✻✳✾❆✮ ❛♥❞ ❛ss❡ss ✇❤❡t❤❡r t❤❡s❡ tr✉♥❝❛t✐♦♥s ❝♦✉•❞ ❢✉♥❝t✐♦♥❛••② r❡s❝✉❡ t❤❡ ❳❈■ ❞❡❢❡❝ts ♦❜s❡r✈❡❞ ✉♣♦♥ t❤❡ •♦ss ♦❢ ❡♥❞♦❣❡♥♦✉s ❙P❊◆ ✭❋✐❣✉r❡ ✻✳✾❇✮✳ ✶✶✾ |❡s✉•ts ❙P❊◆ ❢✉♥❝t✐♦♥ ❛♥❞ ♠❡❝❤❛♥✐s♠s ✐♥ ❳❈■
A B ❋✐❣✉r❡ ✻✳✾ ✕ ❆ ❢✉♥❝t✐♦♥❛• ❝♦♠♣•❡♠❡♥t❛t✐♦♥ str❛t❡❣② t♦ ✐❞❡♥t✐❢② t❤❡ ❞♦♠❛✐♥s ♦❢ ❙P❊◆ ✐♥✈♦•✈❡❞ ✐♥ ❳❈■✳ ✭❆✮ ❉✐✛❡r❡♥t ❙P❊◆ ❝❉◆❆ tr✉♥❝❛t✐♦♥s ❛r❡ ✭❇✮ ❦♥♦❝❦❡❞✲✐♥ ❛t |♦s❛✷✻ ❛♥❞ ❝♦♥st✐t✉t✐✈❡•② ❡①♣r❡ss❡❞ ✐♥ ❙P❊◆✲❞❡❣r♦♥ ♠❊❙❈s✳ ❯♣♦♥ ❳✐st ✉♣r❡❣✉•❛t✐♦♥ ❛♥❞ s✐♠✉•t❛♥❡♦✉s ❞❡♣•❡t✐♦♥ ♦❢ ❡♥❞♦❣❡♥♦✉s ❙P❊◆✱ t❤❡ ❛❜✐•✐t② ♦❢ ❡❛❝❤ ♦❢ t❤❡s❡ tr✉♥❝❛t✐♦♥s t♦ r❡s❝✉❡ ❞❡❢❡❝ts ✐♥ ❳✲•✐♥❦❡❞ ❣❡♥❡ s✐•❡♥❝✐♥❣ ✐s ❛ss❛②❡❞ ✉s✐♥❣ ♣②r♦s❡q✉❡♥❝✐♥❣✳ ❆•• t❤❡s❡ tr✉♥❝❛t✐♦♥s ❛r❡ ❢✉s❡❞ t♦ ❛ ✸①❋▲❆• ❡♣✐t♦♣❡ ❛t t❤❡✐r ◆✲t❡r♠✐♥✉s✱ ❛••♦✇✐♥❣ ♣r♦t❡✐♥ ❞❡t❡❝t✐♦♥ ❛♥❞ •♦❝❛•✐③❛t✐♦♥ r❡•❛t✐✈❡ t♦ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡ ❜② ✐♠♠✉♥♦✢✉♦r❡s❝❡♥❝❡✳ ❆♥ ❛❞❞✐t✐♦♥❛• ◆✲t❡r♠✐♥❛• ❙❱✲✹✵ ◆▲❙ ❡♥s✉r❡s ♥✉❝•❡❛r •♦❝❛•✐③❛t✐♦♥ ♦❢ ❛•• tr✉♥❝❛t✐♦♥s✳ ❚❤❡ ❢✉••✲•❡♥❣t❤ ❙P❊◆ ❝❉◆❆ ✇❛s ♣✉r❝❤❛s❡❞ ❢r♦♠ •❡♥s❝r✐♣t✱ ❛♥❞ ❞✐✛❡r❡♥t ❛♥♥♦t❛t❡❞ ❙P❊◆ ❞♦♠❛✐♥s ✭||▼✶✱ ||▼✷✲✹✱ |■❉ ❛♥❞ ❙P❖❈✮ ✇❡r❡ ✐♥❞✐✈✐❞✉❛••② s♣•✐❝❡❞ ♦✉t ✉s✐♥❣ ♦✈❡r•❛♣ ❡①t❡♥s✐♦♥ P❈| ✐♥ ❝♦••❛❜♦r❛t✐♦♥ ✇✐t❤ ❆❣♥ès ▲❡ ❙❛✉①✱ ❛ r❡s❡❛r❝❤ s❝✐❡♥t✐st ❢r♦♠ t❤❡ •❛❜ ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✸❛✮✳ ❆ ✸①❋▲❆• ❡♣✐t♦♣❡ ✇❛s ✐♥❝•✉❞❡❞ ❛t t❤❡ ◆✲t❡r♠✐♥✉s ♦❢ ❡❛❝❤ ❝❉◆❆ t♦ ❛••♦✇ ♣r♦t❡✐♥ ❞❡t❡❝t✐♦♥ ✭❜♦t❤ ❜② ✇❡st❡r♥ ❜•♦t ❛♥❞ ✐♠♠✉♥♦✢✉♦r❡s❝❡♥❝❡✮✳ ❋✉rt❤❡r♠♦r❡✱ ❛♥ ✧❛rt✐✜❝✐❛•✧ ❙❱✲✹✵ ♥✉❝•❡❛r • ♦❝❛•✐③❛t✐♦♥ s✐❣♥❛• ✭◆▲❙✮ ✇❛s ❛•s♦ ❛❞❞❡❞ t♦ ❝♦♠♣❡♥s❛t❡ ❢♦r t❤❡ r❡♠♦✈❛• ♦❢ ❛♥② ❡♥❞♦❣❡♥♦✉s ◆▲❙s ✐♥ t❤❡ ❣❡♥❡r❛t❡❞ ❙P❊◆ tr✉♥❝❛t✐♦♥s ✭❋✐❣✉r❡ ✻✳✾❆✮✳ ■ t❤❡♥ ❦♥♦❝❦❡❞✲✐♥ ❡❛❝❤ ♦❢ t❤❡s❡ tr✉♥❝❛t✐♦♥s ❛t |♦s❛✷✻ ¶ ¶ ✐♥ ❛♥ ❡♥❞♦❣❡♥♦✉s ❙P❊◆✲ ❞❡❣r♦♥ ♠❊❙❈ ❜❛❝❦❣r♦✉♥❞ ✭❋✐❣✉r❡ ✻✳✾❇✮✱ ❛♥❞ ❝♦♥✜r♠❡❞ t❤❡✐r ❝♦♥st✐t✉t✐✈❡ ❡①♣r❡ss✐♦♥ ✉s✐♥❣ ✇❡st❡r♥ ❜•♦t ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❊①t❡♥❞❡❞ ❉❛t❛ ❋✐❣✳ ✸❜✮✳ ❋♦r ❡❛❝❤ tr✉♥❝❛t✐♦♥✱ ✷ ♦r ✸ ✐♥❞❡♣❡♥❞❡♥t ♠❊❙❈ ❝•♦♥❡s ✇❡r❡ ❣❡♥❡r❛t❡❞ ❛♥❞ t❤❡ ❛❜✐•✐t② ♦❢ ❡❛❝❤ tr✉♥❝❛t✐♦♥ t♦ r❡s❝✉❡ ❳✐st✲♠❡❞✐❛t❡❞ ❣❡♥❡ s✐•❡♥❝✐♥❣ ✉♣♦♥ ❞❡♣•❡t✐♦♥ ♦❢ ❡♥❞♦❣❡♥♦✉s ❙P❊◆ ✇❛s ❛ss❛②❡❞ ✉s✐♥❣ ♣②r♦s❡q✉❡♥❝✐♥❣ ♦❢ ✹ ❳✲•✐♥❦❡❞ ❣❡♥❡s ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✸❜✮✳ ❋✉rt❤❡r♠♦r❡✱ t❤❡ •♦❝❛•✐③❛t✐♦♥ ♦❢ ❡❛❝❤ ❙P❊◆ tr✉♥❝❛t✐♦♥ r❡•❛t✐✈❡ t♦ t❤❡ ✐♥❛❝t✐✈❡ ❳ ❝❤r♦♠♦s♦♠❡ ✇❛s ❛ss❛②❡❞ ✉s✐♥❣ ✐♠♠✉♥♦✢✉♦r❡s❝❡♥❝❡ ✭❍✷❆❑✶✶✾✉❜✶ ✇❛s ✉s❡❞ ❛s ❛ ♠❛r❦❡r ♦❢ t❤❡ ❳✐✱ s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✸❝✮✳ ¶ ¶ ❙♦♠❡ ♦❢ t❤❡s❡ ❦♥♦❝❦✲✐♥ ✧r❡s❝✉❡✧ ❝♦♥str✉❝ts ✇❡r❡ s❤❛r❡❞ ✇✐t❤ t❤❡ •r✐❜♥❛✉ •❛❜ ✐♥ t❤❡ ❝♦♥t❡①t ♦❢ ❛ ♣r♦•❡❝t ✐♥✈❡st✐❣❛t✐♥❣ t❤❡ ❢✉♥❝t✐♦♥ ♦❢ ❙P❊◆ ✐♥ r❡❣✉•❛t✐♥❣ t❤❡ ❡①♣r❡ss✐♦♥ ♦❢ ❳✐st |◆❆✳ ❚❤❡ r❡s✉•ts ❢r♦♠ t❤✐s ♣r♦•❡❝t ❤❛✈❡ ❜❡❡♥ s❤❛r❡❞ ♦♥ t❤❡ ❜✐♦|①✐✈ ♣r❡♣r✐♥t s❡r✈❡r ❬|♦❜❡rt✲❋✐♥❡str❛ ❡t ❛•✳✱ ✷✵✷✵(✱ ❛♥❞ ❛r❡ ❛♣♣❡♥❞❡❞ ❛t s❡❝t✐♦♥ ❆✳✷✳ ✶✷✵ |❡s✉•ts ❙P❊◆ ❢✉♥❝t✐♦♥ ❛♥❞ ♠❡❝❤❛♥✐s♠s ✐♥ ❳❈■ ✻✳✸✳✷✳✷ ❙P❊◆✬s ||▼✷✲✹ ❛♥❞ ❙P❖❈ ❞♦♠❛✐♥s ❛♣♣❡❛r ✈✐t❛• ❢♦r ❳❈■ ❚❤✐s ❛♥❛•②s✐s r❡✈❡❛•❡❞ t❤❛t t❤❡ ||▼✶ ❛♥❞ |■❉ ❞♦♠❛✐♥s ♦❢ ❙P❊◆ ❛r❡ ❞✐s♣❡♥s❛❜•❡ ❢♦r ❙P❊◆ r❡❝r✉✐t♠❡♥t t♦ t❤❡ ❳✐ ❛s ✇❡•• ❛s ❢♦r ❣❡♥❡ s✐•❡♥❝✐♥❣ ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✸❜✱ ❝✮✳ ▲♦ss ♦❢ ||▼✷✲✹ ❤♦✇❡✈❡r r❡s✉•t❡❞ ✐♥ ❢❛✐•✉r❡ t♦ r❡❝r✉✐t ❙P❊◆ t♦ t❤❡ ❳✐ ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✸❜✱ ❝✮✱ ❛ ✜♥❞✐♥❣ ✇❤✐❝❤ ✐s ❝♦♥s✐st❡♥t ✇✐t❤ ♣r❡✈✐♦✉s r❡♣♦rts t❤❛t ||▼✷✲✹ ❞✐r❡❝t•② ❜✐♥❞ ❳✐st ✐♥ ✈✐tr♦ ❬▼♦♥❢♦rt ❡t ❛•✳✱ ✷✵✶✺❀ ▲✉ ❡t ❛•✳✱ ✷✵✶✻(✳ ❋✉rt❤❡r♠♦r❡✱ t❤✐s tr✉♥❝❛t✐♦♥ ❢❛✐•❡❞ t♦ r❡s❝✉❡ ❣❡♥❡ s✐•❡♥❝✐♥❣ ✉♣♦♥ •♦ss ♦❢ ❡♥❞♦❣❡♥♦✉s ❙P❊◆ ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✸❜✱ ❝✮✱ ❢✉rt❤❡r ❝♦♥✜r♠✐♥❣ t❤❛t ❛❝t✐✈❡ ❳✐st ♠❡❞✐❛t❡❞ r❡❝r✉✐t♠❡♥t ♦❢ ❙P❊◆ ✭t❤r♦✉❣❤ ✐ts ||▼✷✲✹ ❞♦♠❛✐♥s✮ ✐s r❡q✉✐r❡❞ ❢♦r ❳ ✐♥❛❝t✐✈❛t✐♦♥✳ ■♠♣♦rt❛♥t•②✱ ✇❤❡♥ ❙P❊◆ ✇❛s tr✉♥❝❛t❡❞ ♦❢ ✐ts ❙P❖❈ ❞♦♠❛✐♥✱ ✐t r❡t❛✐♥❡❞ ✐ts ❛❜✐•✐t② t♦ ❛❝❝✉♠✉•❛t❡ ❛r♦✉♥❞ t❤❡ ❳✐✱ ❜✉t ❢❛✐•❡❞ t♦ r❡s❝✉❡ t❤❡ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❞❡❢❡❝ts ❛ss♦❝✐❛t❡❞ ✇✐t❤ •♦ss ♦❢ ❡♥❞♦❣❡♥♦✉s ❙P❊◆ ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✸❜✱ ❝✮✳ ✻✳✸✳✷✳✸ ❊♥❞♦❣❡♥♦✉s ❙P❖❈✲❞❡•❡t✐♦♥ ❝♦♥✜r♠s ✐ts ❡ss❡♥t✐❛•✐t② ✐♥ ❳❈■ ■ ❢✉rt❤❡r ✈❛•✐❞❛t❡❞ t❤✐s ♦❜s❡r✈❛t✐♦♥ ❜② ♣❡r❢♦r♠✐♥❣ ❛ ❤♦♠♦③②❣♦✉s ❞❡•❡t✐♦♥ ♦❢ t❤❡ ❙P❖❈ ❞♦♠❛✐♥ ❛t t❤❡ ❡♥❞♦❣❡♥♦✉s ❙♣❡♥ •♦❝✉s ✕ r❡♣•❛❝✐♥❣ ✐t ✇✐t❤ ❛ •❋P ✕ ✐♥ ❚❳✶✵✼✷ ♠❊❙❈s ✭❋✐❣✉r❡ ✻✳✶✵✮✳ ❈❤❛r❛❝t❡r✐③❛✐♦♥ ♦❢ ✸ ✐♥❞❡♣❡♥❞❡♥t ❙♣❡♥ ∆❙P❖❈ ✲•❋P ❝•♦♥❡s ❝♦♥✜r♠❡❞ t❤❛t ❙P❖❈ ✐s ❞✐s♣❡♥s❛❜•❡ ❢♦r ♣r♦♣❡r ❳✐st •♦❝❛•✐③❛t✐♦♥ ❛♥❞ ❙P❊◆ r❡❝r✉✐t♠❡♥t t♦ t❤❡ ❳✐ ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❊①t❡♥❞❡❞ ❉❛t❛ ❋✐❣✳ ✸❞✲❢✮✳ 2-3-4 1 RRMs SPOC RID Spen locus SPEN SPEN ∆SPOC -GFP exon15 Stop codon SPOC coding sequence transfection & clone isolation Cas9 exon1 exon12 exon13 exon14 endogenous SPOC deletion 2-3-4 1 RRMs RID GFP targetting vector GFP homology arms GFP exon15 exon1 exon12 exon13 exon14 GFP exon15 exon1 exon12 exon13 exon14 B6 Cast ❋✐❣✉r❡ ✻✳✶✵ ✕ ❙❝❤❡♠❡ ❞❡♣✐❝t✐♥❣ t❤❡ ❣❡♥❡t✐❝ str❛t❡❣② t♦ ❞❡•❡t❡ ❙P❊◆✬s ❙P❖❈ ❞♦♠❛✐♥ ❡♥❞♦❣❡♥♦✉s•② ❛♥❞ r❡♣•❛❝❡ ✐t ✇✐t❤ ❛ •❋P✳ ❆ ❤♦♠♦③②❣♦✉s ❦♥♦❝❦✲✐♥ ♦❢ ❛ •❋P ❝❛ss❡tt❡ ✭❡♥❞✐♥❣ ✇✐t❤ ❛ ❙t♦♣ ❝♦❞♦♥✮ ✇❛s ♣❡r❢♦r♠❡❞ ✐♠♠❡❞✐❛t❡•② ✉♣str❡❛♠ ♦❢ t❤❡ ❙♣❡♥ s❡q✉❡♥❝❡ ❡♥❝♦❞✐♥❣ ✐ts ❙P❖❈ ❞♦♠❛✐♥ ✭s❤♦✇♥ ❛s t❤✐♥ r❡❞ r❡❝t❛♥❣•❡s ♦♥ t❤❡ ❙♣❡♥ •♦❝✉s✮ ✉s✐♥❣ ❤♦♠♦•♦❣②✲❞✐r❡❝t❡❞ r❡♣❛✐r ✭t❤❡ ❤♦♠♦•♦❣② s❡q✉❡♥❝❡ ✐s s❤♦✇♥ ❛s ❛ t❤✐♥ ❞❛r❦ ❣r❡② r❡❝t❛♥❣•❡✮✳ ❚r❛♥s❝r✐♣t♦♠✐❝ ❛♥❛•②s✐s ❢♦••♦✇✐♥❣ ✷✹❤ ♦❢ ❳✐st ✲✐♥❞✉❝t✐♦♥ ✐♥ ❙♣❡♥ ∆❙P❖❈ ✲•❋P ❝❡••s r❡✲ ✶✷✶ |❡s✉•ts ❙P❊◆ ❢✉♥❝t✐♦♥ ❛♥❞ ♠❡❝❤❛♥✐s♠s ✐♥ ❳❈■ ✈❡❛•❡❞ ❞r❛♠❛t✐❝ ❞❡❢❡❝ts ✐♥ ❣❡♥❡ s✐•❡♥❝✐♥❣✱ ❝❤r♦♠♦s♦♠❡ ✇✐❞❡ ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❊①t❡♥❞❡❞ ❉❛t❛ ❋✐❣✳ ✸❣✲✐✮✱ ❞❡♠♦♥str❛t✐♥❣ t❤❛t t❤❡ ❙P❖❈ ❞♦♠❛✐♥ ✐s ❛♥ ❡ss❡♥t✐❛• ❡✛❡❝t♦r ♦❢ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❞✉r✐♥❣ ❳❈■✱ ❛❝t✐♥❣ ❞♦✇♥str❡❛♠ ♦❢ ❳✐st •♦❝❛•✐③❛t✐♦♥✳ |❡♠❛r❦❛❜•②✱ t❤❡ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❞❡❢❡❝t ✇❛s ♠♦❞❡r❛t❡•② ✇❡❛❦❡r t❤❛♥ t❤❡ ♦♥❡ ♦❜s❡r✈❡❞ ✉♣♦♥ •♦ss ♦❢ ❢✉••✲•❡♥❣t❤ ❙P❊◆ ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❊①t❡♥❞❡❞ ❉❛t❛ ❋✐❣✳ ✸•✮✱ s✉❣❣❡st✐♥❣ t❤❛t ♦t❤❡r ✉♥❝❤❛r❛❝t❡r✐③❡❞ r❡❣✐♦♥s ♦❢ ❙P❊◆ st✐•• ❝♦♥tr✐❜✉t❡ ✕ ❛•❜❡✐t ✇❡❛❦•② ✕ t♦ ❳✐st✲♠❡❞✐❛t❡❞ ❣❡♥❡ s✐•❡♥❝✐♥❣ ✭s❡❡ s✉❜s❡❝t✐♦♥ ✾✳✷✳✶ ❢♦r ❢✉rt❤❡r ❞✐s❝✉ss✐♦♥✮✳ ✻✳✸✳✷✳✹ ❙P❖❈ t❡t❤❡r✐♥❣ t♦ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡ ✐s s✉✣❝✐❡♥t t♦ ♠❡❞✐❛t❡ ❣❡♥❡ s✐•❡♥❝✐♥❣ ■ ♥❡①t q✉❡st✐♦♥❡❞ ✇❤❡t❤❡r t❤❡ ❙P❖❈ ❞♦♠❛✐♥ ❛•♦♥❡ ❝♦✉•❞ ❜❡ s✉✣❝✐❡♥t t♦ tr✐❣❣❡r ❣❡♥❡ s✐•❡♥❝✐♥❣ ❛•♦♥❣ t❤❡ ❳✲❝❤r♦♠♦s♦♠❡✳ ❚♦ ❞♦ s♦✱ ■ ♠❛❞❡ ✉s❡ ♦❢ t❤❡ ❇❣• st❡♠✲•♦♦♣ s②st❡♠ ✭s❡❡ s✉❜s✉❜s❡❝t✐♦♥ ✻✳✷✳✹✳✷✮ t♦ ❛rt✐✜❝✐❛••② t❡t❤❡r ❛ ❇❣••✲•❋P✲❙P❖❈ ❢✉s✐♦♥ ♣r♦t❡✐♥ ✭♦r ❇❣••✲•❋P ❛s ❝♦♥tr♦•✮ t♦ ❳✐st |◆❆✱ ✐♥ ❛ ❙P❊◆✲❞❡❣r♦♥ ❜❛❝❦❣r♦✉♥❞ ✭❋✐❣✉r❡ ✻✳✶✶✮✳ SPEN degron

A-repeat Xist

Bgl stem loops +auxin SPEN depletion X-linked gene silencing? (RNA-seq and pyrosequencing)

SPEN degron

Xist

BglG-GFP Rosa26 knock-in SPOC A-repeat BglG-GFP SPOC ❋✐❣✉r❡ ✻✳✶✶ ✕ ❙❝❤❡♠❡ ♦❢ t❤❡ ❙P❖❈ t❡t❤❡r✐♥❣ ❡①♣❡r✐♠❡♥t✳ ❆ ❇❣••✲•❋P✲❙P❖❈ ❢✉s✐♦♥ ♣r♦t❡✐♥ ✐s t❛r❣❡t❡❞ ❛t ❛♥❞ ❝♦♥st✐t✉t✐✈❡•② ❡①♣r❡ss❡❞ ❢r♦♠ t❤❡ |♦s❛✷✻ •♦❝✉s✱ ✐♥ ❛ ❙P❊◆✲❞❡❣r♦♥ ❜❛❝❦❣r♦✉♥❞✳ ❚❤✐s ❢✉s✐♦♥ ♣r♦t❡✐♥ ✐s ✧❛rt✐✜❝✐❛••②✧ r❡❝r✉✐t❡❞ t♦ ❳✐st |◆❆ ✭❛♥❞ ❝♦♥s❡q✉❡♥t✐❛••②✱ t❤❡ ❳✲❝❤r♦♠♦s♦♠❡✮ t❤r♦✉❣❤ ❜✐♥❞✐♥❣ t♦ ❇❣• st❡♠✲•♦♦♣s✳ ❯♣♦♥ ❞❡♣•❡t✐♦♥ ♦❢ ❡♥❞♦❣❡♥♦✉s ❙P❊◆ ❛♥❞ ✉♣r❡❣✉•❛t✐♦♥ ♦❢ ❳✐st |◆❆✱ t❤❡ ❛❜✐•✐t② ♦❢ ❙P❖❈ t♦ ♠❡❞✐❛t❡ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❝❤r♦♠♦s♦♠❡ ✇✐❞❡ ❝❛♥ ❜❡ ♠♦♥✐t♦r❡❞ ✉s✐♥❣ |◆❆✲s❡q ❛♥❞ ♣②r♦s❡q✉❡♥❝✐♥❣✳ |❡♠❛r❦❛❜•②✱ ✐♥ t❤❡ ❛❜s❡♥❝❡ ♦❢ ❡♥❞♦❣❡♥♦✉s ❙P❊◆✱ ❳✐st✲♠❡❞✐❛t❡❞ r❡❝r✉✐t♠❡♥t ♦❢ ❙P❖❈ ✭❜✉t ♥♦t •❋P ❛•♦♥❡✮ •❡❛❞s t♦ ❞r❛st✐❝ s✐•❡♥❝✐♥❣ ♦❢ ♠♦st ❣❡♥❡s ❛•♦♥❣ t❤❡ ❳✲❝❤r♦♠♦s♦♠❡ ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✸❞✲❣✮✳ ❚❤✐s r❡s✉•t s✉❣❣❡sts t❤❛t ♦♥❝❡ r❡❝r✉✐t❡❞ t♦ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡✱ ❙P❖❈ ✐s s✉✣❝✐❡♥t t♦ ✐♥str✉❝t ❛ s✐❣♥✐✜❝❛♥t ❢r❛❝t✐♦♥ ♦❢ ❣❡♥❡ s✐•❡♥❝✐♥❣ ✭s❡❡ s✉❜s❡❝t✐♦♥ ✾✳✷✳✶ ❢♦r ❢✉rt❤❡r ❞✐s❝✉ss✐♦♥✮✳ ✻✳✸✳✸ ■❞❡♥t✐❢②✐♥❣ t❤❡ ♣r♦t❡✐♥ ♣❛rt♥❡rs ♦❢ ❙P❊◆✬s ❙P❖❈ ❞♦♠❛✐♥ ❚♦ ❣❛✐♥ ❢✉rt❤❡r ♠❡❝❤❛♥✐st✐❝ ✐♥s✐❣❤ts ✐♥t♦ ❤♦✇ ❙P❖❈ ✐♥str✉❝ts s✉❝❤ ❣❡♥❡ s✐•❡♥❝✐♥❣✱ ■ ♥❡①t s❡t ♦✉t t♦ ❝❤❛r❛❝t❡r✐③❡ ✐ts ♣r♦t❡✐♥ ♣❛rt♥❡rs ✐♥ ♠❊❙❈s✳ ✶✷✷ |❡s✉•ts ❙P❊◆ ❢✉♥❝t✐♦♥ ❛♥❞ ♠❡❝❤❛♥✐s♠s ✐♥ ❳❈■ ✻✳✸✳✸✳✶ |❛t✐♦♥❛•❡ ❢♦r ❝❤❛r❛❝t❡r✐③✐♥❣ t❤❡ ❙P❖❈✲✐♥t❡r❛❝t♦♠❡ ❑♥♦✇✐♥❣ t❤❛t✿ ✶✳ ❙P❊◆✬s ❙P❖❈ ❞♦♠❛✐♥ ✐s ❛ ✈❡r② ♣♦t❡♥t ✐♥t❡r❛❝t♦r ♦❢ ◆❈♦| ❛♥❞ ❙▼|❚ ✐♥ ❤✉♠❛♥s ❬❙❤✐ ❡t ❛•✳✱ ✷✵✵✶❀ ❆r✐②♦s❤✐ ❛♥❞ ❙❝❤✇❛❜❡✱ ✷✵✵✸❀ ❖s✇❛•❞ ❡t ❛•✳✱ ✷✵✶✻(✱ ✷✳ ❍❉❆❈✸✲♠❡❞✐❛t❡❞ ❤✐st♦♥❡ ❞❡❛❝❡t②•❛t✐♦♥ ♣•❛②s ❛♥ ✐♠♣♦rt❛♥t r♦•❡ ✐♥ ♣r♦♠♦t✐♥❣ ❡❢✲ ✜❝✐❡♥t ❣❡♥❡ s✐•❡♥❝✐♥❣ ❞✉r✐♥❣ ❳❈■ ✐♥ ♠❊❙❈s ❬➏②•✐❝③ ❡t ❛•✳✱ ✷✵✶✾❀ ▼❝❍✉❣❤ ❡t ❛•✳✱ ✷✵✶✺(✱ ✸✳ ❍❉❆❈✸ ❛•♦♥❡ ✐s ♥♦t ❢✉♥❝t✐♦♥❛•✱ ❛♥❞ ✐ts ✐♥t❡r❛❝t✐♦♥ ✇✐t❤ ❙▼|❚ ♦r ◆❈♦| ✕ ❜♦t❤ ♦❢ ✇❤✐❝❤ ❞✐r❡❝t•② ✐♥t❡r❛❝t ✇✐t❤ ❙P❖❈ ✕ ✐s ♥❡❝❡ss❛r② ❢♦r ✐ts ❡♥③②♠❛t✐❝ ❛❝t✐✈✐t② ❬•✉❡♥✲ t❤❡r ❡t ❛•✳✱ ✷✵✵✶(✱ ✐t ❝♦✉•❞ ❛t t❤✐s st❛❣❡ ❜❡ ❛r❣✉❡❞ t❤❛t t❤❡ ❢✉♥❝t✐♦♥ ♦❢ ❙P❊◆✬s ❙P❖❈ ❞♦♠❛✐♥ ❞✉r✐♥❣ ❳❈■ ✐s s♦•❡•② t♦ r❡❝r✉✐t ◆❈♦|✴❙▼|❚ t♦ ❍❉❆❈✸✲❜♦✉♥❞ s✐t❡s✱ t❤❡r❡❜② ❡♥❛❜•✐♥❣ ❍❉❆❈✸ ❛❝t✐✈✐t② ♦♥ t❤❡ ❳✲❝❤r♦♠♦s♦♠❡✱ •❡❛❞✐♥❣ t♦ s✉❜s❡q✉❡♥t ❝❤r♦♠♦s♦♠❡✲✇✐❞❡ ❣❡♥❡ s✐•❡♥❝✐♥❣✳ ■♥ ❢❛❝t✱ ❡✈❡r s✐♥❝❡ t❤❡ ✐❞❡♥t✐✜❝❛t✐♦♥ ♦❢ ❙P❊◆ ❛s ❛ ♣♦t❡♥t✐❛• ❦❡② ♣•❛②❡r ✐♥ ❳❈■✱ t❤✐s ♠♦❞❡• ✇❛s ♣✉t ❢♦rt❤ ❜② t❤❡ •✉tt♠❛♥ •❛❜ ❬▼❝❍✉❣❤ ❡t ❛•✳✱ ✷✵✶✺(✳ ❍♦✇❡✈❡r✱ ❡✈✐❞❡♥❝❡ ❢♦r ❛ ♣❤②s✐❝❛• ✐♥t❡r❛❝t✐♦♥ ❜❡t✇❡❡♥ ❙P❖❈ ❛♥❞ ◆❈♦|✴❙▼|❚ ✐♥ ♠♦✉s❡ t♦ s✉♣♣♦rt t❤✐s ❤②♣♦t❤❡s✐s ✐s ♠✐ss✐♥❣✳ ❋✉rt❤❡r♠♦r❡✱ s❡✈❡r❛• •✐♥❡s ♦❢ ❡✈✐❞❡♥❝❡ ❞❡♠♦♥str❛t❡ t❤❛t ✐♥✈♦❦✐♥❣ ♦♥•② ❛♥ ❍❉❆❈✸✲❛①✐s ✐s ♥♦t s✉✣❝✐❡♥t t♦ ❢✉••② ❡①♣•❛✐♥ ❤♦✇ ❙P❊◆✴❙P❖❈ ❢✉♥❝t✐♦♥s ❞✉r✐♥❣ ❳❈■✿ ✶✳ ❯s✐♥❣ t❤❡ s❛♠❡ ❝❡••✉•❛r ❜❛❝❦❣r♦✉♥❞ ✭❢❡♠❛•❡ ❚❳✶✵✼✷ ♠❊❙❈s✮✱ ❛♥❞ ❛ss❛②✐♥❣ ❳❈■ ✉♥❞❡r ✐❞❡♥t✐❝❛• ❝♦♥❞✐t✐♦♥s ✭✷✹ ❤♦✉rs ♦❢ ❳✐st ✉♣r❡❣✉•❛t✐♦♥✮✱ ■ ✜♥❞ t❤❛t t❤❡ ❣❡♥❡ s✐✲ •❡♥❝✐♥❣ ❞❡❢❡❝ts ♦❜s❡r✈❡❞ ❢♦••♦✇✐♥❣ t❤❡ ❡♥❞♦❣❡♥♦✉s ❞❡•❡t✐♦♥ ♦❢ ❙P❊◆✬s ❙P❖❈ ❞♦♠❛✐♥ ❛r❡ ♠✉❝❤ ♠♦r❡ s❡✈❡r❡ t❤❛♥ t❤♦s❡ r❡♣♦rt❡❞ ✉♣♦♥ t❤❡ •♦ss ♦❢ ❡♥❞♦❣❡♥♦✉s ❍❉❆❈✸ ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❊①t❡♥❞❡❞ ❉❛t❛ ❋✐❣✳ ✸•✱ ❦✮✳ ✷✳ ■♥ ❢❛❝t✱ t❤❡ ♥❛t✉r❡ ♦❢ t❤❡ ❣❡♥❡ s✐•❡♥❝✐♥❣ ♣❤❡♥♦t②♣❡s ❛ss♦❝✐❛t❡❞ ✇✐t❤ ❍❉❆❈✸ ❛♥❞ ❙P❖❈ ♣❡rt✉r❜❛t✐♦♥s ❛♣♣❡❛r t♦ ❜❡ ❞✐✛❡r❡♥t✿ ■♥ ❍❞❛❝✸ ❑❖ ❝❡••s✱ ❳❈■ ✐s ♦♥•② ❞❡•❛②❡❞✿ ♠♦st ❛✛❡❝t❡❞ ❣❡♥❡s ❛r❡ st✐•• s✐❣♥✐✜❝❛♥t•② s✐•❡♥❝❡❞✱ ♦♥•② ♥♦t ❛s q✉✐❝❦•② ❛s ✐♥ ❛ ❲❚ s❝❡♥❛r✐♦✱ ❛♥❞ ✐♥ r❡❛•✐t②✱ ♠❛♥② ❣❡♥❡s ✉♥❞❡r❣♦ s✐•❡♥❝✐♥❣ ✐❞❡♥t✐❝❛••② t♦ ❲❚ ❝❡••s ❬➏②•✐❝③ ❡t ❛•✳✱ ✷✵✶✾(✱ ❤✐♥t✐♥❣ t❤❛t ❍❉❆❈✸ ❛❝ts ❛s ❛ ❝❛t❛•②st✴❢❛❝✐•✐t❛t♦r r❛t❤❡r t❤❛♥ ❛s ❛ ❣❡♥✉✐♥❡ ♠❡❞✐❛t♦r ♦❢ ❳✐st✲❞❡♣❡♥❞❡♥t ❣❡♥❡ s✐•❡♥❝✐♥❣✳ ■♥ t❤❡ ❛❜s❡♥❝❡ ♦❢ ❙P❖❈ ♦♥ t❤❡ ♦t❤❡r ❤❛♥❞✱ ✈✐rt✉❛••② ❝♦♠♣•❡t❡•② ❞❡✜❝✐❡♥t s✐•❡♥❝✐♥❣ ✐s ♦❜s❡r✈❡❞ ❛❝r♦ss ❛•• ❳✲•✐♥❦❡❞ ❣❡♥❡s✳ ✸✳ ❍❉❆❈✸ ♣r❡❞♦♠✐♥❛♥t•② ❛❝ts ❛t ❳✲•✐♥❦❡❞ ❡♥❤❛♥❝❡rs ✭s❡❡ s✉❜s❡❝t✐♦♥ ✺✳✸✳✷✮✱ ✇❤✐•❡ ■ ❢♦✉♥❞ t❤❛t ❙P❊◆ ✐s ❛❝t✐✈❡•② r❡❝r✉✐t❡❞ ♥♦t ♦♥•② t♦ ❡♥❤❛♥❝❡rs ❜✉t ❛•s♦ t♦ ♣r♦♠♦t❡rs ❞✉r✐♥❣ ❳❈■ ✭s❡❡ s✉❜s❡❝t✐♦♥ ✻✳✸✳✶✮✳ ✶✷✸ |❡s✉•ts ❙P❊◆ ❢✉♥❝t✐♦♥ ❛♥❞ ♠❡❝❤❛♥✐s♠s ✐♥ ❳❈■ ❚❛❦❡♥ t♦❣❡t❤❡r✱ t❤❡s❡ ❢❛❝ts ❤✐❣❤•✐❣❤t t❤❡ ♥❡❝❡ss✐t② t♦ ♠❛♣ t❤❡ ❙P❖❈ ♣r♦t❡✐♥ ✐♥t❡r❛❝✲ t♦♠❡ ✭♥♦t❛❜•② ✐♥ ♠❊❙❈s✮ s♦ ❛s t♦ ✐❞❡♥t✐❢② ♦t❤❡r ❙P❖❈✲❛ss♦❝✐❛t❡❞ ❝♦❢❛❝t♦rs ❛♥❞ ♣❛t❤✇❛②s ✐♥✈♦•✈❡❞ ✐♥ ♠❡❞✐❛t✐♥❣ ❳❈■ ✐♥ ♣❛r❛••❡• t♦ ❍❉❆❈✸✳ ✻✳✸✳✸✳✷ ❙P❖❈ ♣r♦t❡♦♠✐❝s ♠❡t❤♦❞♦•♦❣② ■ ❤❡♥❝❡ ♣❡r❢♦r♠❡❞ ✐♠♠✉♥♦♣r❡❝✐♣✐t❛t✐♦♥ ✭■P✮ ♦❢ ❇❣••✲•❋P✲t❛❣❣❡❞ ❙P❖❈✱ ♦r ❇❣••✲ •❋P ❛s ❛ ❝♦♥tr♦•✱ ❜♦t❤ st❛❜•② ✐♥t❡❣r❛t❡❞ ❛t ✭❛♥❞ ❝♦♥st✐t✉t✐✈❡•② ❡①♣r❡ss❡❞ ❢r♦♠✮ t❤❡ |♦s❛✷✻ •♦❝✉s✳ ❈♦✲✐♠♠✉♥♦♣r❡❝✐♣✐t❛t✐♥❣ ♣r♦t❡✐♥s ✇❡r❡ t❤❡♥ ✐❞❡♥t✐✜❡❞ ✉s✐♥❣ ♠❛ss s♣❡❝tr♦♠❡tr② ✭▼❙✱ s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❊①t❡♥❞❡❞ ❉❛t❛ ❋✐❣✳ ✸•✮✳ ❊❛❝❤ ■P ✇❛s ♣❡r❢♦r♠❡❞ ✐♥ ❜✐♦•♦❣✐❝❛• q✉❛❞r✉♣•✐❝❛t❡s✱ ✉s✐♥❣ ❛♥ ❛♥t✐✲•❋P ♥❛♥♦❜♦❞②✲❝♦✉♣•❡❞ ❛✣♥✐t② r❡s✐♥ ✭•❋P✲tr❛♣✮✱ ❛♥❞ ❦❡② ❙P❖❈✲♣❛rt♥❡rs ✇❡r❡ ♦rt❤♦❣♦♥❛••② ✈❛•✐❞❛t❡❞ ✉s✐♥❣ ❝♦✲■P ✇❡st❡r♥ ❜•♦t ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✸❤✮✳ ■♠♣♦rt❛♥t•②✱ ■ ♣❡r❢♦r♠❡❞ t❤❡s❡ ■Ps ✐♥ t❤❡ ❛❜s❡♥❝❡ ♦❢ ❳✐st ✐♥❞✉❝t✐♦♥✱ s♦ ❛s t♦ ✇❛rr❛♥t t❤❛t t❤❡ ❝♦✲✐♠♠✉♥♦♣r❡❝✐♣✐t❛t❡❞ ♣r♦t❡✐♥s ❛r❡ ♥♦t ✐♥❞✐r❡❝t•② ❜r✐❞❣❡❞ t♦ ❙P❖❈ ✈✐❛ ❳✐st |◆❆✳ ✻✳✸✳✸✳✸ ❙P❖❈ ✐♥t❡r❛❝ts ✇✐t❤ ❛ ✈❛r✐❡t② ♦❢ ❝♦♠♣•❡①❡s ✐♥✈♦•✈❡❞ ✐♥ ❞✐r❡❝t tr❛♥✲ s❝r✐♣t✐♦♥ r❡❣✉•❛t✐♦♥✱ |◆❆ ♠❡t❛❜♦•✐s♠✱ ❝❤r♦♠❛t✐♥ ♠♦❞✐✜❝❛t✐♦♥ ❛♥❞ ♥✉❝•❡♦s♦♠❡ r❡♠♦❞❡•✐♥❣ |❡♠❛r❦❛❜•②✱ ❙P❖❈ ❝♦✲✐♠♠✉♥♦♣r❡❝✐♣✐t❛t❡❞ ✇✐t❤ s❡✈❡r❛• ♣r♦t❡✐♥ ❝♦♠♣•❡①❡s ✐♥✈♦•✈❡❞ ✐♥ ♣r♦❝❡ss❡s ♣❡rt❛✐♥✐♥❣ t♦ r❡❣✉•❛t✐♦♥ ♦❢ ❣❡♥❡ ❡①♣r❡ss✐♦♥ ✐♥❝•✉❞✐♥❣✿ tr❛♥s❝r✐♣t✐♦♥✱ |◆❆ ♠❡t❛❜♦•✐s♠✱ ❝❤r♦♠❛t✐♥ ♠♦❞✐✜❝❛t✐♦♥ ❛♥❞ ♥✉❝•❡♦s♦♠❡ r❡♠♦❞❡•✐♥❣✳ ❙P❖❈ ❛♥❞ ◆❈♦|✴❙▼|❚✲❍❉❆❈✸✿ ❊①♣❡❝t❡❞•②✱ ♠♦st ♠❡♠❜❡rs ♦❢ t❤❡ ◆❈♦|✴❙▼|❚ ❝♦♠♣•❡① ✇❡r❡ r❡tr✐❡✈❡❞ ✭❋✐❣✉r❡ ✻✳✶✷✱ s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✸❤ ❛♥❞ ❊①t❡♥❞❡❞ ❉❛t❛ ❋✐❣✳ ✸•✮✳ ❙✉r♣r✐s✐♥❣•②✱ ❍❉❆❈✸ ✇❛s ❢♦✉♥❞ t♦ ✐♠♠✉♥♦♣r❡❝✐♣✐t❛t❡ ✇✐t❤ ❙P❖❈ ✭❋✐❣✉r❡ ✻✳✶✷✱ s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✸❤ ❛♥❞ ❊①t❡♥❞❡❞ ❉❛t❛ ❋✐❣✳ ✸•✮✱ ❛•t❤♦✉❣❤ ✇❡ ♣r❡✈✐♦✉s•② r❡♣♦rt❡❞ t❤❛t ❝♦♥tr❛r② t♦ ❙P❊◆✱ ❍❉❆❈✸ ✐s ♥♦t r❡❝r✉✐t❡❞ t♦ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡ ❞✉r✐♥❣ ❳❈■ ❜✉t ✐s ✐♥st❡❛❞ ♣r❡✲❜♦✉♥❞ t♦ ❳✲•✐♥❦❡❞ ❡♥❤❛♥❝❡rs ✭s❡❡ ❝❤❛♣t❡r ✺✮✳ ❍❡♥❝❡✱ ✐t ✐s ♠♦st •✐❦❡•② t❤❛t ❍❉❆❈✸ ✧s❛t✉r❛t❡s✧ ❝❤r♦♠❛t✐♥ ♦♥ t❤❡ ❳✱ •❡❛✈✐♥❣ •✐tt•❡ r♦♦♠ ❢♦r ✐t t♦ ❜❡ r❡❝r✉✐t❡❞✳ ❈♦♥s❡q✉❡♥t•②✱ ❙P❊◆ r❡❝r✉✐t♠❡♥t ❝♦♥st✐t✉t❡s t❤❡ •✐♠✐t✐♥❣ st❡♣ ✐♥ t❤❡ ❙P❊◆✲❍❉❆❈✸ ❛①✐s ❞✉r✐♥❣ ❳❈■✳ ✶✷✹ |❡s✉•ts ❙P❊◆ ❢✉♥❝t✐♦♥ ❛♥❞ ♠❡❝❤❛♥✐s♠s ✐♥ ❳❈■ Ncor/SMRT complex Gps2 Setd5 Ncor1 Hdac3 Ncor2 Tbl1x Tbl1xr1 Ankrd11 Cdk2Ap1 Mta1/2/
❋✐❣✉r❡ ✻✳✶✷ ✕ ❙❝❤❡♠❡ s❤♦✇✐♥❣ t❤❡ ❞✐✛❡r❡♥t ♣r♦t❡✐♥s ✭❛♥❞ t❤❡ ❝♦♠♣•❡①❡s✴❜✐♦•♦❣✐❝❛• ♣❛t❤✇❛②s t❤❡② ❜❡•♦♥❣ t♦✮ ✐❞❡♥t✐✜❡❞ ✐♥ t❤❡ ❙P❖❈ ✐♥t❡r❛❝t♦♠❡✳ ❙P❖❈ ❛♥❞ |◆❆ ❤♦♠❡♦st❛s✐s✴♠ ✻ ❆ ♠❛❝❤✐♥❡r②✿ |❡♠❛r❦❛❜•②✱ s❡✈❡r❛• ♣r♦t❡✐♥s ✐♥✈♦•✈❡❞ ✐♥ |◆❆ s♣•✐❝✐♥❣ ❛♥❞ •♦❝❛•✐③❛t✐♦♥ ✕ ♥♦t❛❜•② ♠❡♠❜❡rs ♦❢ t❤❡ ❡①♦♥ •✉♥❝t✐♦♥ ❝♦♠♣•❡① ✕ ✇❡r❡ ✐❞❡♥t✐✜❡❞ ✐♥ ♦✉r ❈♦■P✲▼❙ ❛♥❛•②s✐s ✭❋✐❣✉r❡ ✻✳✶✷✱ s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✸❤ ❛♥❞ ❊①t❡♥❞❡❞ ❉❛t❛ ❋✐❣✳ ✸•✮✱ ❤✐♥t✐♥❣ t❤❛t ❙P❊◆ ❝♦✉•❞ ❜❡ ✐♠♣♦r✲ t❛♥t ❢♦r r❡❣✉•❛t✐♥❣ t❤❡ ❢❛t❡ ♦❢ ❳✐st |◆❆ t♦ s♦♠❡ ❡①t❡♥t✱ ❛s ✇❡•• ❛s t❤❛t ♦❢ ♦t❤❡r ❳✲•✐♥❦❡❞ ❣❡♥❡s✳ ❋✉rt❤❡r♠♦r❡✱ ❛•• ♠❡♠❜❡rs ♦❢ t❤❡ ♠ ✻ ❆ |◆❆ ♠❡t❤②•❛t✐♦♥ ♠❛❝❤✐♥❡r② ❛s ✇❡•• ❛s t❤❡ ♥✉❝•❡❛r ♠ ✻ ❆ r❡❛❞❡r (❚❍❉❈✶ ✭s❡❡ s✉❜s❡❝t✐♦♥ ✶✳✶✸✳✸✮ ✇❡r❡ ❛•s♦ ✐❞❡♥t✐✜❡❞ ✭❋✐❣✉r❡ ✻✳✶✷✱ s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✸❤ ❛♥❞ ❊①t❡♥❞❡❞ ❉❛t❛ ❋✐❣✳ ✸•✮✳ ❚❤❡s❡ r❡s✉•ts r❛✐s❡ t❤❡ ♣♦ss✐❜✐•✐t② t❤❛t s✐♠✐•❛r•② t♦ |❇▼✶✺ ✭s❡❡ s✉❜s❡❝t✐♦♥ ✶✳✶✸✳✸✮✱ ❙P❊◆ ❝♦♥st✐t✉t❡s ❛ ♣❤②s✐❝❛• •✐♥❦ ❜❡t✇❡❡♥ ❳✐st ❛♥❞ t❤❡ |◆❆✲♠❡t❤②•❛t✐♦♥ ♣❛t❤✇❛②✳ ❋✉rt❤❡r♠♦r❡✱ t❤✐s ❛♥❛•②s✐s ❞❡✜♥❡s ❙P❖❈ ❛s ❛♥ ♠ ✻ ❆✲✇r✐t✐♥❣ ♠❛❝❤✐♥❡r② ✐♥t❡r❛❝t✐♦♥ ❞♦♠❛✐♥ ✭s❡❡ s✉❜s✉❜s❡❝t✐♦♥ ✾✳✷✳✷✳✹ ❢♦r ❢✉rt❤❡r ❞✐s❝✉ss✐♦♥ ♦❢ t❤❡s❡ r❡s✉•ts✮✳ ❙P❖❈ ❛♥❞ t❤❡ ◆✉|❉ ❝♦♠♣•❡①✿ ❙P❖❈ ❛•s♦ ❝♦✲✐♠♠✉♥♦♣r❡❝✐♣✐t❛t❡❞ ✇✐t❤ ♠♦st ♠❡♠❜❡rs ♦❢ t❤❡ ♥✉❝•❡♦s♦♠❡ r❡♠♦❞❡•✐♥❣ ❞❡❛❝❡t②•❛s❡ ❝♦♠♣•❡① ✭◆✉|❉✮✱ ❛ ♣r♦t❡✐♥ ❝♦♠♣•❡① ❝❛rr②✐♥❣ ❜♦t❤ ❤✐st♦♥❡ ❞❡❛❝❡t②•❛t✐♦♥ ❛♥❞ ❝❤r♦♠❛t✐♥ r❡♠♦❞❡•✐♥❣ ❛❝t✐✈✐t✐❡s ✭❋✐❣✉r❡ ✻✳✶✷✱ s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✸❤ ❛♥❞ ❊①t❡♥❞❡❞ ❉❛t❛ ❋✐❣✳ ✸•✮✳ ❚❤❡ ❙❆▲▲✹ tr❛♥s❝r✐♣t✐♦♥ ❢❛❝t♦r ✭❛♥❞ ♠❛♥② ♦❢ ✐ts ✐♥t❡r❛❝t♦rs✮ ✇❛s ❛•s♦ ❤✐❣❤•② ❡♥r✐❝❤❡❞ ✐♥ t❤❡ ❙P❖❈ ❈♦✲■P ✭❋✐❣✉r❡ ✻✳✶✷✱ s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✸❤ ❛♥❞ ❊①t❡♥❞❡❞ ❉❛t❛ ❋✐❣✳ ✸•✮✱ ❝♦♥s✐st❡♥t ✇✐t❤ ♣r❡✈✐♦✉s r❡♣♦rts t❤❛t ❛ •❛r❣❡ ❢r❛❝t✐♦♥ ♦❢ t❤❡ ◆✉|❉ ♣♦♦• ✐s ❝♦♠♣•❡①❡❞ ✇✐t❤ ❙❆▲▲✹ ✐♥ ♠❊❙❈s ❬▼✐••❡r ❡t ❛•✳✱ ✷✵✶✻❀ ❑•♦❡t ❡t ❛•✳✱ ✷✵✶✺❀ ▲✉ ❡t ❛•✳✱ ✷✵✵✾(✳ ✶✷✺ |❡s✉•ts ❙P❊◆ ❢✉♥❝t✐♦♥ ❛♥❞ ♠❡❝❤❛♥✐s♠s ✐♥ ❳❈■ ❙P❖❈ ❛♥❞ |◆❆P■■✿ ❚❤❡ ◆✉|❉ ❝♦♠♣•❡① ❛❝ts ❛s ❛ ♣♦t❡♥t tr❛♥s❝r✐♣t✐♦♥❛• r❡♣r❡ss♦r✱ ❜② ❞✐s♣•❛❝✐♥❣ |◆❆ ♣♦•②♠❡r❛s❡ ■■ ✭|◆❆P■■✮ ❢r♦♠ tr❛♥s❝r✐♣t✐♦♥ st❛rt s✐t❡s✱ s♣❡❝✐✜❝❛••② t❤r♦✉❣❤ ✐ts ♥✉❝•❡♦s♦♠❡ r❡♠♦❞❡•✐♥❣ ❛❝t✐✈✐t② ❬❇♦r♥❡•ö✈ ❡t ❛•✳✱ ✷✵✶✽(✳ ❚❤❡r❡❢♦r❡✱ ❙P❊◆ ❝♦✉•❞ ♠❡❞✐❛t❡ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❜② ❛✛❡❝t✐♥❣ |◆❆P■■ t❤r♦✉❣❤ t❤❡ ◆✉|❉ ❝♦♠♣•❡①✱ ✇❤✐❝❤ ✇✐•• ❜❡ ❢✉rt❤❡r ❞✐s❝✉ss❡❞ ✐♥ s✉❜s✉❜s❡❝t✐♦♥ ✾✳✷✳✷✳✷✳ |❡♠❛r❦❛❜•②✱ ❙P❖❈ ❛•s♦ ❝♦✲✐♠♠✉♥♦♣r❡❝✐♣✐t❛t❡❞ ✇✐t❤ s❡✈❡r❛• ❢❛❝t♦rs ❞✐r❡❝t•② ✐♥✈♦•✈❡❞ ✐♥ tr❛♥s❝r✐♣t✐♦♥✱ ✐♥❝•✉❞✐♥❣ |◆❆P■■ ✐ts❡•❢ ❛s ✇❡•• ❛s tr❛♥s❝r✐♣t✐♦♥ ✐♥✐t✐❛t✐♦♥ ❛♥❞ ❡•♦♥❣❛t✐♦♥ ❢❛❝t♦rs ✭❋✐❣✉r❡ ✻✳✶✷✱ s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✸❤ ❛♥❞ ❊①t❡♥❞❡❞ ❉❛t❛ ❋✐❣✳ ✸•✮✳ ❍❡♥❝❡✱ ✐t ✐s ♣♦ss✐❜•❡ t❤❛t ❙P❊◆ ❝♦✉•❞ ❛•s♦ r❡♣r❡ss tr❛♥s❝r✐♣t✐♦♥ ❞✐r❡❝t•② t❤r♦✉❣❤ ✐ts ✐♥t❡r❛❝t✐♦♥ ✇✐t❤ t❤❡s❡ ❢❛❝t♦rs✳ ❙✉❝❤ ✐♥t❡r❛❝t✐♦♥ ✇❛s ✐♥✈❡st✐❣❛t❡❞ ✐♥ t❤❡ ❝♦♥t❡①t ♦❢ ❝❤❛♣t❡r ✼ ❛♥❞ ✇✐•• ❜❡ ❢✉rt❤❡r ❞✐s❝✉ss❡❞ ✐♥ s✉❜s✉❜s❡❝t✐♦♥ ✾✳✷✳✷✳✸✳ ■♥t❡❣r❛t✐♥❣ ❙P❊◆ ❈❯❚✫|❯◆ ✇✐t❤ ❙P❖❈✲✐♥t❡r❛❝t♦rs ❜✐♥❞✐♥❣ ♣r♦✜•❡s✿ ❚❤❡ ❢❛❝ts t❤❛t ❙P❖❈ ✐♥t❡r❛❝ts ✇✐t❤ ♦t❤❡r ♣r♦t❡✐♥ ❝♦♠♣•❡①❡s t❤❛♥ ◆❈♦|✴❙▼|❚✱ ❛♥❞ t❤❛t t❤♦s❡ ❛r❡ ❛•s♦ ✐♥✈♦•✈❡❞ ✐♥ tr❛♥s❝r✐♣t✐♦♥❛• r❡❣✉•❛t✐♦♥ ✭♣❛rt✐❝✉•❛r•② r❡♣r❡ss✐♦♥✮✱ str♦♥❣•② s✉❣❣❡st t❤❛t s✉❝❤ ♦t❤❡r ❝♦♠♣•❡①❡s ❝♦♥st✐t✉t❡ ❙P❊◆✴❙P❖❈✲❜r✐❞❣❡❞ s✐•❡♥❝✐♥❣ ♣❛t❤✇❛②s✱ ❛❝t✐♥❣ ✐♥ ♣❛r❛••❡• t♦ t❤❡ ❙P❊◆✲❍❉❆❈✸ ❛①✐s ❞✉r✐♥❣ ❳❈■ ✭s❡❡ s✉❜s✉❜s❡❝✲ t✐♦♥ ✻✳✸✳✸✳✶✮✳ ❚♦ ❝♦♥✜r♠ t❤✐s ❤②♣♦t❤❡s✐s✱ ■ ✐♥t❡❣r❛t❡❞ t❤❡ ❙P❊◆ ❈❯❚✫|❯◆ ❞❛t❛ ✭s❡❡ s✉❜s❡❝✲ t✐♦♥ ✻✳✸✳✶✮ ✇✐t❤ ❈❤■P✲s❡q ❞❛t❛s❡ts ❢♦r s♦♠❡ ♦❢ t❤❡ ♣r♦t❡✐♥ ❢❛❝t♦rs ✐❞❡♥t✐✜❡❞ ❛s ❙P❖❈✲ ✐♥t❡r❛❝t♦rs✳ ❚❤❡s❡ ❞❛t❛s❡ts ✐♥❝•✉❞❡✿ ❼ ❍❉❆❈✸ ❈❤■P✲s❡q✱ ♣❡r❢♦r♠❡❞ ✐♥ t❤❡ s❛♠❡ ❚❳✶✵✼✷ ❢❡♠❛•❡ ♠❊❙❈ ❜❛❝❦❣r♦✉♥❞ ❛s t❤❡ ❙P❊◆ ❈❯❚✫|❯◆✱ ✇✐t❤ ❝❡••s ❝✉•t✉r❡❞ ✉♥❞❡r ✐❞❡♥t✐❝❛• ✷✐✰▲■❋ ❝♦♥❞✐t✐♦♥s ✭s❡❡ ❝❤❛♣t❡r ✺ ❛♥❞ ❬➏②•✐❝③ ❡t ❛•✳✱ ✷✵✶✾(✮✳ ❼ ❈❍❉✹ ❛♥❞ ▼❇❉✸ ❈❤■P✲s❡q ✭t✇♦ ❝♦r❡ ❝♦♠♣♦♥❡♥ts ♦❢ t❤❡ ◆✉|❉ ❝♦♠♣•❡①✮✱ ♣❡r✲ ❢♦r♠❡❞ ✐♥ ♠❛•❡ ❊✶✹ ♠❊❙❈s✱ ❛•s♦ ❝✉•t✉r❡❞ ✉♥❞❡r ✷✐✰▲■❋ ❝♦♥❞✐t✐♦♥s ❬❇♦r♥❡•ö✈ ❡t ❛•✳✱ ✷✵✶✽(✳ ❼ |◆❆P■■ ❈❤■P✲s❡q✱ ♣❡r❢♦r♠❡❞ ✐♥ ♠❛•❡ ❊✶✹ ♠❊❙❈s✱ ❛•s♦ ❝✉•t✉r❡❞ ✉♥❞❡r ✷✐✰▲■❋ ❝♦♥❞✐t✐♦♥s ❬❇♦r♥❡•ö✈ ❡t ❛•✳✱ ✷✵✶✽(✳ ❚❤✐s ❛♥❛•②s✐s r❡✈❡❛•❡❞ t❤❛t ❙P❊◆ ❜✐♥❞✐♥❣ str♦♥❣•② ♦✈❡r•❛♣s t❤❛t ♦❢ ❍❉❆❈✸ ❛t ❡♥✲ ❤❛♥❝❡rs✱ ❜✉t ♥♦t ❛t ♣r♦♠♦t❡rs ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❊①t❡♥❞❡❞ ❉❛t❛ ❋✐❣✳ ✹❤✮✳ ❙✉❝❤ ♦❜s❡r✈❛✲ t✐♦♥ ✐s ✐♥ •✐♥❡ ✇✐t❤ ♦✉r ♣r❡✈✐♦✉s r❡♣♦rt t❤❛t ❍❉❆❈✸ ✐s ❜♦✉♥❞ ♣r❡❞♦♠✐♥❛♥t•② ❛t ❡♥❤❛♥❝❡rs ✭❛♥❞ ♥♦t ♣r♦♠♦t❡rs✱ ❬➏②•✐❝③ ❡t ❛•✳✱ ✷✵✶✾(✮✱ ❛♥❞ ❢✉rt❤❡r s✉♣♣♦rts ❛ ♠♦❞❡• ✇❤❡r❡✐♥ ❳✐st✲ ♠❡❞✐❛t❡❞ r❡❝r✉✐t♠❡♥t ♦❢ ❙P❊◆ ❛❝t✐✈❛t❡s ❍❉❆❈✸✲❞❡♣❡♥❞❡♥t ❞❡❛❝❡t②•❛t✐♦♥ ❛t ❳✲•✐♥❦❡❞ ❡♥❤❛♥❝❡rs✳ ❖♥ t❤❡ ❝♦♥tr❛r②✱ ❙P❊◆ ❜✐♥❞✐♥❣ str♦♥❣•② ♦✈❡r•❛♣♣❡❞ t❤❛t ♦❢ t❤❡ ❈❍❉✹✴▼❇❉✸ ◆✉|❉ ✶✷✻ |❡s✉•ts ❙P❊◆ ❢✉♥❝t✐♦♥ ❛♥❞ ♠❡❝❤❛♥✐s♠s ✐♥ ❳❈■ s✉❜✉♥✐ts ❛t ❣❡♥❡ ♣r♦♠♦t❡rs✱ ❜✉t ♥♦t ❛t ❡♥❤❛♥❝❡rs ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❊①t❡♥❞❡❞ ❉❛t❛ ❋✐❣✳ ✹❤✮✳ ❚❤✐s ❞✐❝❤♦t♦♠② ✐s ✐♥ str♦♥❣ ❢❛✈♦r ♦❢ ❛ ♠♦❞❡• ✇❤❡r❡✐♥ ❙P❊◆✱ t❤r♦✉❣❤ ✐ts ❙P❖❈ ❞♦♠❛✐♥✱ ❜r✐❞❣❡s ❞✐✛❡r❡♥t ♣❛t❤✇❛②s ✭♦♣❡r❛t✐♥❣ ❞✐st✐♥❝t✐✈❡•② ❛t ♣r♦♠♦t❡rs ♦r ❡♥❤❛♥❝❡rs✮ t♦ r♦❜✉st•② s✐•❡♥❝❡ ❳✲•✐♥❦❡❞ tr❛♥s❝r✐♣t✐♦♥ ❞✉r✐♥❣ ❳❈■✳ ❚♦✇❛r❞s ❛ ❙P❖❈✴|◆❆P■■ ✐♥t❡r❛❝t✐♦♥ r❡s❡❛r❝❤ ❛①✐s✿ ❋✉rt❤❡r♠♦r❡✱ ❙P❊◆ ♣r♦✜•❡s ♦✈❡r•❛♣ ♠❛•♦r•② ✇✐t❤ |◆❆P■■✲❜♦✉♥❞ ❝❤r♦♠❛t✐♥ s✐t❡s ♦♥ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡ ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❊①t❡♥❞❡❞ ❉❛t❛ ❋✐❣✳ ✹❤✮✳ ❚❤✐s r❡s✉•t ✐s ❝♦♥s✐s✲ t❡♥t ✇✐t❤ t❤❡ ♦❜s❡r✈❛t✐♦♥ t❤❛t ❙P❊◆ ✐s r❡❝r✉✐t❡❞ ♣r❡❞♦♠✐♥❛♥t•② t♦ ❛❝t✐✈❡•② tr❛♥s❝r✐❜❡❞ ❣❡♥❡ ♣r♦♠♦t❡rs ❛♥❞ ❡♥❤❛♥❝❡rs✱ ❛♥❞ t❤❛t ❙P❊◆✲❜✐♥❞✐♥❣ t♦ ❝❤r♦♠❛t✐♥ ❛♣♣❡❛rs t♦ ❜❡ tr❛♥s❝r✐♣t✐♦♥✲❞❡♣❡♥❞❡♥t ✭s❡❡ s✉❜s❡❝t✐♦♥ ✻✳✸✳✶✮✳ ▼♦st ✐♠♣♦rt❛♥t•②✱ t❤❡② ❝♦rr♦❜♦r❛t❡ t❤❡ ♣r❡•✐♠✐♥❛r② ❡✈✐❞❡♥❝❡ t❤❛t ❙P❖❈ ✐♥t❡r❛❝ts ✇✐t❤ t❤❡ tr❛♥s❝r✐♣t✐♦♥ ♠❛❝❤✐♥❡r②✱ t❤r♦✉❣❤ ✇❤✐❝❤ ✐t ❝♦✉•❞ ❜❡ ❡♥✈✐s✐♦♥♥❡❞ t❤❛t ❙P❊◆ r❡♣r❡ss❡s tr❛♥s❝r✐♣t✐♦♥ ❞✐r❡❝t•② ❞✉r✐♥❣ ❳❈■✳ ■♥ t❤❡ ♥❡①t ❝❤❛♣t❡r ✭❝❤❛♣t❡r ✼✮✱ ■ ✇✐•• ❞❡s❝r✐❜❡ t❤❡ ❜✐♦❝❤❡♠✐❝❛• ❛♥❞ ❜✐♦♣❤②s✐❝❛• ❡①♣❡r✐♠❡♥ts ■ ✉♥❞❡rt♦♦❦ ✐♥ ❝♦••❛❜♦r❛t✐♦♥ ✇✐t❤ ❇r✐❝❡ ▼✉r❝✐❛♥♦✱ ❢r♦♠ ❈❤r✐st♦♣❤ ▼ü••❡r✬s •❛❜ ❛t ❊▼❇▲ ❍❡✐❞❡•❜❡r❣✱ t♦ ❢✉rt❤❡r ❡①♣•♦r❡ t❤❡ ♠♦•❡❝✉•❛r ❜❛s✐s ♦❢ ❛ ♣♦ss✐❜•❡ ✐♥t❡r❛❝t✐♦♥ ❜❡t✇❡❡♥ ❙P❖❈ ❛♥❞ t❤❡ |◆❆P■■ tr❛♥s❝r✐♣t✐♦♥ ♠❛❝❤✐♥❡r②✳ ✻✳✹ ❆rt✐❝•❡ ✷✿ ❙P❊◆ ✐♥t❡❣r❛t❡s tr❛♥s❝r✐♣t✐♦♥❛• ❛♥❞ ❡♣✐✲ ❣❡♥❡t✐❝ ❝♦♥tr♦• ♦❢ ❳✲✐♥❛❝t✐✈❛t✐♦♥ ✭◆❛t✉r❡✳ ✷✵✷✵✮ ❉♦ss✐♥ ❋✱ P✐♥❤❡✐r♦ ■✱ )②•✐❝③ ❏❏✱ |♦❡♥s❝❤ ❏✱ ❈♦••♦♠❜❡t ❙✱ ▲❡ ❙❛✉① ❆✱ ❈❤❡•♠✐❝❦✐ ❚✱ ❆tt✐❛ ▼✱ ❑❛♣♦♦r ❱✱ )❤❛♥ (✱ ❉✐♥❣•✐ ❋✱ ▲♦❡✇ ❉✱ ▼❡r❝❤❡r ❚✱ ❉❡❦❦❡r ❏✱ ❍❡❛r❞ ❊✳ ❙P❊◆ ✐♥t❡❣r❛t❡s tr❛♥s❝r✐♣t✐♦♥❛• ❛♥❞ ❡♣✐❣❡♥❡t✐❝ ❝♦♥tr♦• ♦❢ ❳✲✐♥❛❝t✐✈❛t✐♦♥✳ ◆❛t✉r❡✳ ✷✵✷✵❀ ✺✼✽✭✼✼✾✺✮✿✹✺✺✕✹✻✵✳ ❆|❚■❈▲❊ ❖◆ ◆❊❳❚ P❆•❊ ✶✷✼

SPEN integrates transcriptional and epigenetic control of X-inactivation

François Dossin 1 , Inês Pinheiro 2,8 , Jan J. Żylicz Xist represents a paradigm for the function of long non-coding RNA in epigenetic regulation, although how it mediates X-chromosome inactivation (XCI) remains largely unexplained. Several proteins that bind to Xist RNA have recently been identified, including the transcriptional repressor SPEN [1][2][3] , the loss of which has been associated with deficient XCI at multiple loci [2][3][4][5][6] . Here we show in mice that SPEN is a key orchestrator of XCI in vivo and we elucidate its mechanism of action. We show that SPEN is essential for initiating gene silencing on the X chromosome in preimplantation mouse embryos and in embryonic stem cells. SPEN is dispensable for maintenance of XCI in neural progenitors, although it significantly decreases the expression of genes that escape XCI. We show that SPEN is immediately recruited to the X chromosome upon the upregulation of Xist, and is targeted to enhancers and promoters of active genes. SPEN rapidly disengages from chromatin upon gene silencing, suggesting that active transcription is required to tether SPEN to chromatin. We define the SPOC domain as a major effector of the gene-silencing function of SPEN, and show that tethering SPOC to Xist RNA is sufficient to mediate gene silencing. We identify the protein partners of SPOC, including NCoR/SMRT, the m 6 A RNA methylation machinery, the NuRD complex, RNA polymerase II and factors involved in the regulation of transcription initiation and elongation. We propose that SPEN acts as a molecular integrator for the initiation of XCI, bridging Xist RNA with the transcription machinery-as well as with nucleosome remodellers and histone deacetylases-at active enhancers and promoters.

To assess the importance of SPEN during the initiation of XCI, we used an auxin-inducible degron (AID) 7 that enables controlled and acute depletion of the endogenous SPEN protein. We used our previously described female hybrid (Mus musculus castaneus × C57BL/6) TX1072 8 mouse embryonic stem cells (ES cells), in which a doxycycline (DOX)inducible promoter upstream of the endogenous Xist locus enables conditional Xist RNA expression and XCI (Fig. 1a). In ES cells expressing the Oryza sativa TIR1 (OsTIR1) E3 ligase, we generated a homozygous knock-in that expressed the AID fused to a HaloTag at the C terminus of endogenous SPEN, in order to ensure auxin-dependent SPEN depletion (Extended Data Fig. 1a). Efficient degradation of SPEN occurred within 1 h of auxin treatment (Fig. 1b, Extended Data Fig. 1b, Supplementary Fig. 1) whereas the removal of auxin led to rapid recovery of SPEN (Fig. 1b), demonstrating potent AID-dependent modulation of SPEN levels.

To evaluate the immediate consequences of the loss of SPEN on the initiation of XCI, we acutely depleted SPEN for 4 h before inducing Xist expression for 24 h and performing RNA sequencing. Loss of SPEN had no effect on the formation of Xist RNA clouds (Extended Data Fig. 1c,e), confirming that SPEN is dispensable for Xist localization [2][3][4][5] . However, gene silencing was almost completely abolished along the entire X chromosome in the absence of SPEN (Fig. 1c,d, Supplementary Table 1), whereas auxin had no effect on XCI in wild-type cells (Extended Data Fig. 1d). Clustering analysis highlighted three groups of genes that differed in their silencing defects upon the loss of SPEN (Fig. 1e). Most X-linked genes (80% of 382) were found to be entirely dependent on SPEN for silencing, whereas only a small subset (6%) showed unaltered silencing in the absence of SPEN. This notable defect in XCI was confirmed by pyrosequencing (Fig. 1f) and nascent RNA fluorescence in situ hybridization (FISH) (Extended Data Fig. 1e).

We next assessed the requirement for SPEN in XCI in vivo during mouse early embryogenesis, using allele-specific RNA sequencing in embryonic day (E)3.5 Spen-knockout female embryos 9 harbouring hybrid X chromosomes (Fig. 1g, Extended Data Fig. 1f,g). At this stage in wild-type embryos, imprinted XCI has taken place 10 and only the paternal X chromosome is inactivated (Fig. 1h, Extended Data Fig. 1h). 4 Program in Systems Biology, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA. 5 Institut Curie, PSL Research University, Centre de Recherche, Laboratoire de Spectrométrie de Masse Protéomique, Paris, France. 6 INSERM U1170, Gustave Roussy Institute, Université Paris-Sud, Villejuif, France. 7 Howard Hughes Medical Institute, Chevy Chase, MD, USA. 8 These authors contributed equally: Inês Pinheiro, Jan J. Żylicz. *e-mail: edith.heard@embl.org

Article

In maternal-zygotic Spen knockouts, imprinted XCI is severely hindered although paternal Xist is expressed. Both maternal and paternal X chromosomes are expressed equally, phenocopying Xist-knockout E3.5 embryos 10 (Fig. 1h, Extended Data Fig. 1g, h, Supplementary Table 2). A maternal-only Spen knockout has no effect on imprinted XCI (Fig. 1h), suggesting that the zygotic pool of SPEN is necessary and sufficient for this process. Therefore, the early gene-silencing mechanism(s) involved in imprinted and random XCI are dependent on SPEN.

We next assessed precisely when SPEN is recruited during XCI. HaloTag labelling 11 of SPEN combined with Xist RNA FISH revealed that SPEN associates with Xist RNA rapidly upon Xist coating and throughout XCI (Fig. 2a). To capture early Xist-SPEN dynamics during the short time window in which Xist becomes upregulated, we followed both Xist and SPEN in living cells. We tagged endogenous SPEN with GFP in a background in which Xist RNA is visualized via a BglG-mCherry fusion protein binding to Bgl stem-loops inserted within Xist 12 (Extended Data Fig. 2a,b). Live-cell imaging revealed that SPEN colocalizes with Xist from the very onset of Xist upregulation (Extended Data Fig. 2c,d, Supplementary Video 1). Therefore, SPEN can initiate gene silencing immediately upon Xist coating.

We also found that SPEN robustly accumulated on the inactive X chromosome after differentiation into neural progenitor cells (NPCs, Fig. 2b), in which XCI is epigenetically maintained. The depletion of SPEN for up to two days in independent NPC clones (Fig. 2c) did not lead to reactivation of fully silenced genes (Fig. 2d, Supplementary Table 3); however, we observed moderate but significant upregulation of genes escaping XCI (Fig. 2e,f), which suggests that SPEN buffers the overexpression of X-linked escapee genes in female cells.

Chromosome conformation capture has revealed that, in differentiated cells, the inactive X chromosome is folded into megadomains [13][14][15] and is globally depleted of topologically associating domains except in regions that contain clusters of escapee genes 13 . Xist RNA has been found to have a role in the conformation of the inactive X chromosome 1,14 . To assess whether SPEN is involved, we performed allele-specific Hi-C in NPCs after 48 h of SPEN depletion. No notable conformational changes were observed on the inactive X chromosome (Extended Data Fig. 2e-g); we therefore conclude that the structural effects mediated by Xist RNA in differentiated cells occur independently of SPEN.

In summary, our data suggest that SPEN exerts its role by actively promoting gene silencing during the earliest stages of XCI. However, it has no major role in stabilizing the transcriptionally inactive state of the inactive X chromosome, or in ensuring the maintenance of its conformation.

We next sought to identify which parts of SPEN ensure its function during XCI. SPEN is a very large protein (around 400 kDa) that contains four RNA recognition motifs (RRMs), a nuclear receptor interaction domain (RID) and a SPEN paralogue/orthologue C-terminal (SPOC) domain (Fig. 3a). We overexpressed a series of SPEN complementary DNA truncations, stably targeted into the Rosa26 locus in the SPENdegron mouse ES cell line (Extended Data Fig. 3a, b, Fig. 3a). We then induced Xist expression for 24 h and assessed which SPEN fragments could rescue XCI-initiation function in the context of auxin-mediated depletion of endogenous SPEN. We found that the RRM1 domain and the RID are dispensable for SPEN accumulation on the inactive X chromosome, as well as for X-linked gene silencing (Fig. 3b,c). By contrast, a SPEN truncation lacking the RRM2-4 domains failed to accumulate on the inactive X chromosome and failed to rescue XCI (Fig. 3b,c). SPEN recruitment to the inactive X chromosome is therefore mediated by the RRM2-4 domains and is necessary for gene silencing. This is consistent with studies showing that RRM2-4 directly bind the A-repeat of Xist RNA in vitro 4,16 -a region of Xist that is necessary for gene silencing 17 .

Conversely, a truncation of the SPOC domain enabled efficient SPEN accumulation on the inactive X chromosome, but failed to rescue XCI (Fig. 3b,c). To validate this observation, we performed homozygous deletion of the SPOC domain at the endogenous Spen locus in mouse ES cells (Extended Data Fig. 3c). Deletion of the SPOC domain had no effect either on SPEN recruitment to the inactive X chromosome or on Xist RNA clouds (Extended Data Fig. 3d-f), but resulted in strongly deficient XCI, albeit milder than that in SPEN-depleted cells (Extended Data Fig. 3g-j). Collectively, these results demonstrate that the SPOC domain is essential for XCI. However, other uncharacterized regions of SPEN contribute-albeit to a lesser extent-to ensure its full silencing potential.

To test whether the SPOC domain alone could mediate X-linked gene silencing, we used SPEN-degron ES cells to introduce an array of Bgl stem-loops at the Xist locus (identical to the live-imaging strategy). In this background, we generated several independent ES cell lines expressing a BglG-GFP-SPOC protein fusion (or BglG-GFP as a control) targeted into Rosa26. These proteins would become tethered to Xist-Bgl stem-loop RNA via BglG (Fig. 3d). Notably, upon induction of Xist RNA in the absence of endogenous SPEN, tethering of BglG-GFP-SPOC (but not of BglG-GFP alone) resulted in substantial gene silencing across the X chromosome, with over half of the genes being silenced by more than 50% (Fig. 3e,f). SPOC-specific rescue was confirmed using pyrosequencing (Fig. 3g). Consistent with previous studies [18][19][20] , our results reveal SPOC as a key domain of SPEN that enables gene silencing once recruited to the X chromosome by Xist RNA.

The SPOC domain of SPEN was originally identified as an interactor of the NCoR and SMRT corepressors in human cells 18,21,22 . Given that NCoR and SMRT interact with and activate HDAC3 23 , it was proposed that SPEN triggers XCI via HDAC3 2 , the activity of which is important for Xist-mediated silencing 2,24 . However, XCI is more markedly affected upon the loss of SPEN and SPOC than upon the loss of HDAC3 (Extended Data Fig. 3j,k). These observations suggest that a model involving HDAC3 only partially explains the function of SPEN, and that SPOC must exert its key role in gene silencing also through other, HDAC3independent pathways. To identify such pathways, we characterized the protein interactome of the SPOC domain by performing GFP pull-downs from mouse ES cells that stably expressed BglG-GFP-SPOC (or BglG-GFP as a control, Fig. 3h, Supplementary Table 4), followed by mass spectrometry analysis.

We identified NCoR and SMRT as expected, but we also found HDAC3 (Fig. 3h, Extended Data Fig. 3l), which further supports the proposed model for the function of SPEN in XCI 2 . Notably, we identified the m 6 A methyltransferase complex and the m 6 A reader YTHDC1 (Fig. 3h, Extended Data Fig. 3l), which have been proposed to play a role in XCI 5,20,25 . One of these factors, WTAP, co-purified with Xist RNA in an A-repeat-dependent manner 3 -although, contrary to the case of SPEN, a direct interaction between WTAP and Xist A-repeat has not been reported. Our results therefore suggest that SPOC may participate in the recruitment of m 6 A machinery to Xist RNA. We also identified the NuRD complex-a potent repressor that displaces RNA polymerase II (RNAPII) from transcription start sites through chromatin remodelling 26 -and RNAPII, together with factors that are involved in the regulation of transcription initiation and elongation (Fig. 3h, Extended Data Fig. 3l). Together these findings show that, through its SPOC domain, SPEN bridges Xist to multiple factors that are involved in transcription and chromatin regulation, and together they mediate efficient gene silencing. Given that SPOC immunoprecipitation was performed in the absence of Xist induction, the identified interactions are not mediated by Xist RNA.

We also investigated where SPEN binds to the X chromosome during XCI, and whether it has distinct binding sites or whether it associates with chromatin diffusely across the entire chromosome, as anticipated

Article

from our imaging results. We performed allele-specific, cross-linked CUT&RUN 27 experiments on SPEN during a time course of Xist induction (0 h, 4 h, 8 h, 24 h DOX, or 8 h DOX + auxin as a negative control). We found that there are few binding sites for SPEN across the genome of uninduced ES cells (Extended Data Fig. 4a). Conversely, hundreds of SPEN-binding sites appeared specifically on the X chromosome as early as 4 h after Xist induction (Fig. 4a, Extended Data Fig. 4a). This is consistent with imaging data (Extended Data Fig. 2). We note that SPEN accumulation is seen across the gene body of Xist (Fig. 4b), suggesting that SPEN binds Xist RNA while it is transcribed. In sharp contrast to the Xist locus, SPEN shows focal binding on the rest of the genome, with peaks falling almost exclusively on promoters and enhancers (Fig. 4c,d, Extended Data Figs. 4b, 5a-g).

After Xist induction, recruitment of SPEN to the inactive X chromosome reaches a maximum at 4 h (Fig. 4a, Extended Data Fig. 4c), showing the highest enrichment within regions that were coated earliest by Xist 28 (entry sites, Fig. 4e). SPEN accumulation thus follows the spatial dynamics of Xist spreading. Among promoter targets on the X chromosome, SPEN preferentially binds those of actively expressed genes (Fig. 4f, Extended Data Fig. 4d), which suggests that the ability of SPEN to target chromatin depends on transcriptional activity. Consistently, genes that are classified as fully dependent on SPEN for silencing (Fig. 1e)-which show a greater degree of SPEN binding at their promoters within 4 h of Xist coating than less-dependent genes (Extended Data Fig. 4e)-also show initially higher transcription levels (Fig. 4g).

Furthermore, within 4 h of Xist induction, SPEN binding is greater at the promoters of efficiently silenced genes than at the promoters of less-efficiently silenced genes (Fig. 4h). Similarly, upon Xist coating, efficiently deacetylated enhancers 24 show a higher enrichment of SPEN than less-efficiently deacetylated enhancers (Fig. 4i). Finally, genes that are subject to very little silencing-or those that completely escape XCI in our Xist-inducible system-show a significantly lower SPEN signal at their promoters (Extended Data Figs. 4f,g,). This pattern of SPEN recruitment at discrete sites to the X chromosome that is undergoing XCI indicates that transcriptional silencing is caused by the binding of SPEN to active promoters and enhancers.

To understand how SPEN might function at enhancers and promoters, we integrated CUT&RUN profiles with publicly available data from chromatin immunoprecipitation followed by sequencing (ChIP-seq) experiments for transcription and chromatin-associated factors identified in our mass spectrometry analysis. We included HDAC3 24 , RNAPII 26 and two members of the NuRD complex (MBD3 and CHD4) 26 . SPEN binding strongly overlaps with HDAC3 at enhancers but not at promoters (Extended Data Fig. 4h). Our recent findings revealed that HDAC3 is pre-bound predominantly at enhancers on the X chromosome 24 . Therefore, Xist-mediated recruitment of SPEN to enhancers may activate HDAC3. Conversely, a strong overlap with SPEN binding is observed for the NuRD complex specifically at promoters but not at enhancers (Extended Data Fig. 4h). Furthermore, SPEN peaks extensively overlap with RNAPII phosphorylated on serine 5, which is associated with transcription initiation (Extended Data Fig. 4h). This analysis suggests that SPEN may operate at enhancers and promoters through distinct pathways to promote gene silencing.

Notably, the binding of SPEN to chromatin decreases across the whole X chromosome after 24 h of Xist induction (Fig. 4a, Extended Data Fig. 4c). Clustering of CUT&RUN profiles at SPEN-bound promoters (Extended Data Fig. 4i, Supplementary Table 5) revealed distinct groups of promoters, grouped on the basis of how efficiently SPEN was lost within 24 h of XCI (Fig. 4j). In the 'strong SPEN loss' group, binding was maximal by 4 h but decreased after 8 h, and even more markedly after 24 h (Fig. 4j). Conversely, the 'mild SPEN loss' group showed maximal and persistent SPEN binding at 4 h and 8 h of Xist induction, respectively, with only a mild reduction of SPEN binding by 24 h (Fig. 4j). Finally, a third group-comprising fewer promoters-showed both mild SPEN enrichment at 4 h and low loss at 24 h (Fig. 4j). The group that lost SPEN most efficiently also showed the most pronounced gene silencing by 24 h when compared with the groups that significantly retained SPEN (Fig. 4k, Extended Data Fig. 4j). Altogether this analysis suggests that, once recruited to the X chromosome by Xist RNA, SPEN associates with enhancers and promoters in a transcription-dependent manner. This recruitment leads to gene silencing, after which the favourable transcriptional context for SPEN binding is lost, and SPEN binding to chromatin decreases. Despite loss of the chromatin-bound SPEN fraction, persistent Xist RNA expression and coating ensure that SPEN remains strongly accumulated around the inactive X chromosome (Fig. 2a,b).

Our study demonstrates that SPEN is a crucial factor that collaborates with Xist RNA to initiate gene silencing across the X chromosome, both during XCI in vitro and imprinted XCI in vivo. SPEN becomes dispensable for maintaining gene silencing after XCI has been established, but partially represses escapees, which suggests that Xist may have a silencing role even in somatic cells. Although SPEN coats the X chromosome immediately upon Xist induction, it contacts chromatin only at active promoters and enhancers, which serve as substrates for SPEN-mediated gene silencing. SPEN association with chromatin is favoured by active transcription, as SPEN disengages from chromatin when X-linked genes become silenced. We identify the SPOC domain of SPEN as a potent transcriptional repressor, which is crucial for SPEN-dependent XCI. On the basis of our mass spectrometry analysis, we propose that the SPOC domain is key for bridging Xist with other factors implicated in XCI-such as HDAC3-which we find to be present at most X-linked enhancers to which SPEN is recruited. In particular, the interaction of the SPOC domain with the NuRD complex and the transcription machinery points to a role for SPEN in direct transcriptional repression. We also identify SPOC as an interactor of the m 6 A methyltransferase complex, which has a role in Xist RNA methylation, a modification that is important for Xist-dependent silencing 25 . Methylation of Xist is mediated by RBM15 25 , which interacts with the m 6 A machinery directly through ZC3H13 29 -the most highly enriched m 6 A machinery factor identified in our mass spectrometry experiments. Because RBM15 also carries a SPOC domain, our study raises the possibility that the interaction with the RNA methylation machinery is not restricted solely to the SPOC domain of SPEN, but may instead be a feature that is shared across SPOC-containing proteins.

SPEN binds other non-coding RNAs, including SRA 18 , which is involved in steroid-receptor regulation. Furthermore, another SRA-binding protein-SLIRP-has been shown to bind promoters in an SRA-dependent manner 30 ; this raises the possibility that, similarly to Xist, SRA could guide SPEN to target gene regulatory elements.

In conclusion, our study suggests that RNA-mediated recruitment of SPEN and other SPOC-containing proteins-which are found across fungi, plants and animals-may be a widespread means by which to acutely repress transcription by co-ordinately engaging several layers of epigenetic and transcriptional control. We propose that SPEN bridges Xist to the transcription machinery, histone deacetylases and chromosome remodelling factors to ensure robust and efficient XCI (Extended Data Fig. 4k).

Online content

Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41586-020-1974-9.

Methods

Data reporting and statistical analysis

No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. All statistical tests, resulting P values and observation numbers are indicated in the figure panels or in the figure legends.

Data visualization

All heat maps, violin plots, box plots, density plots, bar graphs and pie charts were generated using ggplot2. Unless stated otherwise, box plots always show the median as the centre line, box limits correspond to upper and lower quartiles, and whiskers cover 1.5× the interquartile range.

Plasmid construction

The plasmids to target OsTIR1 at the TIGRE locus (Addgene plasmid 92141) and the TIGRE-specific guide-RNA-encoding plasmid (Addgene plasmid 92144) were provided by E. Nora. The additional TIGRE-targeting plasmids BglG-mCherry-T2A-OsTir1 (pFD51) and rtTa-VP16-T2A-OsTir1 (pFD68) were cloned using PCR amplification of corresponding gene cassettes followed by traditional cloning into the 92141 backbone.

Targeting constructs (pFD19 and pFD49) to tag endogenous SPEN at its C terminus with AID-HaloTag and AID-GFP, respectively, were generated as follows: 500-bp homology arms (flanking both sides of, but excluding the stop codon of Spen) were amplified from mouse genomic DNA by PCR. One-step Gibson cloning (New England Biolabs) was subsequently used to simultaneously surround the digested AID insert (carrying a puromycin-resistance gene under the control of the PGK promoter) in frame with the homology arms and clone the insert into the pBR322 vector. Synonymous mutations in the PAM/SEED target sequence (located on the 5′ homology arm) were then introduced using the QuickChange II XL site-directed mutagenesis kit (Agilent) to prevent Cas9-mediated cutting of the targeting vector upon transfection and of the AID-tagged allele(s) upon integration. The targeting construct (pFD90) to replace the endogenous SPOC domain of SPEN by GFP was generated using the same strategy. For guide RNA cloning, the pX459 plasmid (a gift from F. Zhang, Addgene 62988) encoding Streptococcus pyogenes Cas9 was digested with BbsI immediately downstream of the U6 promoter, and annealed DNA duplexes corresponding to the target guide RNA sequences were ligated.

Cell culture

Mouse XX ES cells (TX1072) were grown on 0.1% gelatin-coated flasks in 8% CO 2 37 °C incubators. For all experiments, cells were cultured in 2i + LIF, and batch-tested fetal calf serum ES cell medium -DMEM (Sigma), 15% FBS (Gibco), 0.1 mM β-mercaptoethanol, 1,000 U ml -1 leukaemia inhibitory factor (LIF, Chemicon), CHIR99021 (3 μM), PD0325901 (1 μM).

NPC differentiations and subcloning were performed as previously described 13 . NPCs were grown in N2B27 medium supplemented with EGF and FGF (10 ng ml -1 each), on 0.1% gelatin-coated flasks.

Cell transfection and clone isolation

All transgenic insertions were performed using the 4D nucleofector system from Lonza. For each nucleofection, five million cells were electroporated with 2.5 μg each of non-linearized targeting vectors and guide RNA-Cas9 encoding plasmids (MidiPreps). Nucleofected cells were then serially diluted and plated on 10-cm dishes. Forty-eight hours later, antibiotic selection was performed (puromycin, 0.4 μg ml -1 ; hygromycin, 250 μg ml -1 ; blasticidin, 5 μg ml -1 ), except for transfection steps involving flippase-mediated removal of resistance cassettes, during which no selection was applied. One week after the initial plating, 80 to 96 single colonies were picked from dishes showing ideal clonal density and seeded in 96-well plates. These cells were subsequently split into one high-confluency plate used for PCR genotyping, and one low-confluency plate from which desired clones were further expanded until T25 density was reached. At this stage, some cells were kept to reconfirm the correct genotype by PCR, while the remaining cells were frozen.

Cell treatments

Xist expression in TX1072 mouse ES cells was induced upon administration of doxycycline (1 μg ml -1 ). Auxin-mediated depletion of target proteins was achieved by supplementing culture media with auxin (Sigma) at the recommended concentration of 500 μM. Auxincontaining medium was renewed every 24 h. For auxin wash-out, auxincontaining medium was removed, cells were rinsed once with PBS, and exposed to auxin-free medium.

Protein extraction and western blotting

Cells were trypsinized, washed once in medium and once in PBS and then pellets were immediately frozen at -80 °C. Pellets were then resuspended in RIPA buffer (50 mM Tris-HCl pH 8.0-8.5, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) containing protease inhibitors (Roche), incubated for 30 min on ice and sonicated with a Bioruptor (three 10-s pulses). Lysates were then centrifuged for 20 min at 4 °C, and supernatants were kept. Protein concentration was determined using the Bradford (BioRad) assay. Samples were then boiled at 95 °C for 10 min in LDS buffer (Thermo) containing 200 mM DTT. For all western blots except those aimed at detecting SPEN, 4-12% Bis-Tris gels were used. For the detection of SPEN, a high-molecularweight protein (>400 kDa), 3-8% tris-acetate polyacrylamide gels were used. Transfer was performed on a 0.45-μm nitrocellulose membrane using a wet-transfer system, at 350-400 mA for 2 h at 4 °C.

RNA extraction, reverse transcription, pyrosequencing and RNA sequencing

RNA extraction was performed using the RNeasy kit and on-column DNase digestion (Qiagen). Reverse transcription was performed on 1 μg total RNA using SuperScript III (Life Technologies). To quantify allelic skewing, cDNA was amplified using biotinylated primers and subsequently sequenced using Q24 Pyromark (Qiagen). Only samples showing a RNA integrity number greater than 9 were used to prepare RNA sequencing (RNA-seq) libraries (TruSeq). Paired-end 100-nt sequencing was performed on a HiSeq2500 or NovaSeq6000 (Illumina).

RNA FISH

Cells were dissociated using Trypsin (Invitrogen) for ES cells or Accutase (Invitrogen) for NPCs, washed twice in medium, and allowed to attach on poly-l-lysine (Sigma)-coated coverslips for 10 min. Cells were fixed with 3% paraformaldehyde in PBS for 10 min at room temperature, washed in PBS three times, and permeabilized with ice-cold permeabilization buffer (PBS, 0.5% Triton X-100, 2 mM vanadyl-ribonucleoside complex) for 5 min on ice. Coverslips were stored in 70% ethanol at -20 °C. Samples were dehydrated in 4 baths of increasing ethanol concentration (80%, 95%, 100% twice) and air-dried quickly. Probes were prepared from minipreps of intron-spanning bacteria artificial chromosomes (BACs) (clone RP24-157H12 for Huwe1, RP23-260I15 for Atrx) or plasmid (p510 for Xist). Probes were labelled by nick translation (Abbott) using dUTP labelled with spectrum green (Abbott) for Huwe1, spectrum red (Abbott) for Atrx, and Cy5 (Merck) for Xist. Labelled BAC probes were co-precipitated with Cot-1 DNA repeats in the presence of ethanol and salt, resuspended in formamide, denatured at 75 °C for 10 min, and competed at 37 °C for 1 h. Probes were then co-hybridized in FISH hybridization buffer (50% formamide, 20% dextran sulfate, 2X SSC, 1 μg μl -1 BSA, 10 mM vanadyl-ribonucleoside) at 37 °C overnight. The next day, hybridized coverslips were washed three times for 5 min Article with 50% formamide in 2X SSC at 42 °C, and three times for 5 min with 2X SSC. DAPI (0.2 mg ml -1 ) was added to the penultimate wash and coverslips were mounted with Vectashield (Vectorlabs).

HaloTag labelling

HaloTag labelling of the SPEN-Halo fusion protein was performed in live TX1072 ES cells and NPCs. Cells were labelled with HaloTag-ligandconjugated Janelia Fluor 11 ( JF646-HaloTag or JF549-HaloTag, a gift from L. Lavis) at a final concentration of 250 nM in culture medium. Labelling was performed for 1 h at 37 °C, cells were then washed 4 times with generous volumes of PBS, and incubated with unlabelled medium for 15 min before proceeding with downstream experiments. For NPC labelling, cells were washed with unlabelled medium and not PBS, because NPCs detach when exposed to PBS. Auxin and/or doxycycline were kept in the labelling medium when necessary.

HaloTag labelling followed by RNA FISH

For co-detection of SPEN-Halo and Xist RNA, cells were labelled with JF549 as indicated above, and directly processed for fixation and permeabilization as detailed in the section 'RNA FISH'. Importantly, after permeabilization, coverslips were directly washed twice with PBS, twice with 2X SSC and immediately processed for FISH.

Mouse breeding, embryo collection and single-embryo RNA-seq

Timed natural matings were used for all experiments. Noon of the day when the vaginal plugs of mated females were identified was scored as E0.5. For Spen matings a conditional allele was used 9 . For oocyte deletions the published Rosa26:Zp3-Cre allele was used 31 . F 1 hybrid Spen +/-males were obtained by crossing Spen +/+ CAST/EiJ females with Spen +/-C57BL/6J males. For Spen maternally deleted embryos, Spen flox/flox Zp3-Cre +ve C57BL/6J females were crossed with Spen +/-F 1 hybrid males. For Spen control embryos, Spen flox/flox Zp3-Cre -ve C57BL/6J females were crossed with Spen +/-F 1 hybrid males. The care and use of animals in this study was performed in accordance with the recommendations of the European Community (2010/63/UE). All experimental protocols were approved by the ethics committee of Institut Curie CEEA-IC118 under the number APAFIS#8812-2017020611033784v2 given by national authority in compliance with the international guidelines. Single-embryo RNA-seq was performed as previously described 32 . In brief, E3.5 embryos were collected and morphologically assessed to ensure that only viable samples were collected. The zona pellucida was removed by treatment with acidified Tyrode's solution. Single embryos were picked into individual tubes and cDNA was prepared and amplified as previously described 33 . Illumina libraries were prepared as published in ref. 34 . Paired-end 100-nt sequencing was performed with HiSeq2500 (Illumina).

Live-cell imaging and analysis

Cells were seeded on fibronectin-coated 35-mm glass-bottom dishes (Ibidi) 24 h before imaging. Doxycycline was added 1 h before image acquisition. Cells were imaged on the DeltaVision OMX microscope in widefield mode (GE Healthcare) using a 1.4 numerical aperture 100× oil immersion objective. The temperature was controlled at 37 °C and CO 2 at 8% during acquisition.

Images were acquired as z-stacks of 40 slices with 400-nm steps every 10 min for at least 4 h. Movies were deconvolved using Huygens deconvolution with the following parameters: Iteration 4; S/N 5, 10; quality threshold 0.1; and widefield mode 0.7 was used for background estimation. Two channels were registered using TetraSpec microspheres 0.1 μm (Invitrogen) and unwarpJ (Fiji plug-in). For segmentation, z-projected deconvolved registered images were used and pixels were classified as cloud or nuclei using Ilastik. Touching nuclei were sometimes manually separated. Cut-offs on resulting probability maps were set to 0.7. We next performed connected component analysis to obtain integer-labelled images in which each integer label corresponds to a unique nucleus. In the tailor-made Fiji plug-in the inputs are the raw max z-projected time-lapse images of the two channels and the integer labelled time-lapse image of the nuclei. The probability maps of the clouds give the region of interest in the time-lapse sequence in which total intensity is calculated. In the plug-in, clouds are associated with their corresponding nuclei; they are then linked via Kalman filter tracker over time. These unique links constitute track IDs and contain information about the intensity and area measurements for each cell. For each tracked cell, the first time point when a cloud is detected in one channel (Xist or SPEN) is labelled as reference time point 1.

Hi-C

Hi-C was performed as previously described 35 , except that ligated DNA size selection was omitted, and dA-tailing was performed before biotin pull-down. In brief, each Hi-C experiment was performed on 10 million cells (NPCs) per sample. Cells were digested with DpnII at 37 °C overnight. DNA ends were filled with biotin-14-dATP at 23 °C for 4 h. DNA was then ligated with T4 DNA ligase at 16 °C overnight. Binding proteins were removed by treating ligated DNA with proteinase K at 65 °C overnight. Purified proximally ligated molecules were fragmented to obtain an average fragment size of 200 bp. After DNA end repair, dA-tailing and biotin enrichment, DNA molecules were ligated to Illumina TruSeq sequencing adapters at room temperature for 2 h. Final library PCR productions were carried out following the Illumina TruSeq Nano DNA Sample Prep Kit manual. Paired-end 100-nt sequencing was performed on a HiSeq4000 (Illumina).

Genetic engineering strategy for Xist-Bgl stem-loop tagging and SPEN complementation analysis constructs

To tag Xist with Bgl stem-loops 36 , we nucleofected cells with pBS-Ptight-Xist-BglSL 12 (plasmid harbouring 18 repeats of Bgl stem-loops inserted between homology arms to target Xist exon 7, carrying a G418 selection gene, a gift from O. Masui). After G418 selection and FLP-FRT mediated removal of the selection cassette, clones were picked and genotyped. Positive clones were further tested to ensure that the stem-loop-tagged Xist could properly be induced and trigger gene silencing upon addition of doxycycline (data not shown).

Spen cDNA truncations were generated by splicing out different regions of the Spen open reading frame (Genscript, ORF clone OMu11416C) using overlap extension PCR. Each Spen truncation was cloned downstream of a CAGGS promoter into a vector carrying homology arms for targeted insertion at the Rosa26 37 locus as well as a SV40promoter driven hygromycin-resistance gene. The BglG-GFP-SPOC targeting plasmid was designed by inserting a translational fusion between a BglG-GFP cassette and SPEN amino acids 3244-3643 into the same Rosa26 targeting vector. Each of these 'complementation' constructs were independently targeted at Rosa26 in SPEN-degron mouse ES cells. Independent clones were picked and protein expression of each SPEN truncation was assessed by western blot. XCI complementation analysis was then performed in 2-3 independent clones for Spen cDNA truncations, and 4 independent clones for BglG-GFP-SPOC expressing clones. The ability of cells to accumulate BglG-mCherry, BglG-GFP and BglG-GFP-SPOC upon the addition of doxycycline was assessed using microscopy (data not shown).

Immunofluorescence

ES cells were dissociated using trypsin (Invitrogen), washed extensively in medium, and allowed to attach on poly-l-lysine (Sigma)-coated coverslips for 10 min. Cells were then fixed with 3% paraformaldehyde in PBS for 10 min at room temperature, washed in PBS three times, and permeabilized with 0.25% Triton X-100 in PBS for 5 min at room temperature. Coverslips were then washed three times in PBS and blocked for 1 h with blocking buffer (PBS containing 2.5% BSA, 0.1% Tween20 and 10% normal goat serum). Coverslips were then incubated with primary antibodies diluted in blocking buffer at 4 °C overnight, washed three times for 5 min in PBST (0.1% Tween20) the next day, incubated with fluorescently labelled secondary antibodies (1/500 in blocking buffer) for 1 h at room temperature, and washed again three times for 5 min in PBST. DAPI (0.2 mg ml -1 ) was added to the penultimate wash and coverslips were mounted with Vectashield (Vectorlabs).

Immunoprecipitation

Nuclear extracts were prepared by resuspending 50 million fresh cells in ice-cold 10 ml buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl 2 , 0.1% NP-40, c0mplete EDTA free, phosSTOP) and rotating for 10 min at 4 °C. Nuclei were centrifuged at 800g for 10 min at 4 °C and resuspended in 1 ml IP buffer C150 (20 mM HEPES pH 7.9, 150 mM NaCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 0.25% NP-40, cOmplete EDTA free, phosSTOP). Lysates were briefly sonicated followed by Benzonase (Merck) digestion for 30 min at 4 °C. Finally, lysates were cleared through centrifugation at 13,000 rpm for 20 min before being incubated with 15 μl of GFP trap magnetic agarose bead slurry (ChromoTek) overnight at 4 °C. Beads were washed 5 times in IP buffer. For co-immunoprecipitation (Co-IP) western blot, washed beads were directly resuspended in LDS buffer (Thermo) containing 200 mM DTT, and boiled at 95 °C for 10 min.

Proteomics and mass spectrometry analysis

Proteins on magnetic beads were washed twice with 100 μl of 25 mM NH 4 HCO 3 and we performed on-bead digestion with 0.2 μg of trypsin/ LysC (Promega) for 1 h in 100 μl of 25 mM NH 4 HCO 3 . Samples were then loaded onto homemade C18 StageTips for desalting. Peptides were eluted using 40/60 MeCN/H 2 O + 0.1% formic acid and concentrated to dryness under vacuum. Online chromatography was performed with an RSLCnano system (Ultimate 3000, Thermo Scientific) coupled online to a Q Exactive HF-X with a Nanospay Flex ion source (Thermo Scientific). Peptides were first trapped on a C18 column (75 μm inner diameter × 2 cm; nanoViper Acclaim PepMap 100, Thermo Scientific) with buffer A (2/98 MeCN/H 2 O in 0.1% formic acid) at a flow rate of 2.5 μl min -1 over 4 min. Separation was then performed on a 50 cm × 75 μm C18 column (nanoViper Acclaim PepMap RSLC, 2 μm, 100 Å, Thermo Scientific) regulated to a temperature of 50 °C with a linear gradient of 2% to 30% buffer B (100% MeCN in 0.1% formic acid) at a flow rate of 300 nl min -1 over 91 min. Mass spectrometry full scans were performed in the ultra-high-field Orbitrap mass analyser over the range m/z 375-1,500 with a resolution of 120,000 at m/z 200. The top 20 most intense ions were subjected to Orbitrap for further fragmentation via high-energy collision dissociation activation and a resolution of 15,000 with the intensity threshold kept at 1.3 × 10 5 . We selected ions with charge state from 2 + to 6 + for screening. Normalized collision energy was set at 27 and the dynamic exclusion at 40 s. For identification, the data were searched against the M. musculus (UP000000589) Uniprot database using Sequest HF through Proteome Discoverer (v.2.2). Enzyme specificity was set to trypsin and a maximum of two missed-cleavage sites were allowed. Oxidized methionine and N-terminal acetylation were set as variable modifications. Maximum allowed mass deviation was set to 10 ppm for monoisotopic precursor ions and 0.02 Da for MS/ MS peaks. The resulting files were further processed using myProMS 38 v3.6 (work in progress). Calculation of the false discovery rate used Percolator and was set to 1% at the peptide level for the whole study. The label-free quantification was performed by peptide extracted ion chromatograms (XICs) computed with MassChroQ version 2.2 39 . For protein quantification, XICs from proteotypic peptides shared between compared conditions (TopN) with two missed cleavages were used. Median and scale normalization was applied on the total signal to correct the XICs for each biological replicate. To estimate the significance of the change in protein abundance, a linear model (adjusted on peptides and biological replicates) was performed and P values were adjusted with a Benjamini-Hochberg false-discovery rate procedure with a control threshold set to 0.05. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD015699.

Cross-linked CUT&RUN

CUT&RUN against SPEN was performed during a timecourse of Xist induction/SPEN degradation: 0 h DOX, 4 h DOX, 8 h DOX, 24 h DOX and 8 h DOX + auxin. Two biological replicates were performed. The original CUT&RUN protocol 27 was adapted for fixed cells: 10 6 cells in suspension were fixed with 2% formaldehyde diluted in PBS for 10 min at room temperature (2 ml final volume). Fixation was quenched with 125 mM glycine for 5 min and cells were washed twice in 1 ml PBS. Fixed cells were then permeabilized with 1 ml permeabilization buffer (20 mM HEPES pH 7.9, 150 mM NaCl, 0.5 mM spermidine, 0.25% TritonX-100, c0mplete EDTA free) for 5 min and washed twice in 1 ml PBS. Cells were then resuspended in 1 ml washing buffer (20 mM HEPES pH 7.9, 150 mM NaCl, 0.5 mM spermidine, 0.1% BSA, cOmplete EDTA free), bound to activated concanavalin beads (50 μl bead slurry used per 10 million cells) for 10 min, and blocked in 1 ml blocking buffer (wash buffer + 2 mM EDTA) for 5 min. At this stage, cells were resuspended in 500 μl wash buffer containing target antibodies diluted 1/200, transferred to 0.5-ml tubes, and incubated overnight at 4 °C on an end-to-end rotator. Cells were washed three times in 500 μl washing buffer followed by 1-h incubation with pA-MNase (500 μl of washing buffer containing 700 ng ml -1 pA-MNase, produced by the Protein Expression and Purification Core Facility of Institut Curie) and washed again three times in 500 μl washing buffer. After the last wash, cells were resuspended in 150 μl washing buffer, transferred to 1.5-ml tubes, and equilibrated to 0 °C in a metal block for 10 min. To start digestion, CaCl 2 was added to a final concentration of 1.5 mM, taking care to return each sample to 0 °C immediately afterwards. Digestion was performed at 0 °C for 1 h, before being stopped by adding 150 μl of 2X-STOP solution (200 mM NaCl, 20 mM EDTA, 5 mM EGTA, 0.1% NP-40, 40 μg ml -1 glycogen). RNase A was added to a final concentration of 50 μg ml -1 and samples were incubated at 37 °C for 20 min. SDS and proteinase K were then added to final concentrations of 0.1% and 300 μg ml -1 , respectively, and samples were incubated at 56 °C for 2 h followed by 68 °C for 16 h to reverse cross-linking. Total DNA was extracted using phenol/chloroform followed by two rounds of ethanol precipitation and DNA size selection (using 0.55× volume of Ampure XP beads relative to the DNA sample volume) to remove the large predominating undigested DNA fragments. Each time, beads were discarded and the supernatant (containing the selected small fragments resulting from MNase digestion) was precipitated with ethanol. After elution in 50 μl TE buffer, samples were quantified and analysed using Qubit and Tapestation assays. CUT&RUN libraries were prepared from 50 ng DNA per sample, using the Accel-NGS 2S Plus DNA Library Kit (Swift) according to the manufacturer's protocol. Paired-end 100-nt sequencing was performed on a HiSeq2500 (Illumina).

Bioinformatics analyses

All data were mapped to the mouse genome mm10, using the BL6-EiJ/ CAST SNPs from the mouse genome project (v.5 SNP142), and the gene annotation from ensembl (v.92). Analyses were performed in R (v.3.4.2) and Bioconductor (v.3.6). See ref. 24 for more details.

RNA-seq analysis

Reads were trimmed using Trimgalore (v.0.4.4), mapped using STAR (2.5.3a, parameters:-outFilterMultimapNmax 1-outFilterMismatch-Nmax 999-outFilterMismatchNoverLmax 0.06-alignIntronMax 500000-alignMatesGapMax 500000-alignEndsType EndToEnd-out-SAMattributes NH HI NM MD), and removed when mapping to the mitochondrial genome. Remaining reads were split by allele using SNPsplit (v.0.3.2). Allele-specific and the unassigned bam files were sorted, duplicates removed using picard (v.2.18.2, parameters: REMOVE_DUPLI-CATES = true ASSUME_SORTED = true) and pooled as the total reads.

Article

Quantification of expression was performed using featureCount (parameters: -p -t exon -g gene_id, -s 1 for stranded RNA-seq of in vitro cell, -s 0 for non-stranded RNA-seq of single embryo). Data were then analysed in R using DESeq2 (v.1.18.1), calculating the sizeFactor on the count of total reads and applying it to the allele-specific counts.

For all RNA-seq analysis (SPEN-degron mouse ES cells, NPCs, and Spen-knockout embryos), genes showing less than 10 total allelic reads in at least one sample were discarded from the analysis. Allelic ratios were then computed for genes as follows: allelic_ratio = reads B6 / (reads B6 +reads Cast ). Allelic ratios were then averaged between biological replicates.

RNA-seq analysis of SPEN-degron mouse ES cells. In order to define differential dependencies on SPEN for gene silencing during XCI in mouse ES cells, we removed skewed genes (that is, genes showing allelic ratios outside of a [0.15;0.85] interval in control conditions) from the analysis in Fig. 1d. We then defined a silencing index, translating how much a gene is silenced after 24 h of Xist induction with respect to the control condition: silencing_index = 1 -(allelic_ratio DOX /allelic_ratio con- trol ). We next filtered out genes showing less than 10% silencing (that is, silencing_index ≤ 0.1) in SPEN non-depleted conditions.

k-means with three clusters was then performed on the raw allelic_ ratios across control, 24 h DOX and 24 h DOX + auxin conditions. Clustering identified three groups of genes differing by their response to loss of SPEN during Xist induction. To define how dependent on SPEN a gene is for silencing, we expressed the silencing defect observed upon loss of SPEN as a fraction of the total silencing that normally occurs in the presence of SPEN. Computationally, this translates in: Spen_ dependence_index = 1 -(silencing_index DOX + aux /silencing_index DOX ). A Spoc_dependence_index was derived identically.

For integration with Hdac3-knockout RNA-seq during XCI, we integrated the SPEN-degron dataset with an Hdac3-knockout RNA-seq dataset 24 generated from the same mouse ES cell background (TX1072) and at the same time point of Xist induction. The dataset was processed identically, and an Hdac3-dependence index was also computed as follows: Hdac3_dependence_index = 1 -(silencing_index Hdac3KO /silenc-ing_index WT ).

Spen-knockout E3.5 embryo RNA-seq analysis. Integration of Spenknockout and Xist-knockout embryo datasets was performed by integrating our Spen-knockout E3.5 female embryo RNA-seq dataset with a Xist-knockout single-cell RNA-seq (processed as pseudo-bulk for our analysis) dataset from E3.5 female embryos 10 , also generated from a M. musculus domesticus × M. musculus castaneus mouse background.

SPEN-degron NPC RNA-seq analysis. In NPCs, X-linked genes were defined as escapees if their transcript allelic ratio was greater than 0.15 in at least one condition (0 h, 24 h or 48 h of SPEN depletion).

CUT&RUN bioinformatics analysis

Reads were trimmed using Trimgalore (v.0.4.4), mapped using STAR (2.5.3a, parameters:-outFilterMultimapNmax 1-outFilterMismatch-Nmax 999-outFilterMismatchNoverLmax 0.06-alignIntronMax 1-alignMatesGapMax 2000-alignEndsType EndToEnd-outSAMattributes NH HI NM MD), and removed when mapping to the mitochondrial genome. Remaining reads were split by allele using SNPsplit (v.0.3.2). Allele-specific and the unassigned bam files were sorted, duplicates removed using picard (v.2.18.2, parameters: REMOVE_DUPLI-CATES = true ASSUME_SORTED = true) and pooled as the total reads. BigWig of coverage files were performed using DeepTools bamCoverage (parameters:-extendReads-binSize 1, with-extendReads 200 for single end data). A scaling factor was calculated as 10 6 /total number of reads, and the same factor was given as the parameter-scaleFactor for both allelic signals. Peak calling was performed using macs2 (v.2-2.1.2.1, parameters for CUT&RUN:-bw 300 -f BAMPE -q 0.01-keep-dup auto-broad for CUT&RUN and pol2S5 ChIPseq;-bw 300 -f BAMPE -q 0.01-keep-dup auto-call_summits for other ChIPseq). For quantification of signal in peaks, reads were counted using the featureCounts function from Subread (v.1.28.1, parameters: -p -s 0). Data scaling was performed in R using DESeq2 (v.1.18.1), calculating the sizeFactor on the counts of total reads in 10-kb windows and applying it to the allelespecific counts in peaks.

Peak filtering. SPEN-specific peaks were defined as having log 2 Fold-Change ≥ 1 compared to auxin treatment (negative control, SPEN-degraded), and an adjusted P value ≤ 0.001.

Total SPEN enrichment in promoter window. To compare SPEN accumulation among promoters of all X-linked genes in an unbiased manner-including genes that fail to have any peak called at their promoters-we performed DEseq analysis on counts spanning total promoter windows.

Genomic features and integration with RNA-seq. Promoters were defined as ±2-kb windows centred around the transcription start sites of genes. Putative active enhancers and their deacetylation kinetics during XCI were obtained from ref. 24 . Gene-silencing efficiency was determined according to the silencing_index defined in the section 'RNA-seq analysis'. We observed that our silencing_index ranges between 0 and 0.9. Hence, we split this interval in three to define high, medium and low gene-silencing efficiency groups with silencing_index comprising [0.6,0.9], [0.3,0.6[and [0,0.3[respectively.

Integration with publicly available ChIP-seq data. SPEN peaks were intersected with other peaks called from publicly available ChIP-seq data for HDAC3 24 (same cellular background, TX1072, 2i + LIF condition), RNAPII-pS5 26 , CHD4 26 and MBD3 26 (all in the 2i + LIF condition).

Hi-C analysis

Data were processed with HiC-Pro (v.2.11.0) in allele-specific mode. Only pairs with both reads having MAPQ > 30 were kept. Matrices were made using cooler cload (v.0.8.5) at 1-kb or 10-kb resolution, using HiGlass for visualization and snapshots. Topologically associating domains were called using HicExplorer HicFindTads (parameters:-correctFor-MultipleTesting fdr-minDepth 250000-maxDepth 4000000-step 50000-thresholdComparisons 0.1-delta 0). The expected value for the Hi-C signal was calculated on the non-allele-specific signal using cooltool compute-expected. Average scaled matrices of observed/ expected values for allele-specific signal were produced with Coolpup. py (parameters:-local-rescale-rescale_size 299), using non-allelespecific expected values to normalize both alleles to the same expected values. Average heat maps were plotted using plotpup.py. For quantification, the Hi-C signal was averaged over topologically associating domains upper-triangle for each allele-specific matrix (10 kb) using the hicExplorer hicSummarizeScorePerRegion available at https:// github.com/heard-lab/HiCExplorer (parameters:-summarizeType mean-rmDiag 1).

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper. Xist for 24 h (n, number of counted cells). f, RNA FISH for Xist (red) and Huwe1 (green) in SPOC-deletion and wild-type cells treated with DOX for 24 h. g, Violin plot showing the distribution of X-chromosomal transcript allelic ratios (measured by RNA-seq) after 0 h or 24 h DOX treatment in wild-type and SPOCdeletion mouse ES cells. Horizontal lines denote the median, box limits correspond to upper and lower quartiles, averages of three independent clones shown, n = 469 genes, two-sided Student's t-test. h, Bar graph of transcript allelic ratios (obtained from pyrosequencing) for four X-linked genes in SPOCdeletion (blue) or wild-type (grey) cells. Bars show mean values for three independent SPOC-deletion clones (*P < 10 -4 , two-sided Student's t-test).

i, Bar graph showing the proportion of cells expressing Huwe1 monoallelically (white) or biallelically (grey), assayed by RNA FISH, in wild-type cells and in three independent SPOC-deletion clones after induction of Xist for 24 h (n, number of counted cells). j, Density plot showing the distribution of gene silencing defects (see Methods) observed across the X chromosome in RNAseq data from HDAC3-knockout 24 SPEN-degron and SPOC-deletion (this study) ES cells after 24 h of Xist induction. k, Bar graph of normalized allelic ratios (obtained from pyrosequencing) for four X-linked genes in HDAC3-knockout (brown), SPOC-deletion (blue) and wild-type (grey) cells after 24 h of Xist induction. Bars show mean values for two independent HDAC3 clones and three independent SPOC deletion clones; individual data points are shown. l, Volcano plot of fold changes in GFP-pull-down (BglG-GFP-SPOC compared with BglG-GFP) and their adjusted P values (Benjamini-Hochberg procedure, see Methods for statistical analysis). Quantitative label-free mass spectrometric analysis was performed on four independent biological replicates. In b, d, f, experiments were repeated at least twice with similar results. 1e). f, Box plots showing SPEN enrichment after 4 h of Xist induction within promoter windows of genes grouped on the basis of whether or not they are silenced at 24 h of Xist induction (see Methods). In d-f, data were analysed using the two-sided Wilcoxon rank-sum test, horizontal lines denote the median, box limits correspond to upper and lower quartiles. g, UCSC Genome Browser allelespecific track showing SPEN binding around Kdm6a, an escaping gene (blue, Cast-Xa; red, B6-Xi; all tracks are scaled identically). h, Bar graphs showing overlap between SPEN-binding sites and the binding sites of four different factors at X-linked enhancers and promoters. i, j, Heat maps showing normalized SPEN enrichment (log 2 ) at promoters (both replicates are shown) (i) and gene silencing kinetics (allelic ratio) during XCI ( j) within three groups of X-linked genes showing different dynamics of SPEN accumulation and loss. k, Schematic of the function of SPEN in XCI. In a-f, h-j, data are from two biological replicates.

Article

Laboratory animals

We used adult animals (from 6 weeks to 3 months for females, and 7 weeks to 1 year for males) for producing preimplantation embryos (first 4 days post mating). For Spen matings a published conditional allele was used (Yabe at al., 2007). For oocyte deletions published Rosa26:Zp3-Cre allele was used (DeVries et al., 2000). F1 hybrid Spen+/-males were obtained by crossing Spen+/+ CAST/EiJ females with Spen+/-C57BL/6J males. For Spen maternally deleted embryos, Spenflox/flox Zp3-Cre+ve C57BL/6J females were crossed with Spen+/-F1 hybrid males. For Spen control embryos, Spenflox/flox Zp3-Cre-ve C57BL/6J females were crossed with Spen+/-F1 hybrid males.

Wild animals this study does not contain any wild animals

Field-collected samples this study does not contain animals collected from the fields.

Ethics oversight

Animal care and use for this study were performed in accordance with the recommendations of the European community (2010/63/UE). All experimental protocols were approved by the ethics committee of Institut Curie CEEA-IC118 under the number APAFIS#8812-2017020611033784v2 given by national authority in compliance with the international guidelines.

Note that full information on the approval of the study protocol must also be provided in the manuscript. Endogenous retroviruses (ERVs) are abundant and heterogenous groups of integrated retroviral sequences that affect genome regulation and cell physiology throughout their RNA-centred life cycle 1 . Failure to repress ERVs is associated with cancer, infertility, senescence and neurodegenerative diseases 2,3 . Here, using an unbiased genome-scale CRISPR knockout screen in mouse embryonic stem cells, we identify m 6 A RNA methylation as a way to restrict ERVs. Methylation of ERV mRNAs is catalysed by the complex of methyltransferase-like METTL3-METTL14 4 proteins, and we found that depletion of METTL3-METTL14, along with their accessory subunits WTAP and ZC3H13, led to increased mRNA abundance of intracisternal A-particles (IAPs) and related ERVK elements specifically, by targeting their 5′ untranslated region. Using controlled auxin-dependent degradation of the METTL3-METTL14 enzymatic complex, we showed that IAP mRNA and protein abundance is dynamically and inversely correlated with m 6 A catalysis. By monitoring chromatin states and mRNA stability upon METTL3-METTL14 double depletion, we found that m 6 A methylation mainly acts by reducing the half-life of IAP mRNA, and this occurs by the recruitment of the YTHDF family of m 6 A reader proteins 5 . Together, our results indicate that RNA methylation provides a protective effect in maintaining cellular integrity by clearing reactive ERV-derived RNA species, which may be especially important when transcriptional silencing is less stringent.

|❡s✉•ts ❙P❖❈✴❙❡r✺P✲❈❚❉ ✐♥t❡r❛❝t✐♦♥ ❛♥❛•②s✐s ❈❤❛♣t❡r ✼ ❙tr✉❝t✉r❛• ❛♥❞ ❜✐♦♣❤②s✐❝❛• ❛♥❛•②s✐s ♦❢ t❤❡ ❙P❖❈✴❙❡r✺P✲❈❚❉ ✐♥t❡r❛❝t✐♦♥ ✼✳✶ |❛t✐♦♥❛•❡ ❢♦r ✐♥✈❡st✐❣❛t✐♥❣ ❛ ♣♦t❡♥t✐❛• ♣❤♦s♣❤♦❞❡✲ ♣❡♥❞❡♥t ✐♥t❡r❛❝t✐♦♥ ❜❡t✇❡❡♥ ❙P❖❈ ❛♥❞ t❤❡ ❈❚❉ ♦❢ |◆❆P■■ ❆♠♦♥❣ ❛•• ♣r♦t❡✐♥ ✐♥t❡r❛❝t✐♦♥s ❞❡t❡❝t❡❞ ✐♥ ♦✉r ❙P❖❈✲✐♠♠✉♥♦♣r❡❝✐♣✐t❛t✐♦♥ ✭s❡❡ s✉❜✲ s❡❝t✐♦♥ ✻✳✸✳✸✮✱ t❤❡ ♣♦t❡♥t✐❛• ✐♥t❡r❛❝t✐♦♥ ✇✐t❤ ❝♦♠♣♦♥❡♥ts ♦❢ t❤❡ ❣❡♥❡r❛• tr❛♥s❝r✐♣t✐♦♥ ♠❛❝❤✐♥❡r② r❛✐s❡s t❤❡ ❡①❝✐t✐♥❣ ♣♦ss✐❜✐•✐t② t❤❛t ❙P❊◆ ❝♦✉•❞ ❞✐r❡❝t•② r❡❣✉•❛t❡ tr❛♥s❝r✐♣t✐♦♥✳ ■❢ ❝♦♥✜r♠❡❞✱ t❤✐s ❝♦✉•❞ ❡①♣•❛✐♥ ❤♦✇ ❣❡♥❡s ❛r❡ t✉r♥❡❞ ♦✛ s♦ ❡✣❝✐❡♥t•② ✉♣♦♥ ❳✐st ❝♦❛t✐♥❣ ♦❢ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡✳ ❲❤✐•❡ ♣♦✇❡r❢✉• ❢♦r ❞❡t❡❝t✐♥❣ ♣r♦t❡✐♥ ♣❛rt♥❡rs ♦❢ ❙P❖❈✱ t❤❡ ■P✲▼❙ ❢r❛♠❡✇♦r❦ ❞♦❡s ♥♦t ❛••♦✇ t♦ ❝♦♥❝•✉❞❡ ✇❤❡t❤❡r ❛♥② ♦❢ t❤❡ ❞❡t❡❝t❡❞ ♣❛rt♥❡rs ✐♥t❡r❛❝t ❞✐r❡❝t•② ✇✐t❤ ❙P❖❈✱ ❛♥❞ ❤❡♥❝❡ ♣r❡❝•✉❞❡s ❞r❛✇✐♥❣ ❢✉rt❤❡r ♠❡❝❤❛♥✐st✐❝ ❝♦♥❝•✉s✐♦♥s✳ ■♥ ❢❛✈♦r ♦❢ ❛ ❞✐r❡❝t ✐♥t❡r❛❝t✐♦♥ ❜❡t✇❡❡♥ ❙P❖❈ ❛♥❞ |◆❆ ♣♦•②♠❡r❛s❡ ■■✱ ♦✉r ❈❯❚✫|❯◆ ❡①♣❡r✐♠❡♥t r❡✈❡❛•❡❞ t❤❛t✿ ❼ ❙P❊◆ r❡❝r✉✐t♠❡♥t t♦ ❳✲•✐♥❦❡❞ ❝❤r♦♠❛t✐♥ ♣r❡❞♦♠✐♥❛♥t•② ♦✈❡r•❛♣s ✇✐t❤ |◆❆P■■✲ ❜♦✉♥❞ s✐t❡s✱ ❛t tr❛♥s❝r✐♣t✐♦♥❛••② ❛❝t✐✈❡ ❣❡♥❡s ✭s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❊①t❡r♥❛• ❉❛t❛ ❋✐❣✳ ✹❤✮✳ ❼ ❙P❊◆ ❜✐♥❞✐♥❣ t♦ ❝❤r♦♠❛t✐♥ ✐s •♦st ❢♦••♦✇✐♥❣ ❳✲•✐♥❦❡❞ ❣❡♥❡ s✐•❡♥❝✐♥❣ ✭✐✳❡✳ ✉♣♦♥ ❞❡❝r❡❛s✐♥❣ •❡✈❡•s ♦❢ ❝❤r♦♠❛t✐♥✲❜♦✉♥❞ |◆❆P■■✱ s❡❡ ❉♦ss✐♥ ❡t ❛•✳✱ ❋✐❣✳ ✹•✱ ❦✮✳ ❍♦✇❡✈❡r✱ ❙P❊◆ ❜✐♥❞✐♥❣ ✐s r❡str✐❝t❡❞ t♦ ❡♥❤❛♥❝❡r ❛♥❞ ♣r♦♠♦t❡r ❢❡❛t✉r❡s✱ ✇✐t❤ ✈✐rt✉❛••② ✶✺✶ |❡s✉•ts ❙P❖❈✴❙❡r✺P✲❈❚❉ ✐♥t❡r❛❝t✐♦♥ ❛♥❛•②s✐s ♥♦ ✧❞✐✛✉s❡✧ ❜✐♥❞✐♥❣ ❞❡t❡❝t❡❞ ✇✐t❤✐♥ ❣❡♥❡ ❜♦❞✐❡s✱ ❝♦♥tr❛r② t♦ ✇❤❛t ✇♦✉•❞ ❤❛✈❡ ❜❡❡♥ ❡①♣❡❝t❡❞ s❤♦✉•❞ ❙P❊◆✴❙P❖❈ ✐♥t❡r❛❝t ✇✐t❤ |◆❆P■■ ❣❡♥❡r❛••②✳ ❙P❊◆ ❜✐♥❞✐♥❣ ❞♦❡s ❤♦✇❡✈❡r str♦♥❣•② r❡ss❡♠❜•❡ t❤❛t ♦❢ ❛ ♣❡❝✉•✐❛r ❢r❛❝t✐♦♥ ♦❢ |◆❆ ♣♦•②♠❡r❛s❡ ■■✿ t❤❛t ✇❤✐❝❤ ✐s ♣❤♦s♣❤♦r②•❛t❡❞ ♦♥ ❙❡r✐♥❡ ✺ ♦❢ t❤❡ ❈✲t❡r♠✐♥❛• ❞♦♠❛✐♥ ♦❢ P❖▲|✷❆✱ t❤❡ ❡♥③②♠❛t✐❝ ❝♦r❡ ✭❛♥❞ •❛r❣❡st s✉❜✉♥✐t✮ ♦❢ |◆❆P■■✳ ✼✳✶✳✶ ❚❤❡ ❈❚❉ ♦❢ |◆❆P■■ ❛♥❞ ❢✉♥❝t✐♦♥ ♦❢ t❤❡ ❈❚❉ ❝♦❞❡ ❚❤❡ ❈ ✲t❡r♠✐♥❛• ❞♦♠❛✐♥ ✭❈❚❉✮ ♦❢ P❖▲|✷❆ ✐s ✉♥✐q✉❡ ✐♥ t❤❛t ✐t ✐s str✐❦✐♥❣•② ❝♦♠♣♦s❡❞ ♦❢ s❡✈❡r❛• t❛♥❞❡♠ r❡♣❡❛ts ✭✺✷ ✐♥ ♠♦✉s❡ ❛♥❞ ❤✉♠❛♥s ❬❈❤❛♣♠❛♥ ❡t ❛•✳✱ ✷✵✵✽(✮ ♦❢ t❤❡ ❚②r ✶ ❙❡r ✷ Pr♦ ✸ ❚❤r ✹ ❙❡r ✺ Pr♦ ✻ ❙❡r ✼ ✭(❙P❚❙P❙✮ ❝♦♥s❡♥s✉s ❤❡♣t❛♣❡♣t✐❞❡ s❡✲ q✉❡♥❝❡ ✭❋✐❣✉r❡ ✼✳✶❆✮✳ ■♥ ✈✐✈♦✱ t❤❡s❡ r❡♣❡❛ts ❛r❡ s✉❜•❡❝t t♦ ❡①t❡♥s✐✈❡ ♣♦st✲tr❛♥s•❛t✐♦♥❛• ♠♦❞✐✜❝❛t✐♦♥s ✭P❚▼s✮✱ ✇❤✐❝❤ ❝♦♥st✐t✉t❡ ❛ ❜♦♥❛ ✜❞❡ ✧❈❚❉ ❝♦❞❡✧ ❬❑♦♠❛r♥✐ts❦② ❡t ❛•✳✱ ✷✵✵✵❀ ❇✉r❛t♦✇s❦✐✱ ✷✵✵✸(✳ ❚❤❡ ❈❚❉ ❝♦❞❡ ✐s ✐♥tr✐❝❛t❡•② •✐♥❦❡❞ t♦ ❞✐✛❡r❡♥t st❛❣❡s ♦❢ t❤❡ tr❛♥s❝r✐♣t✐♦♥ ❝②❝•❡✱ ❛s ✇❡•• ❛s s❡✈❡r❛• ❝♦✲tr❛♥s❝r✐♣t✐♦♥❛• ❡✈❡♥ts ✐♥❝•✉❞✐♥❣ ♠|◆❆ ❝❛♣♣✐♥❣✱ ♣♦•②❛❞❡♥②•❛t✐♦♥ ❛♥❞ s♣•✐❝✐♥❣✱ ❜✉t ❛•s♦ ❤✐st♦♥❡ ♠♦❞✐✜❝❛t✐♦♥s ❬❍s✐♥ ❛♥❞ ▼❛♥•❡②✱ ✷✵✶✷(✳ ■♥❞❡❡❞✱ ✐t ❤❛s ❜❡❡♥ s❤♦✇♥ t❤❛t t❤❡ ❈❚❉ ♦❢ |◆❆P■■ ❛❝ts ❛s ❛ ❞♦❝❦✐♥❣ ♣•❛t❢♦r♠ ❢♦r s❡✈❡r❛• tr❛♥s❝r✐♣t✐♦♥ ❛♥❞ |◆❆ ♣r♦❝❡ss✐♥❣ ❢❛❝t♦rs✱ ❛s ✇❡•• ❛s ❝❤r♦♠❛t✐♥ ♠♦❞✐✜❡rs✱ ✇❤♦s❡ ❜✐♥❞✐♥❣ ❞❡♣❡♥❞s ♦♥ t❤❡ P❚▼✲st❛t✉s ♦❢ t❤❡ ❈❚❉ ❬❏❡r♦♥✐♠♦ ❡t ❛•✳✱ ✷✵✶✸❀ ❏❛s♥♦✈✐❞♦✈❛ ❛♥❞ ❙t❡✢✱ ✷✵✶✸(✳ ❆♠♦♥❣ ❛•• ❈❚❉✲P❚▼s✱ ♣❤♦s♣❤♦r②•❛t✐♦♥s ❛t ❙❡r✐♥❡ ✷ ✭❙❡r✷P✮✱ ❙❡r✐♥❡ ✺ ✭❙❡r✺P✮ ❛♥❞ ❙❡r✐♥❡ ✼ ✭❙❡r✼P✮ ♦❢ t❤❡ ❤❡♣t❛♣❡♣t✐❞❡ ✭❋✐❣✉r❡ ✼✳✶❆✮ ❤❛✈❡ ❜❡❡♥ ♠♦st ❡①t❡♥s✐✈❡•② st✉❞✐❡❞ ❬❍❡✐❞❡♠❛♥♥ ❡t ❛•✳✱ ✷✵✶✸(✳ ❙❡r✷P ❛♥❞ ❙❡r✺P r❡♣r❡s❡♥t ✼✺✪ ♦❢ t❤❡ t♦t❛• ❢r❛❝t✐♦♥ |◆❆P■■ ♣❤♦s♣❤♦r②•❛t❡❞ ❛t ✐ts ❈❚❉✱ ✐••✉str❛t✐♥❣ t❤❡✐r ❡ss❡♥t✐❛• r♦•❡s ❞✉r✐♥❣ t❤❡ tr❛♥s❝r✐♣t✐♦♥ ♣r♦✲ ❝❡ss ❬❙❝❤ü••❡r ❡t ❛•✳✱ ✷✵✶✻(✳ ❈❤■P ❛♥❛•②s❡s ❬r❡✈✐❡✇❡❞ ✐♥ ❍❡✐❞❡♠❛♥♥ ❡t ❛•✳✱ ✷✵✶✸( r❡✈❡❛•❡❞ t❤❛t ❙❡r✺P✲♠❛r❦❡❞ |◆❆P■■ ✐s ♠♦st ❤✐❣❤•② ❡♥r✐❝❤❡❞ ❛t ❣❡♥❡ ♣r♦♠♦t❡rs✱ ✇✐t❤ •❡✈❡•s s❤❛r♣•② ❞❡❝r❡❛s✐♥❣ ❢✉rt❤❡r ✐♥t♦ t❤❡ ✸✬ ❡♥❞ ♦❢ ❣❡♥❡s ✭❋✐❣✉r❡ ✼✳✶❇✮✳ ❈♦♥✈❡rs❡•②✱ ❙❡r✷P✲♠❛r❦❡❞ |◆❆P■■ s✐❣♥❛• ✐s t②♣✐❝❛••② •♦✇ ❛r♦✉♥❞ t❤❡ ♣r♦♠♦t❡r r❡❣✐♦♥✱ ❜✉t ✐♥❝r❡❛s❡s ❝♦♥t✐♥✉♦✉s•② ✐♥t♦ t❤❡ ✸✬✲❡♥❞ ♦❢ ❣❡♥❡s✱ ❜❡✐♥❣ ♠♦st ❤✐❣❤•② ❡♥r✐❝❤❡❞ ❛r♦✉♥❞ t❤❡ s✐t❡ ♦❢ tr❛♥s❝r✐♣t✐♦♥ t❡r♠✐♥❛t✐♦♥ ✭❋✐❣✉r❡ ✼✳✶❇✮✳ •✐✈❡♥ t❤❡✐r ♣❡❝✉•✐❛r ❞✐str✐❜✉t✐♦♥s ✇✐t❤✐♥ t❤❡ tr❛♥s❝r✐♣t✐♦♥ ✉♥✐t✱ ✐t ✐s ♥♦t s✉r♣r✐s✐♥❣ t❤❛t ❙❡r✺P ❤❛s ❜❡❡♥ ❛ss♦❝✐❛t❡❞ ✇✐t❤ tr❛♥s❝r✐♣t✐♦♥ ✐♥✐t✐❛t✐♦♥ ❛♥❞ t❤❡ tr❛♥s✐t✐♦♥ ✐♥t♦ ❡❛r•② ❡•♦♥❣❛t✐♦♥✱ ✇❤✐•❡ ❙❡r✷P ❤❛s ❜❡❡♥ ❛ss♦❝✐❛t❡❞ ✇✐t❤ ❡✈❡♥ts ♣r♦♠♦t✐♥❣ t❤❡ ♣r♦❞✉❝t✐✈❡ ♣❤❛s❡ ♦❢ tr❛♥s❝r✐♣t✐♦♥ ❡•♦♥❣❛t✐♦♥ ❛s ✇❡•• ❛s t❡r♠✐♥❛t✐♦♥✳ ✶✺✷ |❡s✉•ts ❙P❖❈✴❙❡r✺P✲❈❚❉ ✐♥t❡r❛❝t✐♦♥ ❛♥❛•②s✐s ✼✳✶✳✶✳✶ ❋✉♥❝t✐♦♥ ♦❢ ❙❡r✺ ♣❤♦s♣❤♦r②•❛t✐♦♥ ❙❡r✺ ♣❤♦s♣❤♦r②•❛t✐♦♥ ❬❜② ❦✐♥❛s❡s r❡✈✐❡✇❡❞ ✐♥ ❍❡✐❞❡♠❛♥♥ ❡t ❛•✳✱ ✷✵✶✸( r❡s✉•ts ✐♥ |◆❆P■■ ❞✐ss♦❝✐❛t✐♦♥ ❢r♦♠ t❤❡ ▼❡❞✐❛t♦r ❝♦♠♣•❡① ❬❙ø❣❛❛r❞ ❛♥❞ ❙✈❡•str✉♣✱ ✷✵✵✼(✱ ♣r♦♠♦t✐♥❣ ♣r♦✲ ♠♦t❡r ❡s❝❛♣❡✳ ❋✉rt❤❡r♠♦r❡✱ ❙❡r✺P ✐s r❡q✉✐r❡❞ ❢♦r t❤❡ r❡❝r✉✐t♠❡♥t ♦❢ ♠|◆❆ ✺✬✲❝❛♣♣✐♥❣ ❡♥③②♠❡s ❬❈❤♦ ❡t ❛•✳✱ ✶✾✾✼❀ ▼❝❈r❛❝❦❡♥ ❡t ❛•✳✱ ✶✾✾✼(✱ ❛s ✇❡•• ❛s t❤❡ ❙❊❚✶ ❝♦♠♣•❡① ❬◆❣ ❡t ❛•✳✱ ✷✵✵✸❀ ❑r♦❣❛♥ ❡t ❛•✳✱ ✷✵✵✸( ✕ ✇❤✐❝❤ ❝❛t❛•②s❡s ❍✸❑✹ ♠♦♥♦✲✱ ❞✐✲ ❛♥❞ tr✐♠❡t❤②•❛t✐♦♥✱ ❛ s❡t ♦❢ ♠❛r❦s ✇❤✐❝❤ r❡❝r✉✐t ❤✐st♦♥❡ ❛❝❡t②•❛s❡s✱ ♥✉❝•❡♦s♦♠❡ r❡♠♦❞❡•❡rs ❛♥❞ tr❛♥s❝r✐♣✲ t✐♦♥ ❡•♦♥❣❛t✐♦♥ ❢❛❝t♦rs ❬❑♦✉③❛r✐❞❡s✱ ✷✵✵✼( ✕ ❢✉rt❤❡r s✉♣♣♦rt✐♥❣ ❡♥tr② ✐♥t♦ tr❛♥s❝r✐♣t✐♦♥ ❡•♦♥❣❛t✐♦♥✳ ✼✳✶✳✶✳✷ ❋✉♥❝t✐♦♥ ♦❢ ❙❡r✷ ♣❤♦s♣❤♦r②•❛t✐♦♥ ❙❡r✷ ♣❤♦s♣❤♦r②•❛t✐♦♥ ❬❜② ❦✐♥❛s❡s r❡✈✐❡✇❡❞ ✐♥ ❍❡✐❞❡♠❛♥♥ ❡t ❛•✳✱ ✷✵✶✸( r❡q✉✐r❡s ❛♥✲ t❡❝❡❞❡♥t ❙❡r✺ ♣❤♦s♣❤♦r②•❛t✐♦♥✳ ■♥❝r❡❛s✐♥❣ ❙❡r✷P •❡✈❡•s ❢❛✈♦r ♣r♦❞✉❝t✐✈❡ tr❛♥s❝r✐♣t✐♦♥ ❡•♦♥❣❛t✐♦♥ t❤r♦✉❣❤ t❤❡ ❞✐r❡❝t r❡❝r✉✐t♠❡♥t ♦❢ ❡•♦♥❣❛t✐♦♥ ❢❛❝t♦rs s✉❝❤ ❛s ❙P❚✻ ❬(♦❤ ❡t ❛•✳✱ ✷✵✵✼(✳ ❙✐♠✐•❛r•②✱ ❙❡r✷P ♣r♦♠♦t❡s ❝♦✲tr❛♥s❝r✐♣t✐♦♥❛• ♠|◆❆ ♣r♦❝❡ss✐♥❣ ❜② r❡❝r✉✐t✐♥❣ s♣•✐❝✲ ✐♥❣ ❢❛❝t♦rs ❬▼♦rr✐s ❛♥❞ •r❡❡♥•❡❛❢✱ ✷✵✵✵❀ ❉❛✈✐❞ ❡t ❛•✳✱ ✷✵✶✶(✳ ❋✐♥❛••②✱ ❤✐❣❤ ❙❡r✷P •❡✈❡•s ❛r♦✉♥❞ t❤❡ tr❛♥s❝r✐♣t✐♦♥ t❡r♠✐♥❛t✐♦♥ s✐t❡ ❡♥❛❜•❡ ❡✣❝✐❡♥t ❛♥❞ s✉❝❝❡ss❢✉• tr❛♥s❝r✐♣t✐♦♥ t❡r✲ ♠✐♥❛t✐♦♥✱ ❜② r❡❝r✉✐t✐♥❣ s❡✈❡r❛• tr❛♥s❝r✐♣t✐♦♥ t❡r♠✐♥❛t✐♦♥ ❛♥❞ ♠|◆❆ ✸✬✲❡♥❞ ♣r♦❝❡ss✐♥❣ ✭❡✳❣✳ ♣♦•②❛❞❡♥②•❛t✐♦♥✮ ❢❛❝t♦rs ❬▼❡✐♥❤❛rt ❛♥❞ ❈r❛♠❡r✱ ✷✵✵✹❀ ▲✉♥❞❡ ❡t ❛•✳✱ ✷✵✶✵(✳ ✼✳✶✳✶✳✸ ❋✉♥❝t✐♦♥ ♦❢ ❙❡r✼ ♣❤♦s♣❤♦r②•❛t✐♦♥ ❯♥•✐❦❡ ❙❡r✷ ❛♥❞ ❙❡r✺✱ ❙❡r✼ ♣❤♦s♣❤♦r②•❛t✐♦♥ ❬❜② ❦✐♥❛s❡s r❡✈✐❡✇❡❞ ✐♥ ❍❡✐❞❡♠❛♥♥ ❡t ❛•✳✱ ✷✵✶✸( ❛❝❝♦✉♥ts ❢♦r •❡ss t❤❛♥ ✺✪ ♦❢ t❤❡ t♦t❛• ❢r❛❝t✐♦♥ ♦❢ |◆❆P■■ ♣❤♦s♣❤♦r②•❛t❡❞ ❛t ✐ts ❈❚❉✳ ❚❤❡ ❞✐str✐❜✉t✐♦♥ ♦❢ ❙❡r✼P✲♠❛r❦❡❞ |◆❆P■■ ✐♥✐t✐❛••② r❡ss❡♠❜•❡s t❤❛t ♦❢ ❙❡r✺P✱ ♣❡❛❦✲ ✐♥❣ ❛t t❤❡ tr❛♥s❝r✐♣t✐♦♥❛• st❛rt s✐t❡ ✭❚❙❙✮❀ ❜✉t ✉♥•✐❦❡ ❙❡r✺P✱ ❙❡r✼P •❡✈❡•s r❡♠❛✐♥ ❤✐❣❤ ✉♥t✐• t❤❡ ♣♦•②❛❞❡♥②•❛t✐♦♥ s✐t❡ ✭❋✐❣✉r❡ ✼✳✶❇✮✳ ❲❤✐•❡ ❙❡r✼P ♣•❛②s ❛ ❝r✉❝✐❛• r♦•❡ ✐♥ t❤❡ tr❛♥✲ s❝r✐♣t✐♦♥ ❛♥❞ ♣r♦❝❡ss✐♥❣ ♦❢ |◆❆P■■✲tr❛♥s❝r✐❜❡❞ s♥|◆❆s ❬❊❣•♦✛ ❡t ❛•✳✱ ✷✵✵✼(✱ ✐ts ❢✉♥❝t✐♦♥ ❛t ♣r♦t❡✐♥✲❝♦❞✐♥❣ ❣❡♥❡s ✐s st✐•• ✈❡r② ✉♥❝•❡❛r✳ ✶✺✸ |❡s✉•ts ❙P❖❈✴❙❡r✺P✲❈❚❉ ✐♥t❡r❛❝t✐♦♥ ❛♥❛•②s✐s
♥ ❝②❝•❡ ❛♥❞ ❈❚❉ ♣❤♦s✲ ♣❤♦r②•❛t✐♦♥✳ ❚❙❙✿ tr❛♥s❝r✐♣t✐♦♥ st❛rt s✐t❡✳ ✭❈✮ ❈❯❚✫|❯◆ ♣r♦✜•✐♥❣ ♦❢ ❳✐st ✲♠❡❞✐❛t❡❞ ❙P❊◆ r❡❝r✉✐t♠❡♥t t♦ ❳✲•✐♥❦❡❞ ❝❤r♦♠❛t✐♥ ✭t❤❡ ❊❞❛✷r ❣❡♥❡ ✐s s❤♦✇♥ ❛s ❛♥ ❡①❛♠♣•❡✮✳ ✭❉✮ ❙❝❤❡♠❡ s❤♦✇✐♥❣ t❤❡ ❛♠✐♥♦✲❛❝✐❞ s❡q✉❡♥❝❡ ♦❢ t❤❡ ❙P❖❈✲✐♥t❡r❛❝t✐♥❣ r❡❣✐♦♥s ♦❢ ❙▼|❚ ✭t♦♣✮ ❛♥❞ ◆❈♦| ✭❜♦tt♦♠✮✳ ❉✐r❡❝t ❜✐♥❞✐♥❣ ✇✐t❤ ❙P❖❈ ♦❝❝✉rs t❤r♦✉❣❤ t❤❡ ❝♦♥s❡r✈❡❞ ▲❙❉❙ ♠♦t✐❢ ✭s❤♦✇♥ ✐♥ ♣✉r✲ ♣•❡✮✳ ❚❤❡ t✇♦ s❡r✐♥❡s ✇✐t❤✐♥ t❤✐s ♠♦t✐❢ ✭s❤♦✇♥ ✐♥ ❜♦•❞✮ ❛r❡ r❡❛❞✐•② ♣❤♦s♣❤♦r②•❛t❡❞ ✐♥ ✈✐✈♦✳ ✭❊✮ ❚❛❜•❡ s❤♦✇✐♥❣ t❤❡ ❜✐♥❞✐♥❣ ❛✣♥✐t✐❡s ♠❡❛s✉r❡❞ ❜❡t✇❡❡♥ ❙P❖❈ ❛♥❞ s❡✈❡r❛• ❙▼|❚ ♣❡♣t✐❞❡s ❞✐❢✲ ❢❡r✐♥❣ ❜② t❤❡✐r •❡✈❡• ♦❢ ❙❡r✐♥❡ ♣❤♦s♣❤♦r②•❛t✐♦♥ ✭✈❛•✉❡s t❛❦❡♥ ❞✐r❡❝t•② ❢r♦♠ ❬❖s✇❛•❞ ❡t ❛•✳✱ ✷✵✶✻(✮✳ ✭❋✮ ❊•❡❝tr♦st❛t✐❝ ♣♦t❡♥t✐❛• ❛♥❞ ✭•✮ r✐❜❜♦♥ r❡♣r❡s❡♥t❛t✐♦♥s ♦❢ t❤❡ ❙▼|❚ ❜✐♥❞✐♥❣ s✐t❡ ♦❢ t❤❡ ❙P❖❈ ❞♦♠❛✐♥ ❤✐❣❤•✐❣❤t ❤♦✇ t❤❡ ♣❤♦s♣❤♦r②•❛t❡❞ ❙▼|❚ ♣❡♣t✐❞❡ ✭❣r❡❡♥ •✐❝♦r✐❝❡✮ ✜ts ✐♥t♦ ❛ ❜❛s✐❝ ♣❛t❝❤ ♦❢ ❙P❖❈✱ ✇❤❡r❡ t❤❡ ❙▼|❚ ♣❤♦s♣❤♦s❡r✐♥❡ ✭✇❤✐t❡ ❝✐r❝•❡✮ ✐♥t❡r❛❝ts ✇✐t❤ ❙P❖❈ •②s✐♥❡ ❛♥❞ ❛r❣✐♥✐♥❡ s✐❞❡ ❝❤❛✐♥s✳ ■♠❛❣❡s s❤♦✇♥ ✐♥ ✭❋✮ ❛♥❞ ✭•✮ ✇❡r❡ t❛❦❡♥ ❞✐r❡❝t•② ❢r♦♠ ❬▼✐❦❛♠✐ ❡t ❛•✳✱ ✷✵✶✹( ✳ ✶✺✹ |❡s✉•ts ❙P❖❈✴❙❡r✺P✲❈❚❉ ✐♥t❡r❛❝t✐♦♥ ❛♥❛•②s✐s ✼✳✶✳✷ ❙P❖❈ ✐♥t❡r❛❝t✐♦♥ ✇✐t❤ ◆❈♦|✴❙▼|❚ ✐s ♣❤♦s♣❤♦✲❞❡♣❡♥❞❡♥t ❚❤❡ ❞✐r❡❝t ✐♥t❡r❛❝t✐♦♥ ❜❡t✇❡❡♥ ❙P❖❈ ❛♥❞ ◆❈♦|✴❙▼|❚ ♦❝❝✉rs ❛t ❛ ❤✐❣❤•② ❝♦♥s❡r✈❡❞ ▲❙❉❙ ♠♦t✐❢ ❢♦✉♥❞ ✇✐t❤✐♥ t❤❡ ❈✲t❡r♠✐♥✉s ♦❢ ◆❈♦| ❛♥❞ ❙▼|❚ ✭❋✐❣✉r❡ ✼✳✶❉✮ ❬❆r✐②♦s❤✐ ❛♥❞ ❙❝❤✇❛❜❡✱ ✷✵✵✸(✳ |❡♠❛r❦❛❜•②✱ t❤✐s ✐♥t❡r❛❝t✐♦♥ ❝❛♥ ♦♥•② ♦❝❝✉r ✇❤❡♥ ❛t •❡❛st ♦♥❡ ♦❢ t❤❡ t✇♦ ❙❡r✐♥❡ r❡s✐❞✉❡s ✇✐t❤✐♥ t❤❡ ▲❙❉❙ ♠♦t✐❢ ♦❢ ◆❈♦|✴❙▼|❚ ❛r❡ ♣❤♦s♣❤♦r②•❛t❡❞ ✭❋✐❣✉r❡ ✼✳✶❊✮ ❬❖s✇❛•❞ ❡t ❛•✳✱ ✷✵✶✻(✳ ❚❤❡ str✉❝t✉r❛• ❜❛s✐s ❢♦r t❤✐s ♣❤♦s♣❤♦✲❞❡♣❡♥❞❡♥t ✐♥t❡r❛❝t✐♦♥ ❤❛s ❜❡❡♥ r❡s♦•✈❡❞ ✉s✐♥❣ ◆▼| ❬▼✐❦❛♠✐ ❡t ❛•✳✱ ✷✵✶✹(✱ r❡✈❡❛•✐♥❣ t❤❛t t❤❡ ✜rst ♣❤♦s♣❤♦s❡r✐♥❡ ♦❢ t❤❡ ▲❙❉❙ ♠♦t✐❢ ✜ts t✐❣❤t•② ✐♥t♦ ❛ ❜❛s✐❝ ♣❛t❝❤ ❡①♣♦s❡❞ ❛t t❤❡ s✉r❢❛❝❡ ♦❢ ❙P❖❈ ✭❋✐❣✉r❡ ✼✳✶❋✮✱ ❡♥❣❛❣✐♥❣ ✇✐t❤ ❦❡② ❛r❣✐♥✐♥❡ ❛♥❞ •②s✐♥❡ s✐❞❡ ❝❤❛✐♥s ♦❢ ❙P❖❈ ✭❋✐❣✉r❡ ✼✳✶•✮✳ ❆•t♦❣❡t❤❡r✱ t❤❡s❡ ❜✐♦❝❤❡♠✐❝❛• ❛♥❞ str✉❝t✉r❛• st✉❞✐❡s ❤❛✈❡ ❤✐❣❤•✐❣❤t❡❞ t❤❛t ❙P❊◆✬s ❙P❖❈ ❞♦♠❛✐♥ ❢✉♥❝t✐♦♥s ❛s ❛ ♣❤♦s♣❤♦s❡r✐♥❡✲❜✐♥❞✐♥❣ ✉♥✐t✳ ❚❛❦❡♥ t♦❣❡t❤❡r ✇✐t❤ t❤❡ ♦❜s❡r✈❛t✐♦♥ t❤❛t ❙P❊◆ ❜✐♥❞✐♥❣ ♦♥ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡ ❝•♦s❡•② r❡ss❡♠❜•❡s t❤❛t ♦❢ ❙❡r✺P✲|◆❆P■■ ✭❋✐❣✉r❡ ✼✳✶❇ ❛♥❞ ❈✮✱ ■ ❤②♣♦t❤❡s✐③❡❞ t❤❛t ❙P❊◆ ❝♦✉•❞ s✐♠✐•❛r•② ✐♥t❡r❛❝t ❞✐r❡❝t•② ✇✐t❤ |◆❆✲♣♦•②♠❡r❛s❡ ■■ s♣❡❝✐✜❝❛••② ✇❤❡♥ ✐ts ❈❚❉ ✐s ❙❡r✺ ♣❤♦s♣❤♦r②•❛t❡❞✳ ❚♦ t❡st t❤✐s ❤②♣♦t❤❡s✐s✱ ■ st❛rt❡❞ ❛ ❝♦••❛❜♦r❛t✐♦♥ ✇✐t❤ ❈❤r✐st♦♣❤ ▼ü••❡r✬s •❛❜ ❛t ❊▼❇▲ ❍❡✐❞❡•❜❡r❣ ✭✇❤❡r❡ t❤❡ ❍❡❛r❞ •❛❜ ❤❛❞ ♠♦✈❡❞ ❜② t❤❡♥✮✳ ❈❤r✐st♦♣❤✬s •❛❜ ❤❛s ✇♦r•❞✲ r❡♥♦✇♥❡❞ ❡①♣❡rt✐s❡ ✐♥ ✉s✐♥❣ str✉❝t✉r❛• ❜✐♦•♦❣② ❝♦✉♣•❡❞ t♦ ❜✐♦♣❤②s✐❝❛• ❛♥❞ ❜✐♦❝❤❡♠✐❝❛• t♦♦•s t♦ ❞✐ss❡❝t t❤❡ ♠♦•❡❝✉•❛r ♠❡❝❤❛♥✐s♠s ♦❢ tr❛♥s❝r✐♣t✐♦♥✳ ❆•• t❤❡ ❡①♣❡r✐♠❡♥t❛• ✇♦r❦ ♣r❡s❡♥t❡❞ ✐♥ t❤✐s ❝❤❛♣t❡r ❤❛s ♦♥•② ❜❡❡♥ ♠❛❞❡ ♣♦ss✐❜•❡ t❤r♦✉❣❤ t❡❛♠✐♥❣ ✉♣ ✇✐t❤ ❇r✐❝❡ ▼✉r❝✐❛♥♦ ❢r♦♠ ❈❤r✐st♦♣❤✬s •❛❜✱ ✇❤♦ ❛•s♦ ♦♣❡r❛t❡s t❤❡ ❝r②st❛••✐③❛t✐♦♥ ❢❛❝✐•✐t② ❛t ❊▼❇▲ ❍❡✐❞❡•❜❡r❣✳ ✼✳✷ ❇❛❝t❡r✐❛• ❡①♣r❡ss✐♦♥ ❛♥❞ ♣✉r✐✜❝❛t✐♦♥ ♦❢ t❤❡ ❙P❖❈ ❞♦♠❛✐♥ ❚♦ t❡st ❛ ♣♦t❡♥t✐❛• ❞✐r❡❝t ✐♥t❡r❛❝t✐♦♥ ❜❡t✇❡❡♥ ❙P❖❈ ❛♥❞ |◆❆P■■ ♣❤♦s♣❤♦r②•❛t❡❞ ❛t ❙❡r✺ ♦❢ ✐ts ❈❚❉✱ ✇❡ ✜rst ❛tt❡♠♣t❡❞ t♦ ♣✉r✐❢② ♠✉r✐♥❡ ❙P❊◆✬s ❙P❖❈ ❞♦♠❛✐♥ ✉s✐♥❣ ❛ ❜❛❝t❡r✐❛• ❡①♣r❡ss✐♦♥ s②st❡♠✳ ■ ❞❡s✐❣♥❡❞ ✺ ❞✐✛❡r❡♥t ❝♦♥str✉❝ts ✭•✐st❡❞ ✐♥ ❚❛❜•❡ ✼✳✶✮✱ ❛•• ♦❢ ✇❤✐❝❤ ❡♥❝♦❞❡ ♣r♦t❡✐♥ ❢r❛❣♠❡♥ts ❢✉••② ❡♥❝♦♠♣❛ss✐♥❣ t❤❡ ❙P❖❈ ❞♦♠❛✐♥ ✭❋✐❣✉r❡ ✼✳✷❆✮ ❛s ✇❡•• ❛s ❛ ✈❛r②✐♥❣ ♥✉♠❜❡r ♦❢ ❙P❊◆ ❛♠✐♥♦ ❛❝✐❞s ✉♣str❡❛♠ ♦❢ ❙P❖❈ ✭❋✐❣✉r❡ ✼✳✷❇✮✳ ✶✺✺ |❡s✉•ts ❙P❖❈✴❙❡r✺P✲❈❚❉ ✐♥t❡r❛❝t✐♦♥ ❛♥❛•②s✐s A B beta strand alpha helix PIPPAPPCQPSQLSQPAQPPSGKIPQVSQEAKGTQTGGVEQTRLPAIPTNRPSEPHAQLQRAPVETAQPAHPSPVSVSMKPDLPSPLSSQAAPKQPLFVPAN SGPSTPPGLALPHAEVQPAPKQESSPHGTPQRPVDMVQLLKKYPIVWQGLLALKNDTAAVQLHFVSGNNVLAHRSLPLSEGGPPLRIAQRMRLEASQLEG VARRMTVETDYCLLLALPCGRDQEDVVSQTESLKAAFITYLQAKQAAGIINVPNPGSNQPAYVLQIFPPCEFSESHLSRLAPDLLASISNISPHLMIVIASV pFD99&101 pFD100&102 pFD121 GEARILTVTPSSQLQGLPLTPPVVVTHGVQIVHSSGELFQEYRYGDVRTYHAPAQQLTHTQFPVASSISLASRTKTSAQVPPEGEPLQSTQSAQPAPSTQATQ disordered SPOC SPEN SPOC ❋✐❣✉r❡ ✼✳✷ ✕ ❙❝❤❡♠❡ ❞❡♣✐❝t✐♥❣ t❤❡ ❞✐✛❡r❡♥t ❙P❖❈ ❢r❛❣♠❡♥ts t❡st❡❞ ❢♦r ❝r②st❛••✐③❛✲ t✐♦♥✳ ✭❆✮ ❙❝❤❡♠❛t✐❝ s❤♦✇✐♥❣ t❤❡ ✇❤♦•❡ ❙P❊◆ ♣r♦t❡✐♥✱ ✇✐t❤ ❛ ❢♦❝✉s ♦♥ t❤❡ •❛st ✹✵✼ ❛♠✐♥♦ ❛❝✐❞s✱ ✇❤✐❝❤ ❡♥❝♦♠♣❛ss t❤❡ ❙P❖❈ ❞♦♠❛✐♥✳ ✭❇✮ ❆♠✐♥♦ ❛❝✐❞ s❡q✉❡♥❝❡ ❝♦rr❡s♣♦♥❞✐♥❣ t♦ t❤❡ •❛st ✹✵✼ ❛♠✐♥♦ ❛❝✐❞s ♦❢ ❙P❊◆✳ ❚❤❡ ♥❛♠❡s ♦❢ ❡❛❝❤ ❙P❖❈ ❡①♣r❡ss✐♦♥ ♣•❛s♠✐❞s ✭s❤♦✇♥ ❛❜♦✈❡ t❤❡ s❡✲ q✉❡♥❝❡✱ s❡❡ ❚❛❜•❡ ✼✳✶✮ ❛r❡ ♣♦s✐t✐♦♥❡❞ r❡•❛t✐✈❡ t♦ t❤❡ st❛rt ♦❢ t❤❡ ♣r♦t❡✐♥ ❢r❛❣♠❡♥ts t❤❡② ❡♥❝♦❞❡✳ ❙❡❝♦♥❞❛r② str✉❝t✉r❡s ✕ ♣r❡❞✐❝t❡❞ ✇✐t❤ P❤②r❡✷ ❬❑❡••❡② ❡t ❛•✳✱ ✷✵✶✺(✕ ❛r❡ r❡♣r❡s❡♥t❡❞ ❜❡•♦✇ t❤❡ s❡q✉❡♥❝❡✳ ❆♠✐♥♦ ❛❝✐❞s s❤♦✇♥ ✐♥ ❣r❡② ♠❛♣ t♦ ♣r❡❞✐❝t❡❞ ❞✐s♦r❞❡r❡❞ r❡❣✐♦♥s✱ ✇❤✐•❡ ❛♠✐♥♦ ❛❝✐❞s s❤♦✇♥ ✐♥ ❜♦•❞ r❡❞ ♠❛♣ t♦ t❤❡ ❙P❖❈ ❞♦♠❛✐♥✳ ❊❛❝❤ ❢r❛❣♠❡♥t ✇❛s t❛❣❣❡❞ ✇✐t❤ ✻①❍✐s✲♦♥•② ♦r ❞✉❛• ✻①❍✐s✲❙❯▼❖✸ t❛❣s✱ ✇✐t❤ t❤❡ ✻①❍✐s ♠♦✐❡t② ❜❡✐♥❣ ✉s❡❞ ❢♦r ❛✣♥✐t② ♣✉r✐✜❝❛t✐♦♥✱ ❛♥❞ t❤❡ ❙❯▼❖✸ ♠♦✐❡t② ❛••❡❣❡❞•② ❡♥❤❛♥❝✐♥❣ ♣r♦t❡✐♥ ❡①♣r❡ss✐♦♥ ❛♥❞ s♦•✉❜✐•✐t②✳ •✐✈❡♥ t❤❛t ♦✉r ❙P❖❈✲❡♥❝♦❞✐♥❣ ❉◆❆ ❢r❛❣♠❡♥ts ✇❡r❡ ♥♦t ❝♦❞♦♥✲♦♣t✐♠✐③❡❞ ❢♦r ❜❛❝t❡r✐❛✱ ❛•• t❤❡s❡ ❝♦♥str✉❝ts ✇❡r❡ ❡①♣r❡ss❡❞ ❢r♦♠ t❤❡ |♦s❡tt❛ ✷✭❉❊✸✮♣▲②s❙ ❜❛❝t❡r✐❛• str❛✐♥✱ ✇❤✐❝❤ ❤❛r❜♦rs ❛ ♣|❆|❊ ♣•❛s♠✐❞ ❡①♣r❡ss✐♥❣ s❡✈❡r❛• t|◆❆s r❛r❡•② ❢♦✉♥❞ ✐♥ ❊✳ ❝♦•✐✳ ❝♦♥str✉❝t ❜❛❝❦❜♦♥❡ ❛✣♥✐t② t❛❣ ❛✳❛✳ s✐③❡ ✭❦❉❛✮ ♣■ ②✐❡•❞❡❞ ❝r②st❛•s ♣❋❉✾✾ ♣❊❚▼✶✶ ✻①❍✐s✲❙❯▼❖✸ ✹✵✼ ✹✸✳✸ ✼✳✸ ♥♦ ♣❋❉✶✵✵ ♣❊❚▼✶✶ ✻①❍✐s✲❙❯▼❖✸ ✶✾✻ ✷✶✳✷ ✻✳✼ ♥♦ ♣❋❉✶✵✶ ♣❊❚▼✶✶ ✻①❍✐s ✹✵✼ ✹✸✳✸ ✼✳✸ ♥♦ ♣❋❉✶✵✷ ♣❊❚▼✶✶ ✻①❍✐s ✶✾✻ ✷✶✳✷ ✻✳✼ ♥♦ ♣❋❉✶✷✶ ♣❊❚▼✶✶ ✻①❍✐s✲❙❯▼❖✸ ✶✻✾ ✶✽✳✹ ✻✳✺ ②❡s ❚❛❜•❡ ✼✳✶ ✕ ▲✐st ♦❢ ❙P❖❈ ❝♦♥str✉❝ts t❡st❡❞ ❢♦r ♣✉r✐✜❝❛t✐♦♥ ❛♥❞ ❝r②st❛••✐③❛t✐♦♥✳ ❆•• ❝♦♥str✉❝ts ✇❡r❡ ❝•♦♥❡❞ ✐♥t♦ ❛ ♣❊❚▼✶✶ ❜❛❝❦❜♦♥❡✱ ❛♥❞ ❙P❖❈ ❡①♣r❡ss✐♦♥ ✇❛s ✐♥❞✉❝❡❞ ❛t ✶✽➦❈ ✐♥ |♦s❡tt❛ ✷✭❉❊✸✮♣▲②s❙ ❜❛❝t❡r✐❛• ❝❡••s ✭s❡❡ s❡❝t✐♦♥ ✸✳✷✵✮✳ ❛✳❛✿ ❛♠✐♥♦ ❛❝✐❞s✱ ♣■✿ ✐s♦❡•❡❝tr✐❝ ♣♦✐♥t ❆❞❛♣t✐♥❣ t❤❡ ❝♦♥❞✐t✐♦♥s ♦r✐❣✐♥❛••② ✉s❡❞ t♦ ♣✉r✐❢② ❤✉♠❛♥ ❙P❖❈ ❬❆r✐②♦s❤✐ ❛♥❞ ❙❝❤✇❛❜❡✱ ✷✵✵✸(✱ ♦✉r ♦♣t✐♠✐③❡❞ ♣r♦t♦❝♦• ✭s❡❡ s❡❝t✐♦♥ ✸✳✷✵✮ r❡s✉•t❡❞ ✐♥ ❣r❡❛t ②✐❡•❞s ♦❢ ❤✐❣❤•② ♣✉r✐✜❡❞ ❙P❖❈ ✭❋✐❣✉r❡ ✼✳✸✮✳ ✶✺✻ |❡s✉•ts ❙P❖❈✴❙❡r✺P✲❈❚❉ ✐♥t❡r❛❝t✐♦♥ ❛♥❛•②s✐s
♦♣ ♣❛♥❡•✮ ♦❜t❛✐♥❡❞ ❞✉r✐♥❣ s✐③❡ ❡①❝•✉s✐♦♥ ❝❤r♦♠❛t♦❣r❛♣❤② ♦❢ ◆✐✲◆❚❆ ♣✉r✐✜❡❞ ❙P❖❈ ✭❢r♦♠ t❤❡ ♣❋❉✶✷✶ ❝♦♥str✉❝t✱ s❡❡ ❚❛❜•❡ ✼✳✶✮ ❛♥❞ t❤❡ r❡s✉•t✐♥❣ ❙❉❙✲P❆•❊ ❛♥❛•②s✐s ♦❢ ❡•✉t❡❞ ❢r❛❝t✐♦♥s ✭❜♦tt♦♠ ♣❛♥❡•✮✳ ❋r❛❝t✐♦♥s ✷✷✴✷✸ ✇❡r❡ ✉s❡❞ ❢♦r ■❚❈ ❛♥❞ ❝r②st❛••✐③❛t✐♦♥ ❡①♣❡r✐♠❡♥ts✳ ✼✳✸ ❙tr✉❝t✉r❡ ♦❢ ♠♦✉s❡ ❙P❖❈ ❛t ✶✳✷➴ r❡s♦•✉t✐♦♥ ❲❡ ♥❡①t ❛tt❡♠♣t❡❞ t♦ ❝r②st❛••✐③❡ ❛♥❞ r❡s♦•✈❡ t❤❡ str✉❝t✉r❡ ♦❢ ♦✉r ♣✉r✐✜❡❞ ❙P❖❈ ♣r♦t❡✐♥✳ ▼♦✉s❡ ❛♥❞ ❤✉♠❛♥ ❙P❊◆✬s ❙P❖❈ ❞♦♠❛✐♥s s❤❛r❡ ✾✾✪ s❡q✉❡♥❝❡ ❤♦♠♦•♦❣② ✭❋✐❣✉r❡ ✼✳✹❆✮✱ ❛♥❞ t❤✉s✱ ✇❡ ❛✐♠❡❞ t♦ ❡♥s✉r❡ t❤❛t t❤❡ ✸❉✲str✉❝t✉r❡ ♦❢ ♦✉r ♣✉r✐✜❡❞ ♠♦✉s❡ ❙P❖❈ ♣r♦t❡✐♥ ✇❛s s✐♠✐•❛r t♦ t❤❡ ♣✉❜•✐s❤❡❞ str✉❝t✉r❡ ♦❢ ❤✉♠❛♥ ❙P❖❈ ❬❆r✐②♦s❤✐ ❛♥❞ ❙❝❤✇❛❜❡✱ ✷✵✵✸(✳ ❋♦••♦✇✐♥❣ s❡✈❡r❛• r♦✉♥❞s ♦❢ ❝r②st❛••✐③❛t✐♦♥ s❝r❡❡♥✐♥❣✱ ✇❡ ✇❡r❡ s✉❝❝❡ss❢✉• ❛t ♦❜t❛✐♥✐♥❣ ❞✐✛r❛❝t✐♥❣ ❙P❖❈ ❝r②st❛•s ✭❋✐❣✉r❡ ✼✳✹❇✮✳ ■♠♣♦rt❛♥t•②✱ ♦♥•② t❤❡ ♠✐♥✐♠❛• ❙P❖❈ ❢r❛❣♠❡♥t ✭♣❋❉✶✷✶ ❝♦♥str✉❝t✱ s❡❡ ❚❛❜•❡ ✼✳✶✮ ②✐❡•❞❡❞ ❣♦♦❞ ❞✐✛r❛❝t✐♥❣ ❝r②st❛•s✳ ✶✺✼ |❡s✉•ts ❙P❖❈✴❙❡r✺P✲❈❚❉ ✐♥t❡r❛❝t✐♦♥ ❛♥❛•②s✐s
❲❡ ✇❡r❡ ❛❜•❡ t♦ s♦•✈❡ t❤❡ str✉❝t✉r❡ ♦❢ ♠♦✉s❡ ❙P❖❈ ✭❚❛❜•❡ ✼✳✷ ❛♥❞ ❋✐❣✉r❡ ✼✳✹❈✮✱ ✉s✐♥❣ ♠♦•❡❝✉•❛r r❡♣•❛❝❡♠❡♥t ✇✐t❤ ❤✉♠❛♥ ❙P❖❈ ✭P❉❇ ✶❖❲✶✱ ❬❆r✐②♦s❤✐ ❛♥❞ ❙❝❤✇❛❜❡✱ ✷✵✵✸(✮ ❛s ❛ t❡♠♣•❛t❡✳ ❲❡ ❞❡t❡r♠✐♥❡❞ ❙P❖❈ str✉❝t✉r❡ ❛t ❛ r❡s♦•✉t✐♦♥ ♦❢ ✶✳✷➴✱ ✐♠♣r♦✈✐♥❣ ✐t ❜② ✵✳✻➴ ❝♦♠♣❛r❡❞ t♦ ❬❆r✐②♦s❤✐ ❛♥❞ ❙❝❤✇❛❜❡✱ ✷✵✵✸(✳ ■♠♣♦rt❛♥t•②✱ ♦✉r ♠♦✉s❡ ❙P❖❈ str✉❝t✉r❡ ❛•✐❣♥❡❞ ✈❡r② ✇❡•• ✇✐t❤ t❤❡ ❤✉♠❛♥ ❙P❖❈ str✉❝t✉r❡✱ ✇✐t❤ ❛ r ♦♦t✲♠❡❛♥✲sq✉❛r❡ ❞ ❡✈✐❛t✐♦♥ ✭|▼❙❉✮ ♦❢ ❛t♦♠✐❝ ♣♦s✐t✐♦♥s ❡q✉❛• t♦ ✵✳✶✶✾➴ ✭❋✐❣✉r❡ ✼✳✹❈✮✳ ❙✐♠✐•❛r•② t♦ ❤✉♠❛♥ ❙P❖❈ ❬❆r✐②♦s❤✐ ❛♥❞ ❙❝❤✇❛❜❡✱ ✷✵✵✸(✱ ♠♦✉s❡ ❙P❖❈ ✐s ❝❤❛r❛❝t❡r✐③❡❞ ❜② ❛ s❡✈❡♥✲str❛♥❞❡❞ β✲❜❛rr❡• ✭✐❞❡♥t✐✜❡❞ ❛s β✶✲β✼ ♦♥ ❋✐❣✉r❡ ✼✳✹❈✮ s✉rr♦✉♥❞❡❞ ❜② s✐① α✲❤❡•✐❝❡s ✭✐❞❡♥t✐✜❡❞ ❛s α✶✲α✻ ♦♥ ❋✐❣✉r❡ ✼✳✹❈✮✳ P❛r❛♠❡t❡r |❡s♦•✉t✐♦♥ r❛♥❣❡ ✭➴✮ ✸✵✳✼✽ ✲ ✶✳✷ ✭✶✳✷✹✸ ✲ ✶✳✷✮ ❙♣❛❝❡ ❣r♦✉♣ P ✷ ✶ ✷ ✶ ✷ ✶ ❯♥✐t ❝❡•• ✭➴✮ ❛❂✹✶✳✻✵✹✱ ❜❂✻✶✳✺✵✷✱ ❝❂✻✽✳✹✸✼ ✭α✱β✱γ❂✾✵➦✮ ❚❛❜•❡ ❝♦♥t✐♥✉❡❞ ♦♥ ♥❡①t ♣❛❣❡ ✶✺✽ |❡s✉•ts ❙P❖❈✴❙❡r✺P✲❈❚❉ ✐♥t❡r❛❝t✐♦♥ ❛♥❛•②s✐s ❚❛❜•❡ ✼✳✷ ✕ ❈♦♥t✐♥✉❡❞ ❢r♦♠ ♣r❡✈✐♦✉s ♣❛❣❡ P❛r❛♠❡t❡r ▼♦•❡❝✉•❡s ✭❛✳✉✮ ✶ ❚♦t❛• r❡✢❡❝t✐♦♥s ✻✼✽✵✸✾ ✭✺✷✽✾✺✮ ❯♥✐q✉❡ r❡✢❡❝t✐♦♥s ✺✷✹✺✾ ✭✹✸✺✼✮ ▼✉•t✐♣•✐❝✐t② ✶✷✳✾ ✭✶✷✳✶✮ ❈♦♠♣•❡t❡♥❡ss ✭✪✮ ✾✹✳✷✽ ✭✼✾✳✻✻✮ ▼❡❛♥ ■✴σ✭■✮ ✸✹✳✵✹ ✭✶✸✳✶✻✮ ❲✐•s♦♥ ❇✲❢❛❝t♦r ✶✷✳✹✽ | ♠❡r❣❡ ✵✳✵✹✽✺✸ ✭✵✳✶✼✻✹✮ | ♠❡❛s ✵✳✵✺✵✽✻ ✭✵✳✶✽✹✷✮ | ♣✐♠ ✵✳✵✶✹✼✺ ✭✵✳✵✺✶✼✮ ❈❈ ✶✴✷ ✵✳✾✾✽ ✭✵✳✾✾✹✮ |❡✢❡❝t✐♦♥s ✉s❡❞ ✐♥ r❡✜♥❡♠❡♥t ✺✷✹✺✹ ✭✹✸✺✺✮ |❡✢❡❝t✐♦♥s ✉s❡❞ ❢♦r | ❢r❡❡ ✶✾✾✸ ✭✶✻✼✮ | ✇♦r❦ ✴| ❢r❡❡ ✵✳✶✹✸✾ ✭✵✳✶✷✸✶✮ ✴ ✵✳✶✻✸✺ ✭✵✳✶✼✷✽✮ ❈❈ ✇♦r❦ ✵✳✾✺✷ ✭✵✳✾✼✼✮ ❈❈ ❢r❡❡ ✵✳✾✺✸ ✭✵✳✾✺✹✮ |❛♠❛❝❤❛♥❞r❛♥ ❢❛✈♦r❡❞ ✭✪✮ ✾✼✳✺✻ |❛♠❛❝❤❛♥❞r❛♥ ❛••♦✇❡❞ ✭✪✮ ✷✳✹✹ |❛♠❛❝❤❛♥❞r❛♥ ♦✉t•✐❡rs ✭✪✮ ✵✳✵✵ |♦t❛♠❡r ♦✉t•✐❡rs ✭✪✮ ✵✳✵✵ ❈•❛s❤s❝♦r❡ ✹✳✷✷ ❚❛❜•❡ ✼✳✷ ✕ ❈r②st❛••✐③❛t✐♦♥ ❞❛t❛ ❛♥❞ r❡✜♥❡♠❡♥t st❛t✐st✐❝s ❢♦r ❙P❖❈✳ ❙t❛t✐st✐❝s ❢♦r t❤❡ ❤✐❣❤❡st r❡s♦•✉t✐♦♥ s❤❡•• ❛r❡ s❤♦✇♥ ✐♥ ♣❛r❡♥t❤❡s❡s✳ ✼✳✹ ❙P❖❈ ✐♥t❡r❛❝ts ✇✐t❤ t❤❡ ❈❚❉ ♦❢ |◆❆P■■ s♣❡❝✐✜✲ ❝❛••② ✇❤❡♥ ❙❡r✺ ✐s ♣❤♦s♣❤♦r②•❛t❡❞ ❆•t♦❣❡t❤❡r✱ t❤❡s❡ ♣r❡✈✐♦✉s r❡s✉•ts ❞❡♠♦♥str❛t❡ t❤❛t ♦✉r ❝♦♥❞✐t✐♦♥s ❢♦r ❙P❖❈ ♣✉r✐✜❝❛✲ t✐♦♥ ❢❛✐t❤❢✉••② ♣r❡s❡r✈❡ ✐ts ✸❉✲str✉❝t✉r❡✱ ❛♥❞ ❤❡♥❝❡ ❝❛♥ ❜❡ s✉❜s❡q✉❡♥t•② ✉s❡❞ t♦ ✐♥✈❡st✐❣❛t❡ ❛ ♣♦t❡♥t✐❛• ✐♥t❡r❛❝t✐♦♥ ✇✐t❤ t❤❡ ❈❚❉ ♦❢ |◆❆P■■✳ ❚♦ ❛ss❛② s✉❝❤ ✐♥t❡r❛❝t✐♦♥✱ ✇❡ ✜rst ✉s❡❞ ✐ s♦t❤❡r♠❛• t ✐tr❛t✐♦♥ ❝❛•♦r✐♠❡tr② ✭■❚❈✱ ❋✐❣✉r❡ ✼✳✺✮✳ ■❚❈ ✐s ♦♥❡ ♦❢ t❤❡ ♠♦st s❡♥s✐t✐✈❡ ❛♥❞ q✉❛♥t✐t❛t✐✈❡ ❛♥❛•②t✐❝❛• ♠❡t❤♦❞s t♦ ❝❤❛r❛❝t❡r✐③❡ ♣r♦t❡✐♥✲♣r♦t❡✐♥ ✐♥t❡r❛❝t✐♦♥s✱ ♥♦t❛❜•② t♦ ✐❞❡♥t✐❢② t❤❡ ❞✐ss♦❝✐❛t✐♦♥ ❝♦♥st❛♥t ✭❑ ❞ ✮ ❛♥❞ t❤❡ st♦✐❝❤✐♦♠❡tr②✳ ■♥ ♦✉r ❝❛s❡✱ ■❚❈ ♠❡❛s✉r❡s ✭✉♥❞❡r ❝♦♥st❛♥t ♣r❡ss✉r❡ ❛♥❞ t❡♠♣❡r❛t✉r❡✮ t❤❡ ❤❡❛t ❡①❤❛♥❣❡s r❡s✉•t✐♥❣ ❢r♦♠ ❡①♣♦s✐♥❣ ❛ ❙P❖❈ s♦•✉t✐♦♥ ✭♦❢ ❦♥♦✇♥ ❝♦♥❝❡♥tr❛t✐♦♥✮ t♦ ✐♥❝r❡❛s✐♥❣ q✉❛♥t✐t✐❡s ♦❢ ❛ s②♥t❤❡t✐❝ ❈❚❉ ♣❡♣t✐❞❡✳ ■❚❈ r❡✈❡❛•❡❞ ♥♦ s✐❣♥✐✜❝❛♥t ❜✐♥❞✐♥❣ ❜❡t✇❡❡♥ ❙P❖❈ ❛♥❞ s②♥t❤❡t✐❝ ✉♥♣❤♦s♣❤♦r②•❛t❡❞ ❈❚❉ ♣❡♣t✐❞❡s✱ ❝♦♥t❛✐♥✐♥❣ ❡✐t❤❡r t✇♦ ✭❋✐❣✉r❡ ✼✳✺❆✶✮ ♦r ❢♦✉r ✭❋✐❣✉r❡ ✼✳✺❆✷✮ r❡♣❡❛ts ✶✺✾ |❡s✉•ts ❙P❖❈✴❙❡r✺P✲❈❚❉ ✐♥t❡r❛❝t✐♦♥ ❛♥❛•②s✐s ♦❢ t❤❡ ❝♦♥s❡♥s✉s ❈❚❉ ❤❡♣t❛♣❡♣t✐❞❡✳ ❙✐♠✐•❛r•②✱ ♥♦ ❜✐♥❞✐♥❣ ✇❛s ❞❡t❡❝t❡❞ ❜❡t✇❡❡♥ ❙P❖❈ ❛♥❞ ❙❡r✷✲ ✭❋✐❣✉r❡ ✼✳✺❇✶✮ ♦r ❙❡r✼✲ ✭❋✐❣✉r❡ ✼✳✺❉✶✮ ♣❤♦s♣❤♦r②•❛t❡❞ ❈❚❉ ♣❡♣t✐❞❡s ✱ r❡❣❛r❞•❡ss ♦❢ t❤❡ ♥✉♠❜❡r ♦❢ ❤❡♣t❛♣❡♣t✐❞❡ r❡♣❡❛ts ✭❋✐❣✉r❡ ✼✳✺❇✷ ❛♥❞ ❉✷✮✳
❋✐❣✉r❡ ✼✳✺ ✕ ■s♦t❤❡r♠❛• t✐tr❛t✐♦♥ ❝❛•♦r✐♠❡tr② r❡✈❡❛•s ❛ ❞✐r❡❝t ❛♥❞ s♣❡❝✐✜❝ ✐♥t❡r❛❝✲ t✐♦♥ ❜❡t✇❡❡♥ ❙P❖❈ ❛♥❞ t❤❡ ❙❡r✺✲♣❤♦s♣❤♦r②•❛t❡❞ ❈❚❉ ♦❢ |◆❆P■■✳ |❡♣r❡s❡♥t❛t✐✈❡ t❤❡r♠♦❣r❛♠s ✭❛•• s❝❛•r❡❞ ✐❞❡♥t✐❝❛••②✮ r❡s✉•t✐♥❣ ❢r♦♠ ■❚❈ ♠❡❛s✉r❡♠❡♥ts ♦❢ ❙P❖❈ ❜✐♥❞✐♥❣ t♦ ✭❆✮ ✉♥♣❤♦s♣❤♦r②•❛t❡❞✱ ✭❇✮ ❙❡r✷✲✱ ✭❈✮ ❙❡r✺✲ ♦r ✭❉✮ ❙❡r✼✲♣❤♦s♣❤♦r②•❛t❡❞ ♣❡♣t✐❞❡s ❝♦♥t❛✐♥✐♥❣ ✭✶✮ t✇♦ ♦r ✭✷✮ ❢♦✉r r❡♣❡❛ts ♦❢ t❤❡ |◆❆P■■✲❈❚❉ ❤❡♣t❛♣❡♣t✐❞✐❝ s❡q✉❡♥❝❡✳ ❊❛❝❤ ♣❡♣t✐❞❡ ✇❛s t✐tr❛t❡❞ ❛❣❛✐♥st ❙P❖❈ ✭❞❛t❛ s❤♦✇♥ ✐♥ r❡❞✮ ♦r ❜✉✛❡r ❛s ❛ ❝♦♥tr♦• ✭❞❛t❛ s❤♦✇♥ ✐♥ ❜•❛❝❦✮✳ ❲❤❡♥ ❜✐♥❞✐♥❣ ✐s ♦❜s❡r✈❡❞✱ t❤❡ st❛t✐st✐❝❛• ✜tt✐♥❣ ✕ r❡s✉•t✐♥❣ ❢r♦♠ t❤❡ ♦♥❡✲s✐t❡ ❜✐♥❞✐♥❣ ♠♦❞❡• ✉s❡❞ t♦ ❞❡r✐✈❡ t❤❡ t❤❡r♠♦❞②♥❛♠✐❝ ♣❛r❛♠❡t❡rs r❡♣♦rt❡❞ ✐♥ ❚❛❜•❡ ✼✳✸ ✕ ✐s s❤♦✇♥ ❛s ❛ s✐❣♠♦✐❞ ❝✉r✈❡✳ ❆•• ■❚❈ ❡①♣❡r✐♠❡♥ts ✇❡r❡ r❡♣❡❛t❡❞ ❛t •❡❛st t❤r❡❡ t✐♠❡s✳ ✶✻✵ |❡s✉•ts ❙P❖❈✴❙❡r✺P✲❈❚❉ ✐♥t❡r❛❝t✐♦♥ ❛♥❛•②s✐s |❡♠❛r❦❛❜•② ❤♦✇❡✈❡r✱ ❞✐r❡❝t ❜✐♥❞✐♥❣ ✭❛•❜❡✐t ♠♦❞❡r❛t❡✱ ❑ ❞ ≈✶✵✉▼✱ ❚❛❜•❡ ✼✳✸✮ ✇❛s ♦❜s❡r✈❡❞ ❜❡t✇❡❡♥ ❙P❖❈ ❛♥❞ ❙❡r✺ ♣❤♦s♣❤♦r②•❛t❡❞ ❈❚❉ ♣❡♣t✐❞❡s ✭❋✐❣✉r❡ ✼✳✺❈✶✮✳ ❆•✲ t❤♦✉❣❤ t❤❡ ♠❡❛s✉r❡❞ ❛✣♥✐t② ❞✐❞ ♥♦t ❞✐✛❡r ✇❤❡t❤❡r ✇❡ ✉s❡❞ t✇♦ ♦r ❢♦✉r r❡♣❡❛ts ♦❢ t❤❡ ❙❡r✺ ♣❤♦s♣❤♦r②•❛t❡❞ ❝♦♥s❡♥s✉s ❤❡♣t❛♣❡♣t✐❞❡ ✭❚❛❜•❡ ✼✳✸✮✱ ✇❡ ♥♦t✐❝❡❞ ❤♦✇❡✈❡r t❤❛t ❣r❡❛t❡r ❤❡❛t ✭≈ t✇✐❝❡ ❛s ♠✉❝❤✮ ✇❛s r❡•❡❛s❡❞ ✇❤❡♥ ✉s✐♥❣ t❤❡ ✹✲r❡♣❡❛t ♣❡♣t✐❞❡ ✐♥ •✐❡✉ ♦❢ t❤❡ ✷✲r❡♣❡❛t ♣❡♣t✐❞❡ ✭❋✐❣✉r❡ ✼✳✺❈✶ ❛♥❞ ❈✷✮✳ ❚❤✐s r❡s✉•t s✉❣❣❡sts t❤❛t t❤❡r❡ ❛r❡ t✇✐❝❡ ❛s ♠❛♥② ❙P❖❈ ♠♦•❡❝✉•❡s ❜♦✉♥❞ ♦♥ t❤❡ ✹✲r❡♣❡❛t ♣❡♣t✐❞❡ ❛s t❤❡r❡ ❛r❡ ♦♥ t❤❡ ✷✲r❡♣❡❛t ♣❡♣✐❞❡✳ ❆•t♦❣❡t❤❡r✱ t❤❡s❡ r❡s✉•ts ❞❡♠♦♥str❛t❡ t❤❛t ❙P❖❈ ✐♥t❡r❛❝ts ❞✐r❡❝t•② ✇✐t❤ t❤❡ ❈❚❉ ❞♦♠❛✐♥ ♦❢ |◆❆P■■✱ ♦♥•② ✇❤❡♥ ✐t ✐s ♣❤♦s♣❤♦r②•❛t❡❞ s♣❡❝✐✜❝❛••② ♦♥ ❙❡r✐♥❡ ✺✳ ❋✉rt❤❡r✲ ♠♦r❡✱ t❤❡② ✐♥❞✐❝❛t❡ t❤❛t s❡✈❡r❛• ❙P❖❈ ♠♦•❡❝✉•❡s ❝❛♥ ❜✐♥❞ s✐♠✉•t❛♥❡♦✉s•② t♦ t❤❡ s❛♠❡ ❈❚❉ ♠♦•❡❝✉•❡ s♦ •♦♥❣ ❛s ✐t ❝♦♥t❛✐♥s ❛ s✉✣❝✐❡♥t ♥✉♠❜❡r ♦❢ ❤❡♣t❛♣❡♣t✐❞❡ r❡♣❡❛ts✳ ❚❤✐s ✐♥t❡r❛❝t✐♦♥ ❤❛s ✐♠♣♦rt❛♥t ✐♠♣•✐❝❛t✐♦♥s ❢♦r ❤♦✇ ❙P❊◆ ❝♦✉•❞ s✐•❡♥❝❡ tr❛♥s❝r✐♣t✐♦♥ ❞✉r✐♥❣ ❳❈■✱ ❛♥❞ ✇✐•• ❜❡ ❢✉rt❤❡r ❞✐s❝✉ss❡❞ ✐♥ s✉❜s✉❜s❡❝t✐♦♥ ✾✳✷✳✷✳✸✳ ♣❤♦s♣❤♦r②•❛t✐♦♥ r❡♣❡❛ts ❑ ❞ ✭✉▼✮ ◆ ✭s✐t❡s✮ ∆❍ ✭❦❝❛•✴♠♦•✮ ∆• ✭❦❝❛•✴♠♦•✮ ✉♥♣❤♦s♣❤♦r②•❛t❡❞ ✷ ♥✳❜✳❞✳ ✉♥♣❤♦s♣❤♦r②•❛t❡❞ ✹ ♥✳❜✳❞✳ ❙❡r✷ ✷ ♥✳❜✳❞✳ ❙❡r✷ ✹ ♥✳❜✳❞✳ ❙❡r✺ ✷ ✶✵✳✷±✸✳✸ ✶✳✵✶±✵✳✵✻ ✲✸✳✷±✵✳✸ ✲✻✳✼ ❙❡r✺ ✹ ✶✸✳✹±✵✳✽ ✵✳✺✽±✵✳✵✶ ✲✶✶✳✺±✵✳✹ ✲✻✳✺✹ ❙❡r✼ ✷ ♥✳❜✳❞✳ ❙❡r✼ ✹ ♥✳❜✳❞✳ ❙❡r✷ ❛♥❞ ❙❡r✼ ✷ ♥✳❜✳❞✳ ❙❡r✺ ❛♥❞ ❙❡r✷ ✷ ✹✳✵±✵✳✽ ✶✳✸✸±✵✳✵✸ ✲✹✳✵±✵✳✷ ✲✼✳✷✹ ❙❡r✺ ❛♥❞ ❙❡r✼ ✷ ✷✷✳✷±✹✳✷ ✶✳✸✵±✵✳✵✹ ✲✸✳✼±✵✳✸ ✲✻✳✷✹ ❚❛❜•❡ ✼✳✸ ✕ ❙✉♠♠❛r② ♦❢ ■❚❈ ❞❛t❛ r❡s✉•t✐♥❣ ❢r♦♠ t✐tr❛t✐♥❣ ❞✐✛❡r❡♥t |◆❆P■■✲❈❚❉ ♣❤♦s♣❤♦♣❡♣t✐❞❡s ❛❣❛✐♥st ❙P❖❈✳ ♥✳❜✳❞✳✿ ♥♦ ❜✐♥❞✐♥❣ ❞❡t❡❝t❡❞ ✼✳✺ ❙❡r✷✴✼ ♣❤♦s♣❤♦r②•❛t✐♦♥s ❞♦ ♥♦t ❛•t❡r ❙P❖❈ ❜✐♥❞✲ ✐♥❣ t♦ t❤❡ ❙❡r✺P✲❈❚❉ ♦❢ |◆❆P■■ ❚❤r♦✉❣❤♦✉t t❤❡ tr❛♥s❝r✐♣t✐♦♥ ♣r♦❝❡ss✱ t❤❡ ❈❚❉ ♦❢ |◆❆ ♣♦•②♠❡r❛s❡ ■■ ✐s ♥♦t ❛•✇❛②s ♠❛r❦❡❞ ❜② ♦♥•② ♦♥❡ t②♣❡ ♦❢ ♣❤♦s♣❤♦r②•❛t✐♦♥ ✭✐✳❡✳ ❙❡r✷✲♦♥•②✱ ❙❡r✺✲♦♥•②✱ ❙❡r✼✲♦♥•②✳✳✳✮✳ ■♥✲ ❞❡❡❞✱ s❡✈❡r❛• ❈❚❉ ♣❤♦s♣❤♦r②•❛t✐♦♥✲st❛t❡s ❛r❡ ❢♦✉♥❞ t♦ ❝♦✲♦❝❝✉r ✇✐t❤✐♥ t❤❡ s❛♠❡ |◆❆P■■ ♠♦•❡❝✉•❡ ❬❙❝❤ü••❡r ❡t ❛•✳✱ ✷✵✶✻(✳ ❋♦r ❡①❛♠♣•❡✱ s✐♠✉•t❛♥❡♦✉s ♣❤♦s♣❤♦r②•❛t✐♦♥ ❛t ❙❡r✷ ❛♥❞ ❙❡r✺ ♦❢ t❤❡ |◆❆P■■✲❈❚❉ ✐s ♦❜s❡r✈❡❞ ❛t t❤❡ ❜❡❣✐♥♥✐♥❣ ♦❢ t❤❡ ❡•♦♥❣❛t✐♦♥ ♣❤❛s❡✳ ❚❤✐s ❞♦✉❜•②✲♣❤♦s♣❤♦r②•❛t❡❞ st❛t❡ ❞✐r❡❝t•② r❡❝r✉✐ts ❙❊❚✷ ✭✇❤✐❝❤ ♦♥•② ❜✐♥❞s t♦ ❙❡r✷P✴❙❡r✺P ✶✻✶ |❡s✉•ts ❙P❖❈✴❙❡r✺P✲❈❚❉ ✐♥t❡r❛❝t✐♦♥ ❛♥❛•②s✐s ❈❚❉✮✱ ❛ ❤✐st♦♥❡ •②s✐♥❡ ♠❡t❤②•tr❛♥s❢❡r❛s❡ ✇❤✐❝❤ ❝❛t❛•②s❡s ❍✸❑✸✻♠❡✸ ❛♥❞ ♣•❛②s ❛ ❝r✉❝✐❛• r♦•❡ ✐♥ tr❛♥s❝r✐♣t✐♦♥ ❡•♦♥❣❛t✐♦♥ ❬❑✐③❡r ❡t ❛•✳✱ ✷✵✵✺❀ ❱♦•♥✐❝ ❡t ❛•✳✱ ✷✵✵✻(✳ ❍❡♥❝❡✱ ■ q✉❡st✐♦♥❡❞ ✇❤❡t❤❡r t❤❡ ❛❞❞✐t✐♦♥❛• ♣r❡s❡♥❝❡ ♦❢ ❙❡r✷✲ ♦r ❙❡r✼✲♣❤♦s♣❤♦r②•❛t✐♦♥ ❛•t❡rs ✭♣♦s✐t✐✈❡•② ♦r ♥❡❣❛t✐✈❡•②✮ t❤❡ ❛❜✐•✐t② ♦❢ ❙P❖❈ t♦ ✐♥t❡r❛❝t ✇✐t❤ ❙❡r✺P✲❈❚❉✱ s♦ ❛s t♦ s❤❡❞ •✐❣❤t ♦♥ ✇❤✐❝❤ |◆❆P■■ ♠♦•❡❝✉•❡s ❙P❖❈ ❤❛s t❤❡ ❛❜✐•✐t② t♦ ❡♥❣❛❣❡ ✇✐t❤✳ ❚♦ ❛❞❞r❡ss t❤✐s ♣♦✐♥t✱ ❜✐♥❞✐♥❣ ❜❡t✇❡❡♥ ❞♦✉❜•② ♣❤♦s♣❤♦r②•❛t❡❞ ❈❚❉ ♣❡♣
❋✐❣✉r❡ ✼✳✻ ✕ ■s♦t❤❡r♠❛• t✐tr❛t✐♦♥ ❝❛•♦r✐♠❡tr② r❡✈❡❛•s ❙P❖❈ ✐s ❛❜•❡ t♦ ❜✐♥❞ t❤❡ ❙❡r✺✲ ♣❤♦s♣❤♦r②•❛t❡❞ ❈❚❉ ♦❢ |◆❆P■■ ❡✈❡♥ ✇❤❡♥ ♦t❤❡r ❙❡r✐♥❡s ❛r❡ ♣❤♦s♣❤♦r②•❛t❡❞✳ |❡♣✲ r❡s❡♥t❛t✐✈❡ t❤❡r♠♦❣r❛♠s ✭❛•• s❝❛•❡❞ ✐❞❡♥t✐❝❛••②✮ r❡s✉•t✐♥❣ ❢r♦♠ ■❚❈ ♠❡❛s✉r❡♠❡♥ts ♦❢ ❙P❖❈ ❜✐♥❞✐♥❣ t♦ ✭❆✮ ❙❡r✷❙❡r✼✲✱ ✭❇✮ ❙❡r✷❙❡r✺✲ ♦r ✭❈✮ ❙❡r✺❙❡r✼✲❞♦✉❜•② ♣❤♦s♣❤♦r②•❛t❡❞ ♣❡♣t✐❞❡s ❝♦♥✲ t❛✐♥✐♥❣ t✇♦ r❡♣❡❛ts ♦❢ t❤❡ |◆❆P■■✲❈❚❉ ❤❡♣t❛♣❡♣t✐❞✐❝ s❡q✉❡♥❝❡✳ ❊❛❝❤ ♣❡♣t✐❞❡ ✇❛s t✐tr❛t❡❞ ❛❣❛✐♥st ❙P❖❈ ✭❞❛t❛ s❤♦✇♥ ✐♥ r❡❞✮ ♦r ❜✉✛❡r ❛s ❛ ❝♦♥tr♦• ✭❞❛t❛ s❤♦✇♥ ✐♥ ❜•❛❝❦✮✳ ❲❤❡♥ ❜✐♥❞✐♥❣ ✐s ♦❜s❡r✈❡❞✱ t❤❡ st❛t✐st✐❝❛• ✜tt✐♥❣ ✕ r❡s✉•t✐♥❣ ❢r♦♠ t❤❡ ♦♥❡✲s✐t❡ ❜✐♥❞✐♥❣ ♠♦❞❡• ✉s❡❞ t♦ ❞❡r✐✈❡ t❤❡ t❤❡r♠♦❞②♥❛♠✐❝ ♣❛r❛♠❡t❡rs r❡♣♦rt❡❞ ✐♥ ❚❛❜•❡ ✼✳✸ ✕ ✐s s❤♦✇♥ ❛s ❛ s✐❣♠♦✐❞ ❝✉r✈❡✳ ❆•• ■❚❈ ❡①♣❡r✐♠❡♥ts ✇❡r❡ r❡♣❡❛t❡❞ ❛t •❡❛st t❤r❡❡ t✐♠❡s✳ ❉✐r❡❝t ❜✐♥❞✐♥❣ ✇❛s ♦❜s❡r✈❡❞ ❜❡t✇❡❡♥ ❙P❖❈ ❛♥❞ ❡✐t❤❡r ❙❡r✷❙❡r✺✲ ♦r ❙❡r✺❙❡r✼✲❞♦✉❜•② ♣❤♦s♣❤♦r②•❛t❡❞ ♣❡♣t✐❞❡s ✭❋✐❣✉r❡ ✼✳✻❇✱ ❈ ❛♥❞ ❚❛❜•❡ ✼✳✸✮✳ ■♠♣♦rt❛♥t•②✱ t❤❡ ❜✐♥❞✐♥❣ ❛✣♥✐t✐❡s ❢♦r ❜♦t❤ ♣❡♣t✐❞❡s ❞✐❞ ♥♦t ❞✐✛❡r s✐❣♥✐✜❝❛♥t•② ❢r♦♠ t❤❛t ♦❜s❡r✈❡❞ ✇✐t❤ ❈❚❉ ♣❡♣t✐❞❡s ♣❤♦s♣❤♦r②•❛t❡❞ ♦♥•② ❛t ❙❡r✺ ✭❚❛❜•❡ ✼✳✸✮✳ ❚❛❦❡♥ t♦❣❡t❤❡r✱ t❤❡s❡ r❡s✉•ts s❤♦✇ ✶✻✷ |❡s✉•ts ❙P❖❈✴❙❡r✺P✲❈❚❉ ✐♥t❡r❛❝t✐♦♥ ❛♥❛•②s✐s t❤❛t ❙P❖❈ ❜✐♥❞s ❙❡r✺P✲❈❚❉ ✐rr❡s♣❡❝t✐✈❡•② ♦❢ t❤❡ ♣❤♦s♣❤♦r②•❛t✐♦♥ ❡♥✈✐r♦♥♠❡♥t ♣r❡s❡♥t ❛t ♥❡✐❣❤❜♦r✐♥❣ r❡s✐❞✉❡s✱ s✉❣❣❡st✐♥❣ t❤❛t ❙P❖❈ ✐♥t❡r❛❝ts ✇✐t❤ ❛ •❛r❣❡r ❢r❛❝t✐♦♥ ♦❢ |◆❆P■■ t❤❛♥ t❤❛t ✇❤✐❝❤ ✐s str✐❝t•② ♣❤♦s♣❤♦r②•❛t❡❞ ❛t ❙❡r✐♥❡ ✺✳ ❙✉r♣r✐s✐♥❣•②✱ ❛•t❤♦✉❣❤ ♥♦ s✐❣♥✐✜❝❛♥t ❜✐♥❞✐♥❣ ✇❛s ❞❡t❡❝t❡❞ ❜❡t✇❡❡♥ ❙P❖❈ ❛♥❞ t❤❡ ❙❡r✷❙❡r✼✲❞♦✉❜•② ♣❤♦s♣❤♦r②•❛t❡❞ ♣❡♣t✐❞❡ ✭❛s ✇♦✉•❞ ❜❡ ❡①♣❡❝t❡❞ ❢r♦♠ t❤❡ r❡s✉•ts ♦❜t❛✐♥❡❞ ✐♥ s❡❝t✐♦♥ ✼✳✹✮✱ ❛ ❤❡❛t r❡s♣♦♥s❡ ♣r♦✜•❡ ❞✐✛❡r✐♥❣ ❢r♦♠ t❤❛t ♦❢ t❤❡ ❝♦♥tr♦• ♦♥❡ ✇❛s ♦❜✲ s❡r✈❡❞ ✭❋✐❣✉r❡ ✼✳✻❆✮✳ ❚❤✐s ❝♦✉•❞ ❜❡ ✐♥t❡r♣r❡t❡❞ ❛s ❛ ✈❡r② ✇❡❛❦ ❛♥❞ tr❛♥s✐❡♥t ✐♥t❡r❛❝t✐♦♥ ❜❡t✇❡❡♥ ❙P❖❈ ❛♥❞ t❤✐s ♣❡♣t✐❞❡✱ ✇❤✐❝❤ ❝❛♥ ♦❝❝✉r ♦♥•② ✇❤❡♥ s❡✈❡r❛• r❡s✐❞✉❡s ✇✐t❤✐♥ t❤❡ ❤❡♣t❛♣❡♣t✐❞❡ s❡q✉❡♥❝❡ ❛r❡ ♣❤♦s♣❤♦r②•❛t❡❞✳ ❚❤✐s ✐♥t❡r❛❝t✐♦♥ ✐s ♠♦st •✐❦❡•② ♥♦t ❜✐♦•♦❣✐❝❛••② r❡•❡✈❛♥t ❛s ♥♦ ❑ ❞ ❝♦✉•❞ ❜❡ ❝♦♠♣✉t❡❞ ❢♦••♦✇✐♥❣ ■❚❈ ❛♥❛•②s✐s✳ ✼✳✻ ❚❤❡ ❙P❖❈✴❙❡r✺P✲❈❚❉ ❝♦♠♣•❡① ❛t ✶✳✽➴ r❡s♦•✉t✐♦♥ ❚♦ ❢✉rt❤❡r ❝❛♣t✉r❡ t❤❡ ♠♦•❡❝✉•❛r ❜❛s✐s ♦❢ s✉❝❤ s♣❡❝✐✜❝ ❙P❖❈✴❙❡r✺P✲❈❚❉ ✐♥t❡r❛❝t✐♦♥✱ ✇❡ ♥❡①t r❡s♦•✈❡❞ t❤❡ str✉❝t✉r❡ ♦❢ t❤❡ ❙P❖❈✴❙❡r✺P✲❈❚❉ ❝♦♠♣•❡① ✉s✐♥❣ ❳✲r❛② ❝r②st❛•✲ •♦❣r❛♣❤②✳ ❋♦r t❤✐s ♣✉r♣♦s❡✱ ✇❡ ✐♥❝✉❜❛t❡❞ ♣✉r✐✜❡❞ ❙P❖❈ ✇✐t❤ ❡✐t❤❡r ❛ ✷✲ ♦r ✹✲❢♦•❞ ♠♦•❛r ❡①❝❡ss ♦❢ t❤❡ ❙❡r✺P✲❈❚❉ ♣❡♣t✐❞❡ ✭❝♦♥t❛✐♥✐♥❣ ❢♦✉r ❤❡♣t❛♣❡♣t✐❞❡ r❡♣❡❛ts✮✱ ❛♥❞ s✉❜•❡❝t❡❞ t❤❡ ❝♦♠♣•❡① t♦ ❤✐❣❤✲t❤r♦✉❣❤♣✉t ❝r②st❛••✐③❛t✐♦♥ s❝r❡❡♥✐♥❣ ✭s❡❡ s❡❝t✐♦♥ ✸✳✷✹✮✳ ✐❞ s❝r❡❡♥ r❛t✐♦ s❛•t ❜✉✛❡r ♣r❡❝✐♣✐t❛♥t ✶ ❇❈❙ ✹ ✵✳✶▼ P❖ ✹ ♣❍✺✳✺ ✷✵✪ P❊• ❙♠❡❛r ❍✐❣❤ ✷ ❇❈❙ ✷ ✵✳✶▼ P❖ ✹ ♣❍✺✳✺ ✷✷✪ P❊• ❙♠❡❛r ❍✐❣❤ ✸ ❇❈❙ ✹ ✵✳✶✺▼ ◆❛❈• ✷✽✪ P❊• ❙♠❡❛r ▼❡❞✐✉♠ ✹ ❇❈❙ ✹ ✵✳✶▼ ◆❛❑❍P❖ ✹ ♣❍✺✳✺✱ ✵✳✶▼ |❜❈• ✵✳✶▼ ◆❛❈✐tr❛t❡ ♣❍✺✳✺ ✷✺✪ P❊• ❙♠❡❛r ▼❡❞✐✉♠ ✺ ❇❈❙ ✷ ✵✳✶✺▼ ▼❣✭❈❍ ✸ ❈❖❖✮ ✷ ✯✹❍✷✵ ✵✳✶▼ ◆❛❈✐tr❛t❡ ♣❍✺✳✻ ✷✵✪ P❊• ❙♠❡❛r ❇r♦❛❞ ✻ ❇❈❙ ✹ ✵✳✷▼ ◆❛❑❍P❖ ✹ ♣❍✼✳✺ ✵✳✶▼ ❍❊P❊❙ ♣❍✼✳✺ ✷✷✳✺✪ P❊• ❙♠❡❛r ▼❡❞✐✉♠✱ ✶✵✪ ••②❝❡r♦• ✼ P❈❙ ✹ ✵✳✶▼ ◆❛❈❛❝♦❞②•❛t❡ ♣❍✻ ✶✺✪ P❊• ✹✵✵✵ ✽ P❈❙ ✹ ✵✳✷▼ ❑■ ✵✳✶▼ ▼❊❙ ♣❍✻✳✺ ✷✺✪ P❊• ✹✵✵✵ ✾ P❈❙ ✹ ✵✳✶▼ ◆❛❈✐tr❛t❡ ♣❍✺✳✺ ✶✺✪ P❊• ✻✵✵✵ ✶✵ P❈❙ ✷ ✵✳✷▼ ✭◆❍ ✹ ✮ ✷ ❙❖ ✹ ✵✳✶▼ ▼❊❙ ♣❍✻✳✺ ✷✵✪ P❊• ✽✵✵✵ ✶✶ ❊▼❇▲ ✹ ✵✳✶▼ ◆❛❈✐tr❛t❡ ♣❍✺✳✻ ✶✽✪ ▼P❊• ✺✵✵✵ ✶✷ ❊▼❇▲ ✷ ✵✳✶▼ ❍❊P❊❙ ♣❍✼ ✶✽✪ ▼P❊• ✺✵✵✵ ✶✸ ❊▼❇▲ ✷ ✵✳✶▼ ▼❖P❙ ♣❍✼✳✷ ✶✽✪ ▼P❊• ✺✵✵✵ ✶✹ ❊▼❇▲ ✹ ✵✳✶▼ ❚|■❙ ♣❍✽ ✶✽✪ ▼P❊• ✺✵✵✵ ✶✺ ❊▼❇▲ ✷ ✵✳✶▼ ◆❛❑❍P❖ ✹ ♣❍✻✳✸ ✶✺✪ P❊• ✻✵✵✵ ✶✻ ❊▼❇▲ ✷ ✵✳✶▼ ▼❊❙ ♣❍✻✳✺ ✶✺✪ P❊• ✻✵✵✵ ✶✼ ❊▼❇▲ ✹ ✵✳✶▼ ❇■❙✲❚|■❙ ♣❍✻✳✷ ✶✺✪ P❊• ✽✵✵✵ ✶✽ ❊▼❇▲ ✹ ✵✳✶▼ ◆❛❈❛❝♦❞②•❛t❡ ♣❍✻ ✶✺✪ P❊• ✽✵✵✵ ✶✾ P❊•✶ ✷ ✵✳✶▼ ▼❊❙ ♣❍✻✳✺ ✷✺✪ P❊• ✶✵✵✵ ✷✵ P❊•✶ ✹ ✵✳✶▼ ▼❊❙ ♣❍✻✳✺ ✷✺✪ P❊• ✹✵✵✵ ✷✶ P❊•✶ ✹ ✵✳✶▼ ▼❊❙ ♣❍✻✳✺ ✷✵✪ P❊• ✶✵✵✵✵ ✷✷ P❊•✶ ✹ ✵✳✶▼ ❍❊P❊❙ ♣❍✼✳✺ ✷✺✪ P❊• ✸✵✵✵ ✷✸ P❊•✶ ✹ ✵✳✶▼ ❍❊P❊❙ ♣❍✼✳✺ ✷✵✪ P❊• ✶✵✵✵✵ ✷✹ P❊•✶ ✷ ✵✳✷▼ ◆❛❋ ✷✵✪ P❊• ✸✸✺✵ ✷✺ P❊•✶ ✹ ✵✳✷▼ ◆❛❋ ✷✵✪ P❊• ✸✸✺✵ ✷✻ P❊•✶ ✹ ✵✳✷▼ ◆❛❈• ✷✵✪ P❊• ✸✸✺✵ ❚❛❜•❡ ❝♦♥t✐♥✉❡❞ ♦♥ ♥❡①t ♣❛❣❡ ✶✻✸ |❡s✉•ts ❙P❖❈✴❙❡r✺P✲❈❚❉ ✐♥t❡r❛❝t✐♦♥ ❛♥❛•②s✐s ❚❛❜•❡ ✼✳✹ ✕ ❈♦♥
✐♥t❡r❛❝t✐♦♥ ❛♥❛•②s✐s |❡♠❛r❦❛❜•②✱ ❝r②st❛•s ❣r❡✇ ✇✐t❤✐♥ ❛ ❢❡✇ ❤♦✉rs ✭✉♣ t♦ ✇✐t❤✐♥ ♦♥❡ ❞❛②✮✱ ✉♥❞❡r s❡✈❡r❛• t❡st❡❞ ❝♦♥❞✐t✐♦♥s ✭s❡❡ ❋✐❣✉r❡ ✼✳✼ ❢♦r ❡①❛♠♣•❡s ♦❢ ❝r②st❛•s ❣r♦✇♥ ❛♥❞ ❚❛❜•❡ ✼✳✹ ❢♦r t❤❡✐r ❛ss♦❝✐❛t❡❞ ❝♦♥❞✐t✐♦♥s✮✳ ❲❡ ✇❡r❡ ❛❜•❡ t♦ ❝♦••❡❝t s❡✈❡r❛• ❞❛t❛s❡ts ❢r♦♠ t❤❡s❡ ❞✐✛r❛❝t✐♥❣ ❝r②st❛•s✱ ✇❤✐❝❤ ❛••♦✇❡❞ ✉s t♦ s♦•✈❡ t❤❡ ♠♦•❡❝✉•❛r str✉❝t✉r❡ ♦❢ t❤❡ ❙P❖❈✴❙❡r✺P✲❈❚❉ ❝♦♠♣•❡① ❞♦✇♥ t♦ ✶✳✽➴ r❡s♦•✉t✐♦♥ ✭❚❛❜•❡ ✼✳✺✮✳ P❛r❛♠❡t❡r |❡s♦•✉t✐♦♥ r❛♥❣❡ ✭➴✮ ✹✶✳✼✺ ✲ ✶✳✽ ✭✶✳✽✻✹ ✲ ✶✳✽✮ ❙♣❛❝❡ ❣r♦✉♣ P ✷ ✶ ✷ ✶ ✷ ✶ ❯♥✐t ❝❡•• ✭➴✮ ❛❂✹✶✳✸✵✷✱ ❜❂✶✶✾✳✼✽✽✱ ❝❂✶✸✸✳✻✹✻ ✭α✱β✱γ❂✾✵➦✮ ▼♦•❡❝✉•❡s ✭❛✳✉✮ ✶ ❚♦t❛• r❡✢❡❝t✐♦♥s ✽✵✺✸✻✹ ✭✼✽✹✺✺✮ ❯♥✐q✉❡ r❡✢❡❝t✐♦♥s ✻✷✹✼✼ ✭✻✶✹✶✮ ▼✉•t✐♣•✐❝✐t② ✶✷✳✾ ✭✶✷✳✽✮ ❈♦♠♣•❡t❡♥❡ss ✭✪✮ ✾✽✳✹✷ ✭✾✸✳✾✻✮ ▼❡❛♥ ■✴σ✭■✮ ✷✷✳✺✷ ✭✶✳✶✻✮ ❲✐•s♦♥ ❇✲❢❛❝t♦r ✸✹✳✻✵ | ♠❡r❣❡ ✵✳✵✹✹✼✺ ✭✷✳✸✽✺✮ | ♠❡❛s ✵✳✵✹✻✼ ✭✷✳✹✽✸✮ | ♣✐♠ ✵✳✵✶✸✶✽ ✭✵✳✻✽✻✽✮✮ ❈❈ ✶✴✷ ✶ ✭✵✳✻✻✼✮ ❈❈✯ ✶ ✭✵✳✽✾✺✮ |❡✢❡❝t✐♦♥s ✉s❡❞ ✐♥ r❡✜♥❡♠❡♥t ✻✶✺✶✹ ✭✺✼✼✶✮ |❡✢❡❝t✐♦♥s ✉s❡❞ ❢♦r | ❢r❡❡ ✶✹✶✽ ✭✶✸✵✮ | ✇♦r❦ ✴| ❢r❡❡ ✵✳✷✵✺✶ ✭✵✳✷✽✽✷✮ ✴ ✵✳✷✸✸✸ ✭✵✳✸✸✸✷✮ ❈❈ ✇♦r❦ ✵✳✾✷✼ ✭✵✳✻✻✾✮ ❈❈ ❢r❡❡ ✵✳✾✽✽ ✭✵✳✻✷✼✮ |❛♠❛❝❤❛♥❞r❛♥ ❢❛✈♦r❡❞ ✭✪✮ ✾✼✳✹✵ |❛♠❛❝❤❛♥❞r❛♥ ❛••♦✇❡❞ ✭✪✮ ✷✳✻✵ |❛♠❛❝❤❛♥❞r❛♥ ♦✉t•✐❡rs ✭✪✮ ✵✳✵✵ |♦t❛♠❡r ♦✉t•✐❡rs ✭✪✮ ✵✳✵✵ ❈•❛s❤s❝♦r❡ ✹✳✻✵ ❚❛❜•❡ ✼✳✺ ✕ ❈r②st❛••✐③❛t✐♦♥ ❞❛t❛ ❛♥❞ r❡✜♥❡♠❡♥t st❛t✐st✐❝s ❢♦r t❤❡ ❙P❖❈✴❙❡r✺P✲❈❚❉ ❝♦♠♣•❡①✳ ❙t❛t✐st✐❝s ❢♦r t❤❡ ❤✐❣❤❡st r❡s♦•✉t✐♦♥ s❤❡•• ❛r❡ s❤♦✇♥ ✐♥ ♣❛r❡♥t❤❡s❡s✳ ✼✳✼ ❚❤❡ ❜❛s✐❝ ♣❛t❝❤ ♦❢ ❙P❖❈ ♠❡❞✐❛t❡s ❞✐r❡❝t ✐♥t❡r❛❝✲ t✐♦♥ ✇✐t❤ t❤❡ ❙❡r✺P✲❈❚❉ ♦❢ |◆❆P■■ ❚❤❡ ✸❉✲str✉❝t✉r❡ ♦❢ t❤❡ ❝♦♠♣•❡① s❤♦✇s t❤❛t ❜✐♥❞✐♥❣ ♦❢ t❤❡ ❙❡r✺P✲❈❚❉ ♣❡♣t✐❞❡ t♦ ❙P❖❈ ♦❝❝✉rs ❛t ❛ s✉r❢❛❝❡ s✉rr♦✉♥❞✐♥❣ t❤❡ β✸ str❛♥❞ ✭❋✐❣✉r❡ ✼✳✽✮✱ ✇❤✐❝❤ ❝♦rr❡s♣♦♥❞s t♦ t❤❡ ♦♥•② ❜❛s✐❝ ♣❛t❝❤ ♦❢ ❙P❖❈ ✭❋✐❣✉r❡ ✼✳✾✮✳ ❙tr✉❝t✉r❛• ❛•✐❣♥♠❡♥t ❜❡t✇❡❡♥ t❤❡ ❙P❖❈ ❞♦♠❛✐♥ ❛•♦♥❡ ❛♥❞ t❤❡ ❙P❖❈ ❞♦♠❛✐♥ ✐♥ ✶✻✺ |❡s✉•ts ❙P❖❈✴❙❡r✺P✲❈❚❉ ✐♥t❡r❛❝t✐♦♥ ❛♥❛•②s✐s ❝♦♠♣•❡① ✇✐t❤ ❙❡r✺P✲❈❚❉ ②✐❡•❞s ❛♥ |▼❙❉ ✈❛•✉❡ ♦❢ ✵✳✻✼✻➴✱ ✐♥❞✐❝❛t✐♥❣ t❤❛t ❙P❖❈ ❞♦❡s ♥♦t ✉♥❞❡r❣♦ s✐❣♥✐✜❝❛♥t str✉❝t✉r❛• ❝❤❛♥❣❡s ✉♣♦♥ ❜✐♥❞✐♥❣ t❤❡ ❈❚❉ ♠♦•❡❝✉•❡✳ ❙✐♠✐•❛r•②✱ ♥♦ str✉❝t✉r❛• ❝❤❛♥❣❡s ✐♥ ❙P❖❈ ✇❡r❡ r❡♣♦rt❡❞ ✉♣♦♥ ✐♥t❡r❛❝t✐♥❣ ✇✐t❤ ♣❙▼|❚ ❬▼✐❦❛♠✐ ❡t ❛•✳✱ ✷✵✶✹(✳
✕ ❈❛rt♦♦♥ r❡♣r❡s❡♥t❛t✐♦♥ ♦❢ t❤❡ ❙P❖❈✴❙❡r✺P✲❈❚❉ ❝♦♠♣•❡①✳ ■♥t❡r♠♦•❡❝✲ ✉•❛r ❤②❞r♦❣❡♥ ❜♦♥❞s ❛♣♣❡❛r ❛s ❞♦tt❡❞ ②❡••♦✇ •✐♥❡s✱ ❛♥❞ ❛r❡ ♥✉♠❜❡r❡❞ ✭❜•❛❝❦ ❝✐r❝•❡s✮ ❛s ✐♥ ❚❛❜•❡ ✼✳✻✳ ❣r❡❡♥ ❝❛rt♦♦♥ r❡♣r❡s❡♥t❛t✐♦♥✿ ❙P❖❈✱ ♦r❛♥❣❡ •✐❝♦r✐❝❡ r❡♣r❡s❡♥t❛t✐♦♥✿ ❙❡r✺P✲❈❚❉ ♣❡♣t✐❞❡ ❆•t❤♦✉❣❤ ❙P❖❈ ❜✐♥❞s t❤❡ ❈❚❉ ♣❡♣t✐❞❡ ♦♥•② ✇❤❡♥ t❤❡ •❛tt❡r ✐s ♣❤♦s♣❤♦r②•❛t❡❞ ♦♥ ❙❡r✺ ✭s❡❡ s❡❝t✐♦♥ ✼✳✹✮✱ str✉❝t✉r❛• ❛♥❛•②s✐s r❡✈❡❛•s t❤❛t ❙P❖❈ ❢♦r♠s s❡✈❡r❛• ✐♥t❡r♠♦•❡❝✲ ✉•❛r ✐♥t❡r❛❝t✐♦♥s ✇✐t❤ ❈❚❉ r❡s✐❞✉❡s •♦❝❛t❡❞ ✉♣str❡❛♠ ✭✐✳❡✳ ◆✲t❡r♠✐♥❛••②✮ ♦❢ t❤❡ ❙❡r✺✲ ♣❤♦s♣❤♦s❡r✐♥❡ ✐♥❝•✉❞✐♥❣ ❚②r ✶ ✱ ❙❡r ✷ ❛♥❞ ❚❤r ✹ ✱ ❛s ✇❡•• ❛s ❙❡r ✼ ♦❢ t❤❡ ♣r❡❝❡❞✐♥❣ ❤❡♣t❛♣❡♣✲ t✐❞❡ ✭❋✐❣✉r❡ ✼✳✽ ❛♥❞ ❚❛❜•❡ ✼✳✻✮✳ ❚❤✐s ♦❜s❡r✈❛t✐♦♥ ✐s r❡♠✐♥✐s❝❡♥t ♦❢ t❤❡ ❙P❖❈✴♣❙▼|❚ ✐♥t❡r❛❝t✐♦♥ ✐♥ ✇❤✐❝❤ s❡✈❡r❛• r❡s✐❞✉❡s ♦❢ t❤❡ ♣❙▼|❚ ♣❤♦s♣❤♦♣❡♣t✐❞❡✱ •♦❝❛t❡❞ ✉♣str❡❛♠ ♦❢ t❤❡ ♣❤♦s♣❤♦s❡r✐♥❡✱ ❛r❡ r❡q✉✐r❡❞ ❢♦r ❜✐♥❞✐♥❣ ♦❢ ♣❙▼|❚ t♦ ❙P❖❈ ❬▼✐❦❛♠✐ ❡t ❛•✳✱ ✷✵✶✹(✳ ✐❞ ❙P❖❈ r❡s✐❞✉❡ ❢✉♥❝t✐♦♥❛• ❣r♦✉♣ ❈❚❉ r❡s✐❞✉❡ ❢✉♥❝t✐♦♥❛• ❣r♦✉♣ ✶ ◗ ✸✺✸✾ ✭α✸✮ α✲❛♠✐❞❡ ✭❈❂❖ ❞✐♣♦•❡✱ ❜❛❝❦❜♦♥❡✮ ❙❡r ✼❛ α✲❛♠✐❞❡ ✭◆✲❍ ❞✐♣♦•❡✱ ❜❛❝❦❜♦♥❡✮ ✷ ◗ ✸✻✵✼ ✭β✻✮ γ✲❛♠✐❞❡ ✭◆✲❍ ❞✐♣♦•❡✮ ❚②r ✶❜ β✲❤②❞r♦①②♣❤❡♥②• ✸ | ✸✺✸✷ ✭β✸✧✮ α✲❛♠✐❞❡ ✭❈❂❖ ❞✐♣♦•❡✱ ❜❛❝❦❜♦♥❡✮ ❙❡r ✷❜ α✲❛♠✐❞❡ ✭◆✲❍ ❞✐♣♦•❡✱ ❜❛❝❦❜♦♥❡✮ ✹ | ✸✺✸✷ ✭β✸✧✮ α✲❛♠✐❞❡ ✭◆✲❍ ❞✐♣♦•❡✱ ❜❛❝❦❜♦♥❡✮ ❙❡r ✷❜ α✲❛♠✐❞❡ ✭❈❂❖ ❞✐♣♦•❡✱ ❜❛❝❦❜♦♥❡✮ ✺ ❆ ✸✺✸✵ ✭•♦♦♣ β✸✬✲β✸✧ ✮ α✲❛♠✐❞❡ ✭❈❂❖ ❞✐♣♦•❡✱ ❜❛❝❦❜♦♥❡✮ ❚❤r ✹❜ α✲❛♠✐❞❡ ✭◆✲❍ ❞✐♣♦•❡✱ ❜❛❝❦❜♦♥❡✮ ✻ ❑ ✸✹✾✻ ✭•♦♦♣ β✶✲β✷ ✮ ǫ✲❛♠✐♥♦ ♣❙❡r ✺❜ β✲♣❤♦s♣❤♦♥♦♦①② ✭❖ ✶❆ ✮ ✼ ( ✸✺✽✷ ✭α✹✮ β✲❤②❞r♦①②♣❤❡♥②• ♣❙❡r ✺❜ β✲♣❤♦s♣❤♦♥♦♦①② ✭❖ ✷❆ ✮ ✽ | ✸✺✸✷ ✭β✸✧✮ δ✲❣✉❛♥✐❞✐♥♦ ♣❙❡r ✺❜ β✲♣❤♦s♣❤♦♥♦♦①② ✭❖ ✷❆ ✮ ✽✬ | ✸✺✸✷ ✭β✸✧✮ δ✲❣✉❛♥✐❞✐♥♦ ♣❙❡r ✺❜ β✲♣❤♦s♣❤♦♥♦♦①② ✭❖ ✸❆ ✮ ✾ ❑ ✸✺✽✻ ✭•♦♦♣ α✹✲β✺ ✮ ǫ✲❛♠✐♥♦ ♣❙❡r ✺❜ β✲♣❤♦s♣❤♦♥♦♦①② ✭❖ ✸❆ ✮ ❚❛❜•❡ ✼✳✻ ✕ ❙✉♠♠❛r② ♦❢ ✐♥t❡r♠♦•❡❝✉•❛r ❤②❞r♦❣❡♥ ❜♦♥❞s ❜❡t✇❡❡♥ ❙P❖❈ ❛♥❞ t❤❡ ❙❡r✺P✲❈❚❉ ♣❡♣t✐❞❡✳ ❍②❞r♦❣❡♥ ❜♦♥❞s ❛r❡ •❛❜❡•❡❞ ❛❝❝♦r❞✐♥❣ t♦ ❋✐❣✉r❡ ✼✳✽✱ ❛♥❞ ♦①②❣❡♥ ❛t♦♠s ✇✐t❤✐♥ t❤❡ ♣❤♦s♣❤❛t❡ ❣r♦✉♣ ❛r❡ ♥❛♠❡❞ ❢♦••♦✇✐♥❣ ■❯P❆❈ r❡❝♦♠♠❡♥❞❛t✐♦♥s ❬❇•❛❝❦❜✉r♥ ❡t ❛•✳✱ ✷✵✶✼(✳ ✶✻✻ |❡s✉•ts ❙P❖❈✴❙❡r✺P✲❈❚❉ ✐♥t❡r❛❝t✐♦♥ ❛♥❛•②s✐s ❚❤❡ ❙❡r ✺ ♣❤♦s♣❤♦s❡r✐♥❡ ✐s ❞♦❝❦❡❞ ❞❡❡♣•② ✐♥t♦ t❤❡ ❜❛s✐❝ ♣❛t❝❤ ♦❢ ❙P❖❈✱ ✇✐t❤ t❤❡ ♣❤♦s♣❤❛t❡ ❣r♦✉♣ ♦r✐❡♥t❡❞ ✐♥✇❛r❞s✱ t♦✇❛r❞ t❤❡ ❞❡♣t❤ ♦❢ ❛ ❝❛✈✐t② ❢♦r♠❡❞ ❜② t❤❡ ❑ ✸✹✾✻ ✱ | ✸✺✸✷ ❛♥❞ ❑ ✸✺✽✻ ❙P❖❈ r❡s✐❞✉❡s ✭❋✐❣✉r❡ ✼✳✽ ❛♥❞ ❋✐❣✉r❡ ✼✳✾✮✳ ❚❤❡s❡ r❡s✐❞✉❡s ❛r❡ ♠♦st •✐❦❡•② ❝r✐t✐❝❛• ❢♦r t❤❡ ❙P❖❈ s♣❡❝✐✜❝✐t② t♦✇❛r❞s t❤❡ ❙❡r✺P✲❈❚❉✱ ❛s t❤❡② ❛r❡ ❛•• ✐♥✈♦•✈❡❞ ✐♥ ❤②❞r♦❣❡♥ ❜♦♥❞s ✇✐t❤ t❤❡ ♣❤♦s♣❤♦♥♦♦①② ❣r♦✉♣ ♦❢ t❤❡ ❙❡r ✺ ♣❤♦s♣❤♦s❡r✐♥❡ ✭❋✐❣✉r❡ ✼✳✽ ❛♥❞ ❋✐❣✉r❡ ✼✳✾✮✳ |❡♠❛r❦❛❜•②✱ ❜✐♥❞✐♥❣ ♦❢ ♣❤♦s♣❤♦r②•❛t❡❞ ❙▼|❚ ✭♣❙▼|❚✮ ♦❝❝✉rs ✇✐t❤✐♥ t❤❡ s❛♠❡ r❡✲ ❣✐♦♥ ♦❢ ❙P❖❈ ❛♥❞ ✐♥✈♦•✈❡s t❤❡ s❛♠❡ ❙P❖❈ r❡s✐❞✉❡s ✭s❡❡ s✉❜s❡❝t✐♦♥ ✼✳✶✳✷ ❛♥❞ ❬▼✐❦❛♠✐ ❡t ❛•✳✱ ✷✵✶✹(✮✱ s✉❣❣❡st✐♥❣ t❤❛t ◆❈♦|✴❙▼|❚ ❛♥❞ t❤❡ ❙❡r✺P✲❈❚❉ ♦❢ |◆❆P■■ ♠❛② ❝♦♠♣❡t❡ ❢♦r ✐♥t❡r❛❝t✐♥❣ ✇✐t❤ ❙P❖❈ ✐♥ ✈✐✈♦✳ 180°R3532 K3496 K3586 Y3582 S2 pS5 8'
♦r ♦❢ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❞✉r✐♥❣ ❳❈■ ❲❤✐•❡ s❡✈❡r❛• st✉❞✐❡s ❤❛❞ s❤♦✇♥ t❤❛t ❙♣❡♥ ❦♥♦❝❦❞♦✇♥ •❡❛❞s t♦ s✐•❡♥❝✐♥❣ ❞❡❢❡❝ts ❛t ❛ ❢❡✇ ❳✲•✐♥❦❡❞ ❣❡♥❡s ❬▼♦✐♥❞r♦t ❡t ❛•✳✱ ✷✵✶✺❀ ▼♦♥❢♦rt ❡t ❛•✳✱ ✷✵✶✺❀ ❈❤✉ ❡t ❛•✳✱ ✷✵✶✺❀ ▼❝❍✉❣❤ ❡t ❛•✳✱ ✷✵✶✺(✱ ♥♦♥❡ ❛❞❞r❡ss❡❞ t❤❡ ❡✛❡❝t ♦❢ ❙P❊◆ •♦ss ♦♥ ❝❤r♦♠♦s♦♠❡ ✇✐❞❡ ❣❡♥❡ s✐•❡♥❝✐♥❣✳ ■♥ t❤✐s ✇♦r❦✱ ■ s❤♦✇ t❤❛t ❙P❊◆ ✐s t❤❡ ♠❛st❡r r❡❣✉•❛t♦r ♦❢ ❣❡♥❡ s✐•❡♥❝✐♥❣ ✐♥✐t✐❛t✐♦♥ ❞✉r✐♥❣ ❳❈■✱ ❜♦t❤ ✐♥ ✈✐tr♦ ❛♥❞ ✐♥ ✈✐✈♦✳ ■♠♣♦rt❛♥t•②✱ ❳✐st r❡❝r✉✐ts ❙P❊◆ ✐♠♠❡❞✐❛t❡•② ✉♣♦♥ ✐ts ✉♣r❡❣✉•❛t✐♦♥✱ ❛♥❞ ❙P❊◆ r❡♠❛✐♥s ❛ss♦❝✐❛t❡❞ ✇✐t❤ t❤❡ ❳✐st ❞♦♠❛✐♥ t❤r♦✉❣❤♦✉t t❤❡ ❳❈■ ♣r♦❝❡ss✳ ✽✳✶ ▼♦st ❳✲•✐♥❦❡❞ ❣❡♥❡s ❛r❡ s✐•❡♥❝❡❞ ❡♥t✐r❡•② t❤r♦✉❣❤ ❙P❊◆✲❞❡♣❡♥❞❡♥t ♠❡❝❤❛♥✐s♠s✳ ❯s✐♥❣ ❙P❊◆✲❞❡❣r♦♥ ♠❊❙❈s ❛♥❞ |◆❆s❡q✱ ■ ❢♦✉♥❞ t❤❛t ❞❡♣•❡t✐♥❣ ❙P❊◆ t❤r♦✉❣❤♦✉t ✷✹ ❤♦✉rs ♦❢ ❳✐st ✉♣r❡❣✉•❛t✐♦♥ r❡s✉•ts ✐♥ ❞r❛♠❛t✐❝ ❞❡❢❡❝ts ✐♥ ❳✲•✐♥❦❡❞ ❣❡♥❡ s✐•❡♥❝✐♥❣✱ ❝❤r♦✲ ♠♦s♦♠❡ ✇✐❞❡✳ ❆♠♦♥❣ t❤❡ ✸✽✷ ❳✲•✐♥❦❡❞ ❣❡♥❡s ♠♦♥✐t♦r❡❞ ❜② |◆❆s❡q✱ ✼✽✪ ♦❢ t❤❡♠ s❤♦✇ ♥♦ s✐❣♥✐✜❝❛♥t s✐❣♥s ♦❢ ❳✐st✲♠❡❞✐❛t❡❞ ❣❡♥❡ s✐•❡♥❝✐♥❣ ✉♣♦♥ ❙P❊◆ ❞❡♣•❡t✐♦♥✱ ❞❡♠♦♥str❛t✐♥❣ t❤❛t t❤❡ ♠❛•♦r✐t② ♦❢ ❣❡♥❡s ♦♥ t❤❡ ❳ ❛r❡ ❢✉••② ❞❡♣❡♥❞❡♥t ♦❢ ❙P❊◆ ❢♦r s✐•❡♥❝✐♥❣✳ ■♠♣♦rt❛♥t•②✱ ✶✽✪ ♦❢ t❤❡ ✸✽✷ ❣❡♥❡s ♦♥•② s❤♦✇ ♣❛rt✐❛• ❞❡❢❡❝ts ✐♥ ❳✐st✲♠❡❞✐❛t❡❞ ❣❡♥❡ s✐•❡♥❝✐♥❣ ✉♣♦♥ ❙P❊◆ ❞❡♣•❡t✐♦♥✳ ❖♥ ❛✈❡r❛❣❡✱ t❤❡s❡ ❣❡♥❡s ❛r❡ s✐•❡♥❝❡❞ ❜② ✺✵✪ ✭❝♦♠♣❛r❡❞ t♦ ❛ ✇✐•❞✲t②♣❡ s❝❡♥❛r✐♦✮✱ s✉❣❣❡st✐♥❣ t❤❛t t❤❡✐r s✐•❡♥❝✐♥❣ ♣❛rt✐❛••② r❡•✐❡s ♦♥ ❙P❊◆✲❞❡♣❡♥❞❡♥t ♠❡❝❤❛♥✐s♠s✱ ❛♥❞ t❤❛t ♦t❤❡r ♠❡❝❤❛♥✐s♠s ❡♥s✉r❡ t❤❛t t❤❡s❡ ❣❡♥❡s ❛r❡ s✐•❡♥❝❡❞ t♦ t❤❡✐r ❢✉•• ♣♦t❡♥t✐❛•✳ ❋✉t✉r❡ st✉❞✐❡s ❝♦✉•❞ ✐❞❡♥t✐❢② s✉❝❤ ♠❡❝❤❛♥✐s♠s ❜② s❡tt✐♥❣ ✉♣ ❣❡♥❡t✐❝ s❝r❡❡♥s ✶✻✾ ❉✐s❝✉ss✐♦♥ ❙P❊◆ ❢✉♥❝t✐♦♥ ❞✉r✐♥❣ ❳❈■ ❢♦r ❳❈■ ✉s✐♥❣ s♦♠❡ ♦❢ t❤❡s❡ ❣❡♥❡s ❛s t❤❡✐r ♣❤❡♥♦t②♣✐❝ r❡♣♦rt❡rs✳ ✽✳✷ ❆ ❢❡✇ ❳✲•✐♥❦❡❞ ❣❡♥❡s ❛r❡ s✐•❡♥❝❡❞ t❤r♦✉❣❤ ❙P❊◆✲ ✐♥❞❡♣❡♥❞❡♥t ♠❡❝❤❛♥✐s♠s✳ ❲❤✐•❡ ♠♦st ❣❡♥❡s ✭✾✻✪✮ s❤♦✇ ❞r❛st✐❝ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❞❡❢❡❝ts ✉♣♦♥ •♦ss ♦❢ ❙P❊◆✱ ■ ♦❜s❡r✈❡❞ t❤❛t ❛ ❢❡✇ ❣❡♥❡s ✭✶✻ ✐♥ t♦t❛•✮ ✇❡r❡ ❢✉••② s✐•❡♥❝❡❞✱ ✐♥❞❡♣❡♥❞❡♥t•② ♦❢ ❙P❊◆✳ ❆♠♦♥❣ t❤❡s❡ ✶✻ ❙P❊◆✲✐♥❞❡♣❡♥❞❡♥t ❣❡♥❡s ✭❚❛❜•❡ ✽✳✶✮✿ ❼ ✺ ✇❡r❡ ❛•s♦ s✐•❡♥❝❡❞ ✉♣♦♥ ❞❡•❡t✐♦♥ ♦❢ ❳✐st ❆✲r❡♣❡❛t ❬➏②•✐❝③ ❡t ❛•✳✱ ✷✵✶✾(✱ s✉❣❣❡st✐♥❣ t❤❛t t❤❡✐r s✐•❡♥❝✐♥❣ ❞❡♣❡♥❞s ❡♥t✐r❡•② ♦♥ ♠❡❝❤❛♥✐s♠s ✐♥❞❡♣❡♥❞❡♥t ♦❢ t❤❡ ❙P❊◆✴❆✲ r❡♣❡❛t ❛①✐s✳ ❖♥❡ s✉❝❤ ♠❡❝❤❛♥✐s♠ ❝♦✉•❞ ❜❡ ❳✐st ❇✲r❡♣❡❛t ♠❡❞✐❛t❡❞ r❡❝r✉✐t♠❡♥t ♦❢ P|❈✶✱ ✇❤✐❝❤ ❤❛s r❡❝❡♥t•② ❜❡❡♥ s❤♦✇♥ t♦ ♣•❛② ❛ r♦•❡ ✐♥ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❞✉r✐♥❣ ❳❈■ ❬P✐♥t❛❝✉❞❛ ❡t ❛•✳✱ ✷✵✶✼❀ ◆❡st❡r♦✈❛ ❡t ❛•✳✱ ✷✵✶✾(✳ ❼ ✶✶ ❢❛✐•❡❞ t♦ ❜❡ s✐•❡♥❝❡❞ ✉♣♦♥ ❞❡•❡t✐♦♥ ♦❢ ❳✐st ❆✲r❡♣❡❛t ❬➏②•✐❝③ ❡t ❛•✳✱ ✷✵✶✾(✱ s✉❣✲ ❣❡st✐♥❣ t❤❛t t❤❡ ❆✲r❡♣❡❛t r❡❝r✉✐ts ❙P❊◆✲✐♥❞❡♣❡♥❞❡♥t ♠❡❝❤❛♥✐s♠s t♦ s✐•❡♥❝❡ t❤❡s❡ ❢❡✇ ❣❡♥❡s✳ ❆♣❛rt ❢r♦♠ ❙P❊◆✱ t✇♦ ♦t❤❡r ♣r♦t❡✐♥s✱ ❲❚❆P ❛♥❞ |◆❋✷✵✱ ❤❛✈❡ ❜❡❡♥ s❤♦✇♥ t♦ ❝♦✲♣r❡❝✐♣✐t❛t❡ ✇✐t❤ ❳✐st |◆❆ ✐♥ ❛♥ ❆✲r❡♣❡❛t ❞❡♣❡♥❞❡♥t ♠❛♥♥❡r ❬❈❤✉ ❡t ❛•✳✱ ✷✵✶✺(✳ ❲❚❆P ✐s ❛ ♠❡♠❜❡r ♦❢ t❤❡ |◆❆ ♠ ✻ ❆ |◆❆ ♠❡t❤②•❛t✐♦♥ ♠❛❝❤✐♥❡r②✱ ❛♥❞ ❤❛s ❜❡❡♥ s✉❣❣❡st❡❞ t♦ ♣•❛② ❛ r♦•❡ ✐♥ ❳✐st✲♠❡❞✐❛t❡❞ ❣❡♥❡ s✐•❡♥❝✐♥❣ ✭s❡❡ s✉❜s❡❝✲ t✐♦♥ ✶✳✶✸✳✸ ❛♥❞ ❬▼♦✐♥❞r♦t ❡t ❛•✳✱ ✷✵✶✺❀ P❛t✐• ❡t ❛•✳✱ ✷✵✶✻(✮✳ |◆❋✷✵ ✐s ❛♥ ❊✸ •✐❣❛s❡ ✇❤✐❝❤ ♠❡❞✐❛t❡s ♠♦♥♦✲✉❜✐q✉✐t②•❛t✐♦♥ ♦❢ ❤✐st♦♥❡ ❍✷❇ ❛t ▲②s✐♥❡ ✶✷✵ ✭❍✷❇✶✷✵✉❜✶✮ ❬)❤✉ ❡t ❛•✳✱ ✷✵✵✺❀ ❑✐♠ ❡t ❛•✳✱ ✷✵✵✺(✳ •✐✈❡♥ t❤❛t |◆❋✷✵ ✐s ✐♥✈♦•✈❡❞ ✐♥ ❞✐r❡❝t tr❛♥✲ s❝r✐♣t✐♦♥❛• ❛❝t✐✈❛t✐♦♥ ❬)❤✉ ❡t ❛•✳✱ ✷✵✵✺❀ ❑✐♠ ❡t ❛•✳✱ ✷✵✵✺(✱ ✐t ✐s ✉♥•✐❦❡•② t❤❛t t❤✐s ❢❛❝t♦r ✐s ✐♥✈♦•✈❡❞ ✐♥ ♠❡❞✐❛t✐♥❣ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❞✉r✐♥❣ ❳❈■✳ |❡♠❛r❦❛❜•② ❤♦✇❡✈❡r✱ ✸ ❣❡♥❡s ✇❡r❡ ❞❡♣❡♥❞❡♥t ♦♥ ❙P❊◆✱ ❜✉t ♥♦t ♦♥ t❤❡ ❆✲r❡♣❡❛t ❢♦r t❤❡✐r s✐•❡♥❝✐♥❣ ✭❚❛❜•❡ ✽✳✶✮✳ ❚❤✐s ✐♥tr✐❣✉✐♥❣ r❡s✉•t s✉❣❣❡sts t❤❛t s♦♠❡ r❡s✐❞✉❛• ❙P❊◆✲ r❡❝r✉✐t♠❡♥t ❜② ❳✐st ♠❛② st✐•• ❤❛♣♣❡♥ ✐♥❞❡♣❡♥❞❡♥t•② ♦❢ t❤❡ ❆✲r❡♣❡❛t✱ s✉♣♣♦rt✐♥❣ s✐•❡♥❝✐♥❣ ♦❢ ❛ ✈❡r② ♠✐♥♦r ❢r❛❝t✐♦♥ ♦❢ ❣❡♥❡s✳ •✐✈❡♥ t❤❛t t❤❡ ♥✉♠❜❡r ♦❢ s✉❝❤ ❣❡♥❡s ✐s ✈❡r② •♦✇✱ t❤✐s ♦❜✲ s❡r✈❛t✐♦♥ ♥❡❡❞s t♦ ❜❡ ❢✉rt❤❡r r❡♣r♦❞✉❝❡❞ ✐♥ s❡✈❡r❛• ♦t❤❡r ❙P❊◆✲❞❡❣r♦♥ ❝•♦♥❡s ❛♥❞ ✉♥❞❡r ♥♦♥✲❛rt✐✜❝✐❛••② ✐♥❞✉❝❡❞ ❳✐st ❡①♣r❡ss✐♦♥ ✭✐✳❡✳ ✐♥ t❤❡ ❝♦♥t❡①t ♦❢ ❝❡••✉•❛r ❞✐✛❡r❡♥t✐❛t✐♦♥✮✳ ❝❛t❡❣♦r② ❣❡♥❡ ♥❛♠❡s ❙P❊◆✲✐♥❞❡♣❡♥❞❡♥t ♦♥•② ✸✽✸✵✹✶✼❆✶✸|✐❦✱ ❈❞❦•✺✱ ❈♥❦sr✷✱ ❍♠❣♥✺✱ ▼❞♠✹✲♣s✱ ◆❛♣✶•✷✱ P❝s❦✶♥✱ |♣❣r✱ ❙✶✵✵❣✱ ❙❤✸❜❣r•✱ ❚♠❡♠✷✾ ❆✲r❡♣❡❛t ✐♥❞❡♣❡♥❞❡♥t ♦♥•② ❋❤•✶✱ P❤❦❛✶✱ Prr❣✸ ❙P❊◆✴❆✲r❡♣❡❛t ✐♥❞❡♣❡♥❞❡♥t ❆♠♦t✱ ❈♦•✹❛✺✱ ❋❣❢✶✸✱ ❋♥❞❝✸❝✶✱ |♥❢✶✷✽ ❚❛❜•❡ ✽✳✶ ✕ ▲✐st ♦❢ ❙P❊◆ ❛♥❞✴♦r ❆✲r❡♣❡❛t✲✐♥❞❡♣❡♥❞❡♥t ❣❡♥❡s✳ ✶✼✵ ❉✐s❝✉ss✐♦♥ ❙P❊◆ ❢✉♥❝t✐♦♥ ❞✉r✐♥❣ ❳❈■ ✽✳✸ ❙P❊◆ ✐s ❝r✐t✐❝❛• ❢♦r s✐•❡♥❝✐♥❣ ❤✐❣❤•② ❡①♣r❡ss❡❞ ❣❡♥❡s✳ ◆❡✈❡rt❤❡•❡ss✱ ♦✉r ❛♥❛•②s✐s r❡✈❡❛•s t❤❛t t❤❡ ✈❛st ♠❛•♦r✐t② ♦❢ ❣❡♥❡s ❛r❡ s✐•❡♥❝❡❞ t❤r♦✉❣❤ t❤❡ ❙P❊◆✴❆✲r❡♣❡❛t ❛①✐s✳ ■♠♣♦rt❛♥t•②✱ ✇❡ ❢♦✉♥❞ t❤❛t t❤❡ ❡①♣r❡ss✐♦♥ •❡✈❡• ♦❢ t❤❡s❡ ❣❡♥❡s ✭♣r✐♦r t♦ ❳❈■✮ ✐s ❤✐❣❤•② ❝♦rr❡•❛t❡❞ ✇✐t❤ ❤♦✇ ♠✉❝❤ ❙P❊◆ ✐s r❡q✉✐r❡❞ ❢♦r t❤❡✐r s✐•❡♥❝✐♥❣✳ ◆❡st❡r♦✈❛ ❡t ❛•✳ ❛•s♦ r❡♣♦rt❡❞ t❤❛t ❤✐❣❤•② ❡①♣r❡ss❡❞ ❣❡♥❡s s❡❡♠ t♦ r❡•② ♠♦r❡ ♦♥ ❙P❊◆ ❢♦r s✐•❡♥❝✐♥❣ t❤❛♥ •❡ss ❡①♣r❡ss❡❞ ❣❡♥❡s✱ ❛♥❞ ❢✉rt❤❡r ♣r♦♣♦s❡❞ ❛ ♠♦❞❡• ✇❤❡r❡✐♥ P♦•②❝♦♠❜✲ ❞❡♣❡♥❞❡♥t ♠❡❝❤❛♥✐s♠s ✇♦✉•❞ ❜❡ ✐♥✈♦•✈❡❞ ✐♥ s✐•❡♥❝✐♥❣ t❤❡s❡ •❡ss ❡①♣r❡ss❡❞ ❣❡♥❡s ❬◆❡s✲ t❡r♦✈❛ ❡t ❛•✳✱ ✷✵✶✾(✳ ❚❤❡s❡ ♦❜s❡r✈❛t✐♦♥s ❛r❡ ❝♦♥s✐st❡♥t ✇✐t❤ ♦✉r ❈❯❚✫|❯◆ ❞❛t❛✱ ✇❤✐❝❤ ❞❡♠♦♥str❛t❡s t❤❛t ❙P❊◆ ✐s ♣r❡❢❡r❡♥t✐❛••② r❡❝r✉✐t❡❞ t♦ s✐t❡s ♦❢ ❛❝t✐✈❡ tr❛♥s❝r✐♣t✐♦♥✳ •✐✈❡♥ t❤❛t ❙P❊◆ ✐s q✉✐❝❦•② r❡❝r✉✐t❡❞ t♦ t❤❡ ✐♥❛❝t✐✈❛t✐♥❣ ❳ ✭s❡❡ s✉❜s❡❝t✐♦♥ ✽✳✻✳✸✮✱ t❤✐s ♠❛② ❜❡ ❛ ✇❛② t♦ ❣✉❛r❛♥t❡❡ ❡✣❝✐❡♥t s✐•❡♥❝✐♥❣ ♦❢ ❤✐❣❤•② ❡①♣r❡ss❡❞ ❣❡♥❡s✱ ✇❤♦s❡ ❞♦s❛❣❡ ♠✐❣❤t ❜❡ ♠♦r❡ ✐♠♣♦rt❛♥t t♦ ❝♦♠♣❡♥s❛t❡ ❢♦r✳ ■t ❛•s♦ ❤✐❣❤•✐❣❤ts ❙P❊◆ ❛s ❛ ♣♦t❡♥t s✐•❡♥❝❡r✱ ❝❛♣❛❜•❡ t♦ r❡♣r❡ss ❤✐❣❤•② ❡①♣r❡ss❡❞ ❣❡♥❡s✳ ✽✳✹ ❙P❊◆ ✐s ❡ss❡♥t✐❛• ❢♦r ✐♠♣r✐♥t❡❞ ❳❈■ ✐♥ ✈✐✈♦ |❡♠❛r❦❛❜•②✱ ✇❡ ❛•s♦ ❢♦✉♥❞ ❙P❊◆ t♦ ❜❡ ❡ss❡♥t✐❛• ❢♦r ♠❡❞✐❛t✐♥❣ ❣❡♥❡ s✐•❡♥❝✐♥❣ ✐♥ ✈✐✈♦✱ ❞✉r✐♥❣ ✐♠♣r✐♥t❡❞ ❳❈■✳ ■♥❞❡❡❞✱ ❛•t❤♦✉❣❤ ❳✐st ✇❛s st✐•• ❤✐❣❤•② ❡①♣r❡ss❡❞✱ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❛❝r♦ss t❤❡ ♣❛t❡r♥❛• ❳ ❝❤r♦♠♦s♦♠❡ ✇❛s ❝♦♠♣•❡t❡•② ❤✐♥❞❡r❡❞ ✐♥ ❙♣❡♥ ❑❖ ❡♠❜r②♦s✳ ❚❤✐s ✜♥❞✐♥❣ ✐s ✐♠♣♦rt❛♥t✱ ❛s ✐t ❞❡♠♦♥str❛t❡s t❤❛t t❤❡ ♠❡❝❤❛♥✐s♠s r❡s♣♦♥s✐❜•❡ ❢♦r ✐♥✐t✐❛t✐♥❣ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❛r❡ s✐♠✐•❛r ❞✉r✐♥❣ ❜♦t❤ ✇❛✈❡s ♦❢ ❳❈■ ✭✐♠♣r✐♥t❡❞ ❛♥❞ r❛♥❞♦♠✱ t❤❡ •❛tt❡r ❜❡✐♥❣ ♠♦❞❡•❡❞ ✐♥ ♦✉r ♠❊❙❈ s②st❡♠✮✱ ❛♥❞ •❛r❣❡•② ❞❡♣❡♥❞❡♥t ♦♥ ❙P❊◆✳ ❈♦♥✈❡rs❡•②✱ s❡✈❡r❛• st✉❞✐❡s ❤❛✈❡ s❤♦✇♥ t❤❛t t❤❡ ♠❡❝❤❛♥✐s♠s ✐♥✈♦•✈❡❞ ✐♥ ♠❛✐♥t❛✐♥✐♥❣ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❛❢t❡r ❳❈■ ✐s ❡st❛❜•✐s❤❡❞ ❞✐✛❡r ❜❡t✇❡❡♥ ✐♠♣r✐♥t❡❞ ❛♥❞ r❛♥❞♦♠ ❳❈■ ✭s❡❡ s❡❝t✐♦♥ ✶✳✾✮✳ ❲❡ ❢♦✉♥❞ t❤❛t t❤❡ ✐❳❈■ tr❛♥s❝r✐♣t✐♦♥❛• ♣❤❡♥♦t②♣❡ ♦❢ ❙♣❡♥ ❑❖ ❡♠❜r②♦s ✐s ✐❞❡♥t✐❝❛• t♦ t❤❛t ♦❢ ❳✐st ❑❖ ❡♠❜r②♦s ❬❇♦r❡♥s③t❡✐♥ ❡t ❛•✳✱ ✷✵✶✼❜(✳ ❋❡♠❛•❡ ❳✐st ❑❖ ❡♠❜r②♦s s❤♦✇ s❡✈❡r❡ ❣r♦✇t❤ ❞❡❢❡❝ts st❛rt✐♥❣ ❛t ❊✻✳✺✱ ❛♥❞ •❡t❤❛•✐t② ❜② ❊✶✵✳✺ ❛s ❛ ❝♦♥s❡q✉❡♥❝❡ ♦❢ ✐❳❈■ ❢❛✐•✉r❡ ✐♥ ❡①tr❛❡♠❜r②♦♥✐❝ t✐ss✉❡s ❬▼❛r❛❤r❡♥s ❡t ❛•✳✱ ✶✾✾✼(✳ ❑✉r♦❞❛ ❡t ❛•✳ ❤♦✇❡✈❡r r❡♣♦rt❡❞ t❤❛t ❙♣❡♥ ❑❖ ❡♠❜r②♦s ❛♣♣❡❛r s✐♠✐•❛r t♦ ✇✐•❞✲t②♣❡ ❡♠❜r②♦s ✕ ✐♥ s✐③❡ ❛♥❞ ♠♦r♣❤♦•♦❣② ✕ ❛t ❊✶✵✳✺❀ ❛♥❞ •❡t❤❛•✐t② ✐s ♦❜s❡r✈❡❞ ♦♥•② •❛t❡r✱ ❛t ❊✶✸✳✺ ❬❑✉r♦❞❛ ❡t ❛•✳✱ ✷✵✵✸(✳ ❚❤✐s ❞✐✛❡r❡♥❝❡ ✐♥ t✐♠✐♥❣ ♦❢ •❡t❤❛•✐t② ✐s s✉r♣r✐s✐♥❣ ❝♦♥s✐❞❡r✐♥❣ t❤❛t ✐❳❈■ ✐s ❢✉••② ❞❡✜❝✐❡♥t ✐♥ ❙♣❡♥ ❑❖ ❡♠❜r②♦s✳ ❑✉r♦❞❛ ❡t ❛•✳ ❤♦✇❡✈❡r ♦♥•② ❡①❛♠✐♥❡❞ ❛ ❢❡✇ ❙♣❡♥ ❑❖ ❡♠❜r②♦s ❛r♦✉♥❞ ❊✶✵✳✺ ✭♥❂✼✮ ❛♥❞ ❞✐❞ ♥♦t ❢♦••♦✇ s❡① r❛t✐♦s ✐♥ t❤❡✐r ❡①♣❡r✐♠❡♥ts✳ ❍❡♥❝❡✱ ✐t ✐s ♠♦st •✐❦❡•② t❤❛t ❙♣❡♥ ❑❖ ❢❡♠❛•❡ ❡♠❜r②♦s ❞✐❡ ❛t ❊✶✵✳✺ ❞✉❡ t♦ ✐❳❈■ ❢❛✐•✉r❡✱ ✇❤✐•❡ ❙♣❡♥ ❑❖ ♠❛•❡ ❡♠❜r②♦s ❞✐❡ ✶✼✶ ❉✐s❝✉ss✐♦♥ ❙P❊◆ ❢✉♥❝t✐♦♥ ❞✉r✐♥❣ ❳❈■ ❛t ❊✶✸✳✺✱ ❞✉❡ t♦ ♦t❤❡r ❙P❊◆✲❞❡♣❡♥❞❡♥t ♣❤❡♥♦t②♣❡s✳ ❋✉t✉r❡ st✉❞✐❡s s❤♦✉•❞ ✐♥✈❡st✐❣❛t❡ s✉❝❤ •✐❦❡•② ❢❡♠❛•❡✲s♣❡❝✐✜❝ •❡t❤❛•✐t② ♦❢ ❙♣❡♥ ❑❖ ❡♠❜r②♦s✳ ✽✳✺ ❙P❊◆ ✐s ❞✐s♣❡♥s❛❜•❡ t♦ ♠❛✐♥t❛✐♥ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❜✉t ❜✉✛❡rs ❡①♣r❡ss✐♦♥ ♦❢ ❡s❝❛♣❡❡s ✐♥ s♦♠❛t✐❝ ❝❡••s ◆♦ ♣r✐♦r st✉❞✐❡s ❤❛❞ ❡①♣•♦r❡❞ ✇❤❡t❤❡r ❙P❊◆ ✐s ✐♠♣♦rt❛♥t t♦ ♠❛✐♥t❛✐♥ ❳✲•✐♥❦❡❞ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❛❢t❡r ✐t ❤❛s ❜❡❡♥ ❡st❛❜•✐s❤❡❞✳ ❚❤❡ ♦❜s❡r✈❛t✐♦♥ t❤❛t ❙P❊◆ ✐s ❡♥r✐❝❤❡❞ ♦♥ t❤❡ ❳✐ ✐♥ ❞✐✛❡r❡♥t✐❛t❡❞ ❝❡••s s✉❣❣❡st❡❞ ✐t ❝♦✉•❞ ♣•❛② ❛ r♦•❡ ✐♥ s✉❝❤ ♣r♦❝❡ss✳ ❚❤❛♥❦s t♦ t❤❡ ❞❡❣r♦♥ ❛♣♣r♦❛❝❤✱ ■ ✇❛s ❛❜•❡ t♦ ❞❡♣•❡t❡ ❙P❊◆ ✐♥ ❞✐✛❡r❡♥t✐❛t❡❞ ❝❡••s ✭◆P❈s✮✱ ❛❢t❡r ❳❈■ ❤❛❞ ❜❡❡♥ ❡st❛❜•✐s❤❡❞ ❛♥❞ ♠❛✐♥t❛✐♥❡❞✳ ❚r❛♥s❝r✐♣t♦♠✐❝ ❛♥❛•②s✐s r❡✈❡❛•❡❞ ♥♦ s✐❣♥ ♦❢ r❡❛❝t✐✲ ✈❛t✐♦♥ ♦❢ ❢✉••② ✐♥❛❝t✐✈❛t❡❞ ❣❡♥❡s✱ ❞❡♠♦♥str❛t✐♥❣ t❤❛t ❙P❊◆ ❜❡❝♦♠❡s ❞✐s♣❡♥s❛❜•❡ ❞✉r✐♥❣ ♠❛✐♥t❡♥❛♥❝❡ ♦❢ ❳❈■✳ ❚❤✐s ♦❜s❡r✈❛t✐♦♥ ✐s ♥♦t ✉♥❡①♣❡❝t❡❞✱ ❣✐✈❡♥ t❤❛t ♣r❡✈✐♦✉s st✉❞✐❡s ❤✐❣❤•✐❣❤t❡❞ t❤❛t ♦t❤❡r ♣❛t❤✇❛②s ✭♥❛♠❡•② P♦•②❝♦♠❜ ❛♥❞ ❉◆❆ ♠❡t❤②•❛t✐♦♥✮ ❛r❡ ✐♥✈♦•✈❡❞ ✐♥ ♠❛✐♥t❛✐♥✐♥❣ ❣❡♥❡ s✐•❡♥❝✐♥❣ ♦♥ t❤❡ ❳✐ ✭s❡❡ s❡❝t✐♦♥ ✶✳✾✮✳ ❚❤❡ ✐♠♣❛❝t ♦❢ ❙P❊◆ •♦ss ♦♥ ♠❛✐♥t❡♥❛♥❝❡ ♦❢ ❳❈■ s❤♦✉•❞ ❜❡ ❢✉rt❤❡r st✉❞✐❡❞ ✐♥ ♦t❤❡r s♦♠❛t✐❝ ❝❡•• t②♣❡s✳ ■♥❞❡❡❞✱ ✐t ✐s t❡♠♣t✐♥❣ t♦ ❤②♣♦t❤❡s✐③❡ t❤❛t s♦♠❡ ❝❡•• t②♣❡s ♠❛② ❜❡ ♠♦r❡ ❡♣✐❣❡♥❡t✐❝❛••② •❛❜✐•❡ t❤❛♥ ♦t❤❡rs✱ ❛♥❞ ❤❡♥❝❡ ♠♦r❡ s✉s❝❡♣t✐❜•❡ t♦ ❳✲•✐♥❦❡❞ ❣❡♥❡ r❡❛❝✲ t✐✈❛t✐♦♥ ✉♣♦♥ •♦ss ♦❢ ❙P❊◆✳ ❋♦r ❡①❛♠♣•❡✱ ✐t ✐s ❦♥♦✇♥ t❤❛t ♠❛•❡ ❝❡••s ♦❢ t❤❡ ❤❡♠❛t♦♣♦✐❡t✐❝ ❝♦♠♣❛rt♠❡♥t ❝❛♥ ✉♥❞❡r❣♦ ❳✐st✲♠❡❞✐❛t❡❞ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❬❙❛✈❛r❡s❡ ❡t ❛•✳✱ ✷✵✵✻( ❛♥❞ t❤❛t ❳✐st✲❞❡✜❝✐❡♥t ❢❡♠❛•❡ ❤❡♠❛t♦♣♦✐❡t✐❝ ❝❡••s s❤♦✇ ❳✲r❡❛❝t✐✈❛t✐♦♥✱ ✉•t✐♠❛t❡•② •❡❛❞✐♥❣ t♦ ❝❛♥✲ ❝❡r ❬(✐•❞✐r✐♠ ❡t ❛•✳✱ ✷✵✶✸(✳ ■♥ t❤❡s❡ ❝❡••s✱ r❡❣✉•❛t✐♦♥ ♦❢ ❙P❊◆ ❡①♣r❡ss✐♦♥ ❝♦✉•❞ ❜❡ ❛ ♠❡❛♥s t♦ ❛✛❡❝t ❳✲•✐♥❦❡❞ ❣❡♥❡ ❞♦s❛❣❡✳ ❱❡r② r❡❝❡♥t•②✱ ❛ st✉❞② r❡♣♦rt❡❞ t❤❛t ❙♣❡♥ •♦ss ♦❢ ❢✉♥❝t✐♦♥ ✐♥ ❇✲❝❡••s r❡s✉•t❡❞ ✐♥ s✐❣♥✐✜❝❛♥t r❡❛❝t✐✈❛t✐♦♥ ♦❢ t❤❡ ❳✐ ❬(✉ ❡t ❛•✳✱ ✷✵✷✶(✱ t❤❡r❡❜② ❝♦♥✜r♠✐♥❣ t❤❛t ❙P❊◆✬s ✐♠♣♦rt❛♥❝❡ ❞✉r✐♥❣ ❳❈■ ♠❛✐♥t❡♥❛♥❝❡ ❞❡♣❡♥❞s ♦♥ ❝❡••✉•❛r ❝♦♥t❡①t✳ ◆♦♥❡t❤❡•❡ss✱ ♦✉r r❡s✉•ts ✐♥ ❊❙❈s ❛♥❞ ◆P❈s ❤✐♥t t♦✇❛r❞s ❛ ♠♦❞❡• ✐♥ ✇❤✐❝❤ ❙P❊◆ ✐s ✐♠♣♦rt❛♥t ❢♦r ❣❡♥❡ s✐•❡♥❝✐♥❣ ♦♥•② ✇❤❡♥ ❣❡♥❡s ❛r❡ tr❛♥s❝r✐♣t✐♦♥❛••② ❖◆✳ ❈♦♥s✐st❡♥t ✇✐t❤ s✉❝❤ ♠♦❞❡•✱ ■ ✜♥❞ t❤❛t ❙P❊◆ s✐❣♥✐✜❝❛♥t•② ❞❛♠♣❡♥s ❡①♣r❡ss✐♦♥ ♦❢ ❡s❝❛♣❡❡s ✭✇❤✐❝❤ ❛r❡ tr❛♥s❝r✐♣t✐♦♥❛••② ❡①♣r❡ss❡❞ ❢r♦♠ t❤❡ ❳✐✮ ✐♥ ◆P❈s✳ ❚❤✐s tr❛♥s❝r✐♣t✐♦♥❛• ❡✛❡❝t ♦❝❝✉rs ❣•♦❜❛••②✱ t❤r♦✉❣❤♦✉t ❛•♠♦st ❛•• ❡s❝❛♣❡❡s✱ s✉❣❣❡st✐♥❣ t❤❛t ❞♦s❛❣❡ ♦❢ ❡s❝❛♣❡❡ ❡①♣r❡ss✐♦♥ ❝♦✉•❞ ❜❡ ❜✉✛❡r❡❞ ❞✐r❡❝t•② t❤r♦✉❣❤ ❙P❊◆✳ ❍♦✇❡✈❡r✱ ✐t ✐s ✐♠♣♦rt❛♥t t♦ ♥♦t❡ t❤❛t t❤❡ ♠❛❣♥✐t✉❞❡ ♦❢ ❡s❝❛♣❡❡ tr❛♥s❝r✐♣t✐♦♥❛• ❝❤❛♥❣❡s ✉♣♦♥ ❙P❊◆✲•♦ss ✐s ❢❛✐r•② •♦✇ ✭•❡ss t❤❛♥ t✇♦✲❢♦•❞✮✱ ❛♥❞ ✇❡ ❞✐❞ ♥♦t ❛ss❡ss ✇❤❡t❤❡r t❤❡✐r r❡s♣❡❝t✐✈❡ ♣r♦t❡✐♥ •❡✈❡•s ✇❡r❡ ❛✛❡❝t❡❞✳ ❚❤❡s❡ ✜♥❞✐♥❣s ❝♦✉•❞ ❤♦•❞ ❝•✐♥✐❝❛• r❡•❡✈❛♥❝❡ ✐♥ t❤❡ ❝♦♥t❡①t ♦❢ ❝❡rt❛✐♥ ❞✐s❡❛s❡s ✐♥ ✇♦♠❡♥✳ ❋♦r ❡①❛♠♣•❡✱ ✐t ♠❛② ❤❡•♣ ✉♥❞❡rst❛♥❞ t❤❡ •✐♥❦ ❜❡t✇❡❡♥ ❙♣❡♥ •♦ss ♦❢ ❢✉♥❝t✐♦♥ ♠✉t❛t✐♦♥s ✶✼✷ ❉✐s❝✉ss✐♦♥ ❙P❊◆ ❢✉♥❝t✐♦♥ ❞✉r✐♥❣ ❳❈■ ❛♥❞ ❜r❡❛st ❝❛♥❝❡r ✭s❡❡ s❡❝t✐♦♥ ✷✳✼✮✳ ■t ✐s t❡♠♣t✐♥❣ t♦ ❤②♣♦t❤❡s✐③❡ t❤❛t ✐♥ ❙♣❡♥✲♠✉t❛t❡❞ ❝♦♥t❡①ts✱ t✉♠♦r♦❣❡♥❡s✐s ♠❛② ❜❡ ♣r♦♠♦t❡❞ ❜② ♦✈❡r❛•• ✐♥❝r❡❛s❡s ✐♥ ❡①♣r❡ss✐♦♥ ♦❢ ❞♦s❛❣❡✲ s❡♥s✐t✐✈❡ ❡s❝❛♣❡❡s✱ ♦r ❡✈❡♥ ♣❛rt✐❛• r❡❛❝t✐✈❛t✐♦♥ ♦❢ s❡✈❡r❛• ❳✲•✐♥❦❡❞ ❣❡♥❡s✳ ✽✳✻ ❳✐st r❡❝r✉✐ts ❙P❊◆ t♦ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡ ✽✳✻✳✶ ❙P❊◆ ❛❝ts ❞♦✇♥str❡❛♠ ♦❢ ❳✐st ❝♦❛t✐♥❣ ■♥ ❊❙ ❝❡••s✱ •♦ss ♦❢ ❙P❊◆ ❞♦❡s ♥♦t ❛✛❡❝t ❳✐st |◆❆ ❝♦❛t✐♥❣ ✭✐✳❡✳ ♠♦r♣❤♦•♦❣② ❛♥❞ ❢r❡q✉❡♥❝② ♦❢ ❛♣♣❡❛r❛♥❝❡ ♦❢ ❳✐st ❝•♦✉❞s✮ ✇❤❡♥ ❛ss❛②❡❞ ✉s✐♥❣ |◆❆ ❋■❙❍✱ s✉❣❣❡st✐♥❣ t❤❛t ❙P❊◆ ♣•❛②s ♥♦ ♠❛•♦r r♦•❡ ✐♥ ♦r❣❛♥✐③✐♥❣ t❤❡ ❳✐st ❞♦♠❛✐♥✳ ❚❤✐s ♦❜s❡r✈❛t✐♦♥ ✐s ♠❛❞❡ ✉s✐♥❣ ♦✉r ❞♦①✲✐♥❞✉❝✐❜•❡ s②st❡♠✱ ✐♥ ✇❤✐❝❤ ❢♦r❝❡❞ ❳✐st ❡①♣r❡ss✐♦♥ ♠❛② ❤✐❞❡ s♦♠❡ ❡✛❡❝ts ❙P❊◆ ❝♦✉•❞ ❤❛✈❡ ♦♥ ❳✐st ❝♦❛t✐♥❣✳ ❋✉rt❤❡r♠♦r❡✱ ❳✐st ❝♦❛t✐♥❣ ❝❛♥♥♦t ❜❡ ♠♦♥✐t♦r❡❞ ✇✐t❤ ❣r❡❛t r❡s♦•✉t✐♦♥ ✉s✐♥❣ ❋■❙❍✳ P❡r❢♦r♠✐♥❣ ❳✐st |◆❆ ❛♥t✐s❡♥s❡ ♣✉r✐✜❝❛t✐♦♥ ❢♦••♦✇❡❞ ❜② ❣❡♥♦♠✐❝ s❡q✉❡♥❝✐♥❣ ✭|❆P✲s❡q✮ ❬❊♥❣r❡✐t③ ❡t ❛•✳✱ ✷✵✶✸( ✉♥❞❡r ❙P❊◆ ❞❡♣•❡t❡❞ ❝♦♥❞✐t✐♦♥s ✇♦✉•❞ ❤❡•♣ ✉♥r❛✈❡• ✇❤❡t❤❡r ❙P❊◆ ❛✛❡❝ts ❳✐st s♣r❡❛❞✐♥❣ ❛t ❣❡♥♦♠✐❝ r❡s♦•✉t✐♦♥✳ ◆♦♥❡t❤❡•❡ss✱ ■ ♣r♦♣♦s❡ t❤❛t t❤❡ ❙P❊◆ ❞❡❣r♦♥ ❝❛♥ ❜❡ ❛ ♥✐❝❡ ♠♦❞❡• t♦ ✉♥❝♦✉♣•❡ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❢r♦♠ ♦t❤❡r ❢✉♥❝t✐♦♥s ♠❡❞✐❛t❡❞ ❜② ❳✐st |◆❆✱ ✇✐t❤♦✉t ❤❛✈✐♥❣ t♦ ✐♥t❡r❢❡r❡ ✇✐t❤ ❳✐st✬s s❡q✉❡♥❝❡ ✐ts❡•❢✳ ✽✳✻✳✷ ❙P❊◆ •♦ss ❛✛❡❝ts ❳✐st ❡①♣r❡ss✐♦♥ ❞✐✛❡r❡♥t•② ✐♥ ✈✐tr♦ ❛♥❞ ✐♥ ✈✐✈♦ ■♥tr✐❣✉✐♥❣•②✱ ■ ♥♦t✐❝❡❞ ✉s✐♥❣ |◆❆✲s❡q ❛♥❞ qP❈| t❤❛t ❳✐st •❡✈❡•s ❢♦••♦✇✐♥❣ ❞♦①✲ ♠❡❞✐❛t❡❞ ✐♥❞✉❝t✐♦♥ ✐♥ ❙P❊◆✲❞❡♣•❡t❡❞ ❊❙ ❝❡••s ❛r❡ t✇✐❝❡ ❛s •♦✇ ❛s ✐♥ ✇✐•❞✲t②♣❡ ❝❡••s ✭❞❛t❛ ♥♦t s❤♦✇♥✮✳ ❚❤❡ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❞❡❢❡❝ts ♦❜s❡r✈❡❞ ✉♣♦♥ ❙P❊◆ •♦ss ❛r❡ ✉♥•✐❦❡•② t♦ ❜❡ •✐♥❦❡❞ t♦ s✉❝❤ •♦✇❡r ❳✐st ❡①♣r❡ss✐♦♥✳ ■♥❞❡❡❞✱ ❳✐st st✐•• r❡♠❛✐♥s ✈❡r② ❤✐❣❤•② ❡①♣r❡ss❡❞✱ ❢♦r♠✐♥❣ str♦♥❣ ❞♦♠❛✐♥s ✐♥ ❙P❊◆✲❞❡♣•❡t❡❞ ❝❡••s✱ ❛♥❞ ❛ ♣r❡✈✐♦✉s st✉❞② s❤♦✇❡❞ t❤❛t ❳✐st ❡①♣r❡ss✐♦♥ •❡✈❡•s ♣♦♦r•② ❝♦rr❡•❛t❡ ✇✐t❤ ❝❤r♦♠♦s♦♠❡✲✇✐❞❡ s✐•❡♥❝✐♥❣ ♦✉t❝♦♠❡s ❬▲♦❞❛ ❡t ❛•✳✱ ✷✵✶✼(✳ ❚❤❡ •♦✇❡r ❳✐st •❡✈❡•s ♦❜s❡r✈❡❞ ✐♥ t❤❡ ❛❜s❡♥❝❡ ♦❢ ❙P❊◆ ✐♥ ✈✐tr♦ ❝❛♥ ❜❡ ❡①♣•❛✐♥❡❞ ❜② s❡✈❡r❛• ❤②♣♦t❤❡s❡s✿ ❋✐rst✱ ✐t ❤❛s ❜❡❡♥ r❡♣♦rt❡❞ t❤❛t ✇❤❡♥ ❳✐st ✐s ❞❡•❡t❡❞ ♦❢ ✐ts ❆✲r❡♣❡❛t ✭✇❤✐❝❤ ✐s r❡q✉✐r❡❞ ❢♦r ❣❡♥❡ s✐•❡♥❝✐♥❣✮✱ ❳✐st ❝❛♥♥♦t s♣r❡❛❞ ♦✈❡r ❛❝t✐✈❡ r❡❣✐♦♥s ♦❢ t❤❡ ❳✲❝❤r♦♠♦s♦♠❡ ❬❊♥❣r❡✐t③ ❡t ❛•✳✱ ✷✵✶✸(✳ •✐✈❡♥ t❤❛t ❙P❊◆ ❜✐♥❞s t❤❡ ❆✲r❡♣❡❛t ❛♥❞ ✐s ❡ss❡♥t✐❛• ❢♦r ♠❡❞✐❛t✐♥❣ ❣❡♥❡ ✶✼✸ ❉✐s❝✉ss✐♦♥ ❙P❊◆ ❢✉♥❝t✐♦♥ ❞✉r✐♥❣ ❳❈■ s✐•❡♥❝✐♥❣✱ ✐t ✐s ♠♦st •✐❦❡•② t❤❛t ❙P❊◆ •♦ss ❛•s♦ ❤✐♥❞❡rs ❳✐st s♣r❡❛❞✐♥❣ ✐♥t♦ ❛❝t✐✈❡ r❡❣✐♦♥s✳ ❙✉❝❤ ❤②♣♦t❤❡s✐s ❝♦✉•❞ ❜❡ t❡st❡❞ ❜② ♣❡r❢♦r♠✐♥❣ |❆P✲s❡q ❬❊♥❣r❡✐t③ ❡t ❛•✳✱ ✷✵✶✸( ✐♥ ❙P❊◆✲ ❞❡♣•❡t❡❞ ❝❡••s✳ ▲♦✇❡r ❳✐st •❡✈❡•s ✇♦✉•❞ t❤❡♥ ❜❡ ❛ ❝♦♥s❡q✉❡♥❝❡ ♦❢ ✐ts ❞❡st❛❜✐•✐③❛t✐♦♥ ❜② ♥✉❝•❡❛r |◆❆s❡s ✉♣♦♥ ❢❛✐•✉r❡ t♦ s♣r❡❛❞ ♣r♦♣❡r•② ❛❝r♦ss t❤❡ ❳ ❝❤r♦♠♦s♦♠❡✳ ❙❡❝♦♥❞✱ ■ ♦❜s❡r✈❡❞ t❤❛t ❚s✐① ✐s ♥♦t ❛s s✐•❡♥❝❡❞ ✐♥ ❙P❊◆✲❞❡♣•❡t❡❞ ❝❡••s ❛s ✐t ✐s ✐♥ ✇✐•❞✲t②♣❡ ❝❡••s✳ ❲❤✐•❡ t❤✐s s✉❣❣❡sts t❤❛t ❚s✐① s✐•❡♥❝✐♥❣ ✐s ❛t •❡❛st ♣❛rt✐❛••② ❞❡♣❡♥❞❡♥t ♦♥ ❙P❊◆✱ ✐t ❛•s♦ ❤✐♥ts t❤❛t t❤✐s ❤✐❣❤❡r ❚s✐① ❡①♣r❡ss✐♦♥ ❝♦✉•❞ •❡❛❞ t♦ ❳✐st r❡♣r❡ss✐♦♥ ✭s❡❡ s❡❝t✐♦♥ ✶✳✻✮✱ t❤✉s ❡①♣•❛✐♥✐♥❣ t❤❡ •♦✇❡r ❳✐st •❡✈❡•s ♦❜s❡r✈❡❞ ✐♥ ❙P❊◆✲❞❡♣•❡t❡❞ ❝❡••s✳ ■♥ ❢❛❝t✱ ❛ r❡❝❡♥t st✉❞② t♦ ✇❤✐❝❤ ■ ❝♦♥tr✐❜✉t❡❞ ✭s❡❡ s❡❝t✐♦♥ ✷✳✶✵ ❛♥❞ s❡❝t✐♦♥ ❆✳✷✮ r❡♣♦rts t❤❛t ✐♥ t❤❡ ❝♦♥t❡①t ♦❢ ❞✐✛❡r❡♥t✐❛t✐♦♥✲❝♦✉♣•❡❞ ❳❈■✱ ❙P❊◆✲♠❡❞✐❛t❡❞ s✐•❡♥❝✐♥❣ ♦❢ ❚s✐① ✐s ❛❜s♦•✉t❡•② r❡q✉✐r❡❞ ❢♦r ❳✐st ✉♣r❡❣✉•❛t✐♦♥ ❬|♦❜❡rt✲❋✐♥❡str❛ ❡t ❛•✳✱ ✷✵✷✵(✳ ❈♦♥✈❡rs❡•②✱ ✐♥ ✈✐✈♦ ❞✉r✐♥❣ ✐♠♣r✐♥t❡❞ ❳❈■✱ ✇❡ ❢♦✉♥❞ t❤❛t ❙♣❡♥ ❑❖ ❡♠❜r②♦s s❤♦✇ ♠✉❝❤ ❤✐❣❤❡r ❳✐st ❡①♣r❡ss✐♦♥ t❤❛♥ ✇✐•❞✲t②♣❡ ❡♠❜r②♦s✳ ❚❤✐s ❝♦✉•❞ ❜❡ ❛ ❝♦♠♣❡♥s❛t♦r② r❡s♣♦♥s❡ t♦ ❢❛✐•✉r❡ t♦ ✐♥✐t✐❛t❡ ❳❈■✱ s✐♥❝❡ ✐♠♣r✐♥t❡❞ ❳❈■ ✐s ❡ss❡♥t✐❛• ❢♦r ❞❡✈❡•♦♣♠❡♥t✳ ❖♥ t❤❡ ♦t❤❡r ❤❛♥❞✱ ♠❊❙❈s ♣❡r❢❡❝t•② t♦•❡r❛t❡ t♦ ❤❛✈❡ t✇♦ ❛❝t✐✈❡ ❳ ❝❤r♦♠♦s♦♠❡s✱ ✇❤✐❝❤ ❝♦✉•❞ ❡①♣•❛✐♥ ✇❤② t❤❡r❡ ✐s ♥♦ s❡•❡❝t✐✈❡ ♣r❡ss✉r❡ t♦ ✐♥❝r❡❛s❡ ❳✐st ❡①♣r❡ss✐♦♥ ✉♣♦♥ ❙P❊◆ •♦ss ✐♥ ♦✉r ✐♥ ✈✐tr♦ s②st❡♠✳ ✽✳✻✳✸ ❳✐st ✉♣r❡❣✉•❛t✐♦♥ ✐♠♠❡❞✐❛t❡•② tr✐❣❣❡rs ❙P❊◆ r❡❝r✉✐t♠❡♥t ❲❤✐•❡ ✐t ✇❛s r❡♣♦rt❡❞ t❤❛t ❙P❊◆ ❞✐r❡❝t•② ✐♥t❡r❛❝ts ✇✐t❤ ❳✐st |◆❆ ❬❈❤✉ ❡t ❛•✳✱ ✷✵✶✺❀ ▼♦♥❢♦rt ❡t ❛•✳✱ ✷✵✶✺❀ ▼❝❍✉❣❤ ❡t ❛•✳✱ ✷✵✶✺(✱ ✇❤❡t❤❡r ❙P❊◆ ✐s ❛❝t✐✈❡•② r❡❝r✉✐t❡❞ t♦ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡ ❞✉r✐♥❣ ❳❈■ r❡♠❛✐♥❡❞ ✉♥❡①♣•♦r❡❞✳ ❯s✐♥❣ ✜①❡❞ ❛♥❞ •✐✈❡ ❝❡•• ✐♠❛❣✐♥❣ ❞✉r✲ ✐♥❣ ❛ t✐♠❡❝♦✉rs❡ ♦❢ ❳✐st ❡①♣r❡ss✐♦♥✱ ■ ❢♦✉♥❞ t❤❛t ✐♠♠❡❞✐❛t❡•② ✉♣♦♥ ❳✐st ✉♣r❡❣✉•❛t✐♦♥✱ ❙P❊◆ ❢♦r♠s ✏❝•♦✉❞✲•✐❦❡✑ str✉❝t✉r❡s ✐❞❡♥t✐❝❛• t♦ ❛♥❞ ♦✈❡r•❛♣♣✐♥❣ ❳✐st ❝•♦✉❞s✳ ❚❤✐s ✜♥❞✲ ✐♥❣ ❞❡♠♦♥str❛t❡s t❤❛t ❙P❊◆ ❝❛♥ tr✐❣❣❡r s✐•❡♥❝✐♥❣ ✈❡r② ❡❛r•② ♦♥ ✉♣♦♥ ❳✐st ✉♣r❡❣✉•❛t✐♦♥✱ ❢✉rt❤❡r ❤✐❣❤•✐❣❤t✐♥❣ ✐ts ❡ss❡♥t✐❛•✐t② ❞✉r✐♥❣ ❳❈■✳ ❈♦♥s✐st❡♥t ✇✐t❤ ❛ ✈❡r② ❡❛r•② r❡❝r✉✐t♠❡♥t ♦❢ ❙P❊◆ ❜② ❳✐st✱ ❈❯❚✫|❯◆ r❡✈❡❛•❡❞ ❙P❊◆ ❢♦♦t♣r✐♥ts ❛❝r♦ss t❤❡ ❳✐st ❣❡♥❡ ❜♦❞②✱ s✉❣❣❡st✐♥❣ t❤❛t ❙P❊◆ ❜✐♥❞s ❳✐st |◆❆ ✇❤✐•❡ ✐t ✐s ♥❛s❝❡♥t•② tr❛♥s❝r✐❜❡❞✳ ■♥ ❢❛❝t✱ t❤❡ ❙P❊◆ ❢♦♦t♣r✐♥t ✐s ♠❛r❦❡❞•② ✐♥❝r❡❛s❡❞ ❛t t❤❡ ✺✬ r❡❣✐♦♥ ♦❢ ❳✐st✱ ✇❤❡r❡ t❤❡ ❆✲r❡♣❡❛t ✕ t♦ ✇❤✐❝❤ ❙P❊◆ ❜✐♥❞s ✕ ✐s ❡♥❝♦❞❡❞✳ ■t ✐s ❤❛r❞ t♦ r❡❝♦♥❝✐•❡ t❤❡♥✱ ❤♦✇ ❳✐st ❡✈❛❞❡s tr❛♥s❝r✐♣t✐♦♥❛• s✐•❡♥❝✐♥❣ ❞❡s♣✐t❡ s✉❝❤ str♦♥❣ ❙P❊◆ r❡❝r✉✐t♠❡♥t ❛t ✐ts •♦❝✉s✳ ❖♥❡ ♣♦ss✐❜•❡ ❡①♣•❛♥❛t✐♦♥ ❝♦✉•❞ ❜❡ t❤❛t ❳✐st |◆❆ tr❛♣s ❙P❊◆ ❛♥❞ ✏❝❛rr✐❡s✑ ✐t ❛✇❛② ❢r♦♠ ✐ts ♣r♦♠♦t❡r ❛s t❤❡ |◆❆ ✐s tr❛♥s❝r✐❜❡❞✱ t❤❡r❡❜② ♣r❡✈❡♥t✐♥❣ ❙P❊◆ ❢r♦♠ ❡①❡rt✐♥❣ ✐ts s✐•❡♥❝✐♥❣ ❢✉♥❝t✐♦♥✳ ✶✼✹ ❉✐s❝✉ss✐♦♥ ❙P❊◆ ❢✉♥❝t✐♦♥ ❞✉r✐♥❣ ❳❈■ ✽✳✻✳✹ ❙P❊◆ r❡❝r✉✐t♠❡♥t ✐s r❡q✉✐r❡❞ ❢♦r ❣❡♥❡ s✐•❡♥❝✐♥❣ ❛♥❞ ❞❡✲ ♣❡♥❞s ♦♥ ||▼✷✲✹ ❯s✐♥❣ ❛ ❝♦♠♣•❡♠❡♥t❛t✐♦♥ str❛t❡❣②✱ ■ ✐❞❡♥t✐✜❡❞ t❤❛t t❤r❡❡ ♦❢ t❤❡ ❢♦✉r ||▼ ❞♦♠❛✐♥s ♦❢ ❙P❊◆ ✭||▼✷✱ ||▼✸ ❛♥❞ ||▼✹✮ ❛r❡ r❡q✉✐r❡❞ ❢♦r ✐ts r❡❝r✉✐t♠❡♥t t♦ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡ ✐♥ •✐✈❡ ❝❡••s✳ ❚❤✐s ✜♥❞✐♥❣ ✐s ❝♦♥s✐st❡♥t ✇✐t❤ ♣r❡✈✐♦✉s r❡♣♦rts ✇❤✐❝❤ ❞❡♠♦♥str❛t❡❞ t❤❛t t❤❡ ||▼✷✲✹ ❞♦♠❛✐♥s ✐♥t❡r❛❝t ❞✐r❡❝t•② ✇✐t❤ ❳✐st ✐♥ ✈✐tr♦✳ ■♠♣♦rt❛♥t•②✱ ■ ❢♦✉♥❞ t❤❛t ✇❤❡♥ ❙P❊◆ ✇❛s ❞❡•❡t❡❞ ♦❢ ✐ts ||▼✷✲✹ ❞♦♠❛✐♥s✱ ✐t ❢❛✐•❡❞ t♦ s✐•❡♥❝❡ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡✱ ❛•t♦❣❡t❤❡r ❞❡♠♦♥str❛t✐♥❣ t❤❛t ❙P❊◆ r❡❝r✉✐t♠❡♥t ✭❜② ❳✐st✮ ✐s ❛♥ ❡ss❡♥t✐❛• st❡♣ ✐♥ t❤❡ ❣❡♥❡ s✐•❡♥❝✐♥❣ ♣r♦❝❡ss ❞✉r✐♥❣ ❳❈■✳ ✶✼✺ ❉✐s❝✉ss✐♦♥ ❙P❊◆ ♠❡❝❤❛♥✐s♠s ❈❤❛♣t❡r ✾ ▼❡❝❤❛♥✐s♠s ♦❢ ❙P❊◆✲♠❡❞✐❛t❡❞ tr❛♥s❝r✐♣t✐♦♥❛• s✐•❡♥❝✐♥❣ ✾✳✶ ❙P❊◆ ✐♥t❡r❛❝ts ✇✐t❤ ❝❤r♦♠❛t✐♥ t♦ ♣r♦♠♦t❡ tr❛♥✲ s❝r✐♣t✐♦♥❛• s✐•❡♥❝✐♥❣ ✾✳✶✳✶ ❙P❊◆ t❛r❣❡ts ♣r♦♠♦t❡rs ❛♥❞ ❡♥❤❛♥❝❡rs ❈❯❚✫|❯◆ ♣r♦✜•✐♥❣ ♦❢ ❙P❊◆ r❡✈❡❛•❡❞ ✈✐rt✉❛••② ♥♦ ❝❤r♦♠❛t✐♥✲❜✐♥❞✐♥❣ s✐t❡s ✐♥ ♠❊❙❈s✱ ❛ ✜♥❞✐♥❣ t❤❛t ✐s ❝♦♥s✐st❡♥t ✇✐t❤ t❤❡ ♦❜s❡r✈❛t✐♦♥ t❤❛t ✈✐rt✉❛••② ♥♦ ❣❡♥❡s ❛r❡ ❞✐✛❡r❡♥t✐❛••② ❡①♣r❡ss❡❞ ✉♣♦♥ ❙P❊◆ ❞❡♣•❡t✐♦♥ ✐♥ ♠❊❙❈s ✭❞❛t❛ ♥♦t s❤♦✇♥✮✳ ❍♦✇❡✈❡r✱ ❤✉♥❞r❡❞s ♦❢ ❜✐♥❞✐♥❣ s✐t❡s ❛♣♣❡❛r❡❞ s♣❡❝✐✜❝❛••② ♦♥ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡ ❢♦••♦✇✐♥❣ ❳✐st ❡①♣r❡ss✐♦♥✳ ❚❛❦❡♥ t♦❣❡t❤❡r✱ t❤❡s❡ ❞✐s❝♦✈❡r✐❡s str♦♥❣•② ✐♥❞✐❝❛t❡ t❤❛t ❙P❊◆ ❞♦❡s ♥♦t t❛r❣❡t ❉◆❆✴❝❤r♦♠❛t✐♥ ♦♥ ✐ts ♦✇♥✱ ❝♦♥tr❛r② t♦ ✇❤❛t ❤❛❞ ❜❡❡♥ ♣r♦♣♦s❡❞ ✐♥ ❛♥ ♦•❞ r❡♣♦rt ❬◆❡✇❜❡rr② ❡t ❛•✳✱ ✶✾✾✾(✳ ■♥st❡❛❞✱ ❙P❊◆ r❡q✉✐r❡s ❛♥ |◆❆✲✐♥t❡r♠❡❞✐❛t❡ t♦ ❜❡ ❣✉✐❞❡❞ t♦ ✐ts t❛r❣❡t •♦❝✐✳ ❋♦••♦✇✐♥❣ ❳✐st ❡①♣r❡ss✐♦♥✱ ❙P❊◆✲❜✐♥❞✐♥❣ ♦♥ t❤❡ ❳ ✐s r❡str✐❝t❡❞ t♦ ♣r♦♠♦t❡rs ❛♥❞ ❡♥✲ ❤❛♥❝❡rs ♦❢ ❳✲•✐♥❦❡❞ ❣❡♥❡s✱ ✇✐t❤ ❜✐♥❞✐♥❣ ❜❡✐♥❣ ❤✐❣❤❡st ❛t r❛♣✐❞•② s✐•❡♥❝❡❞ ❣❡♥❡s✱ ❛♥❞ •♦✇❡st ✭♥❛② ✉♥❞❡t❡❝t❛❜•❡✮ ❛t ❡s❝❛♣✐♥❣ ❣❡♥❡s✳ ❚❤✐s ❞✐s❝♦✈❡r② ❞❡♠♦♥str❛t❡s t❤❛t ❡♥❤❛♥❝❡rs ❛♥❞ ♣r♦♠♦t❡rs ❝♦♥st✐t✉t❡ t❤❡ s✉❜str❛t❡s ♦❢ ❙P❊◆ ❢♦r ✐ts ❢✉♥❝t✐♦♥ ✐♥ tr❛♥s❝r✐♣t✐♦♥❛• s✐•❡♥❝✐♥❣✳ ■t r❡♠❛✐♥s t♦ ❜❡ ❞❡t❡r♠✐♥❡❞ ❤♦✇ ❙P❊◆ ✐s ♣❤②s✐❝❛••② ❞✐r❡❝t❡❞ t♦ t❤❡s❡ s✐t❡s✳ ▼♦st ♣r♦❜✲ ❛❜•②✱ ✇❤❡r❡ ❳✐st ❛❝❝✉♠✉•❛t❡s ❝♦♥st✐t✉t❡s t❤❡ ✜rst •✐♠✐t✐♥❣ st❡♣ ✐♥ ❣♦✈❡r♥✐♥❣ ✇❤❡r❡ ❙P❊◆ ❜✐♥❞s✱ ❛♥❞ ❝♦♥s✐st❡♥t•②✱ ✐t ✐s ♦❜s❡r✈❡❞ t❤❛t ❙P❊◆ s♣❛t✐❛• ❞②♥❛♠✐❝s ♦❢ r❡❝r✉✐t♠❡♥t ❝•♦s❡•② ❢♦••♦✇ t❤♦s❡ ♦❢ ❳✐st ❝♦❛t✐♥❣✳ ❙P❊◆✲❜✐♥❞✐♥❣ t♦ ❝❤r♦♠❛t✐♥ ✐s t❤❡♥ s❡❝♦♥❞❛r✐•② ❞✐❝t❛t❡❞ t❤r♦✉❣❤ ✐♥t❡r❛❝t✐♦♥s ✇✐t❤ ✐ts ❝❤r♦♠❛t✐♥✲❜♦✉♥❞ ♣r♦t❡✐♥ ♣❛rt♥❡rs ✭s❡❡ s✉❜s❡❝t✐♦♥ ✾✳✷✳✷✮✳ ✶✼✻ ❉✐s❝✉ss✐♦♥ ❙P❊◆ ♠❡❝❤❛♥✐s♠s ❇② ❞❡✈❡•♦♣✐♥❣ ❛♥ ♦♣t✐♠✐③❡❞ ❈❯❚✫|❯◆ ♠❡t❤♦❞♦•♦❣②✱ ✇❤✐❝❤ s✉♣♣♦rts ♣r♦✜•✐♥❣ ♦❢ ❳✐st |◆❆✲❞❡♣❡♥❞❡♥t ❝❤r♦♠❛t✐♥ r❡❝r✉✐t♠❡♥t✱ ■ r❡♣♦rt t❤❡ ✜rst ❣❡♥♦♠❡✲❜✐♥❞✐♥❣ ♠❛♣ ♦❢ ❛ ❳✐st✲r❡❝r✉✐t❡❞ ♣r♦t❡✐♥✳ ❚❤✐s ❛♣♣r♦❛❝❤ s❤♦✉•❞ ❜❡ ❛♣♣•✐❡❞ t♦ ♦t❤❡r ❳✐st✲❜✐♥❞✐♥❣ ♣r♦t❡✐♥s ✭s❡❡ s❡❝t✐♦♥ ✶✳✶✸✮ ❛♥❞ ❝♦✉•❞ ❛•s♦ ❜❡ ❡①t❡♥❞❡❞ t♦ st✉❞② ❝❤r♦♠❛t✐♥ ❛ss♦❝✐❛t❡❞ |◆❆s ✐♥ ❣❡♥❡r❛• ❬▲✐ ❛♥❞ ❋✉✱ ✷✵✶✾(✳ |❡♠❛r❦❛❜•②✱ t❤❡ ❞✐s❝r❡t❡ ♣r♦✜•❡ ♦❢ ❙P❊◆ ❜✐♥❞✐♥❣ t♦ ❳✲•✐♥❦❡❞ ❝❤r♦♠❛t✐♥ ✐s ✉♥❡①♣❡❝t❡❞ ❣✐✈❡♥ t❤❛t ✐♠❛❣✐♥❣ r❡✈❡❛•s ❙P❊◆ ❛❝❝✉♠✉•❛t❡s ❣•♦❜❛••② ❛♥❞ ♠❛ss✐✈❡•② ❛r♦✉♥❞ t❤❡ ❳ ❝❤r♦✲ ♠♦s♦♠❡✳ ❚❤✐s ✐♥❞✐❝❛t❡s t❤❛t ❈❯❚✫|❯◆ ✐s s✉✣❝✐❡♥t•② r❡s♦•✉t✐✈❡ t♦ ♦♥•② ❝❛♣t✉r❡ t❤❡ ❢r❛❝t✐♦♥ ♦❢ ❙P❊◆ t❤❛t ✐s ✐♥ t❤❡ ✈❡r② ❝•♦s❡ ✈✐❝✐♥✐t② ♦❢ ❝❤r♦♠❛t✐♥✱ ❞✐s❝r✐♠✐♥❛t✐♥❣ ✐t ❢r♦♠ t❤❡ r❡♠❛✐♥✐♥❣ ❙P❊◆ ❢r❛❝t✐♦♥ ✇❤✐❝❤ ✐s ❛❝❝✉♠✉•❛t❡❞ ❛r♦✉♥❞ t❤❡ ❳ ✭t❤r♦✉❣❤ ❳✐st✮ ❜✉t ♥♦t ✐♥ ❝♦♥t❛❝t ✇✐t❤ ❝❤r♦♠❛t✐♥✳ ✾✳✶✳✷ ❆❝t✐✈❡ tr❛♥s❝r✐♣t✐♦♥ ✐s r❡q✉✐r❡❞ ❢♦r ❙P❊◆ ❜✐♥❞✐♥❣ ❆•t❤♦✉❣❤ ❙P❊◆ ❜✐♥❞s t♦ ♣r♦♠♦t❡rs ❛♥❞ ❡♥❤❛♥❝❡rs✱ ✐t ❞♦❡s s♦ ♦♥•② ❛t t❤♦s❡ ♦❢ ❛❝t✐✈❡ ❣❡♥❡s✳ ❈♦♥s✐st❡♥t•②✱ ❤✐❣❤•② ❡①♣r❡ss❡❞ ❣❡♥❡s s❤♦✇ ❤✐❣❤❡r ❙P❊◆ ❜✐♥❞✐♥❣ ❛♥❞ ❛r❡ ❝♦♥s❡✲ q✉❡♥t•② ♠♦r❡ ❞❡♣❡♥❞❡♥t ♦♥ ❙P❊◆ ❢♦r s✐•❡♥❝✐♥❣ t❤❛♥ •❡ss ❡①♣r❡ss❡❞ ♦♥❡s ✭s❡❝t✐♦♥ ✽✳✸✮✳ ❋✉rt❤❡r♠♦r❡✱ ✉♣♦♥ ❣❡♥❡ s✐•❡♥❝✐♥❣✱ ❙P❊◆ ✧❞✐s❡♥❣❛❣❡s✧ ❢r♦♠ ❝❤r♦♠❛t✐♥ ✭❛s ✇✐t♥❡ss❡❞ ❜② ❛ s❤❛r♣ ❞❡❝r❡❛s❡ ✐♥ ❈❯❚✫|❯◆ s✐❣♥❛•✮✳ ❍✐❣❤ r❡s♦•✉t✐♦♥ ✐♠❛❣✐♥❣ ❛♣♣r♦❛❝❤❡s t♦ ❞✐r❡❝t•② ✈✐s✉❛•✐③❡ ♠♦•❡❝✉•❡s ♦❢ ❙P❊◆✱ |◆❆P■■ ❛♥❞ ❳✲•✐♥❦❡❞ ❝❤r♦♠❛t✐♥✱ ✐♥ s✐♥❣•❡ ❝❡••s✱ s❤♦✉•❞ ♥♦✇ ❜❡ ❡♠♣•♦②❡❞ t♦ ❢✉rt❤❡r ❡①♣•♦r❡ t❤❡ ❞②♥❛♠✐❝s ♦❢ s✉❝❤ ❙P❊◆ ✧❞✐s❡♥❣❛❣❡♠❡♥t✧✳ ◆❡✈❡rt❤❡•❡ss✱ t❤❡s❡ r❡s✉•ts ✐♥❞✐❝❛t❡ t❤❛t ❛❝t✐✈❡ tr❛♥s❝r✐♣t✐♦♥ ✐s r❡q✉✐r❡❞ ❢♦r ❙P❊◆ ❜✐♥❞✐♥❣✳ ❇✐♦•♦❣✐❝❛••②✱ t❤✐s ❣✉❛r❛♥t❡❡s t❤❛t ❛•• r❡ss♦✉r❝❡s ❛r❡ t❛r❣❡t❡❞ ♦♥•② t♦ ❣❡♥❡s t❤❛t ♥❡❡❞ t♦ ❜❡ ❞♦s❛❣❡ ❝♦♠♣❡♥s❛t❡❞ ✭✐✳❡✳ tr❛♥s❝r✐❜❡❞ ♦♥❡s✮✳ ▼❡❝❤❛♥✐st✐❝❛••②✱ t❤❡ ❜❛s✐s ❢♦r s✉❝❤ ❛ •✐♥❦ ❜❡t✇❡❡♥ ❙P❊◆ ❛♥❞ ❛❝t✐✈❡ tr❛♥s❝r✐♣t✐♦♥ ❝♦✉•❞ ❜❡ ❡①♣•❛✐♥❡❞ ❜② t❤❡ ❞✐r❡❝t ✐♥t❡r❛❝t✐♦♥ ❜❡t✇❡❡♥ ❙P❊◆✬s ❙P❖❈ ❞♦♠❛✐♥ ❛♥❞ t❤❡ tr❛♥s❝r✐♣t✐♦♥ ♠❛❝❤✐♥❡r②✱ ❛s ✐s ❞✐s❝✉ss❡❞ •❛t❡r ✐♥ s✉❜s✉❜s❡❝t✐♦♥ ✾✳✷✳✷✳✸✳ ✶✼✼ ❉✐s❝✉ss✐♦♥ ❙P❊◆ ♠❡❝❤❛♥✐s♠s ✾✳✷ ❙P❖❈✲❝❡♥t❡r❡❞ ❣❡♥❡ s✐•❡♥❝✐♥❣ ♠❡❝❤❛♥✐s♠s ❞✉r✐♥❣ ❳❈■ ✾✳✷✳✶ ❙P❊◆✬s ❙P❖❈ ❞♦♠❛✐♥ ✐s ♥❡❝❡ss❛r② ❛♥❞ s✉✣❝✐❡♥t ❢♦r ❣❡♥❡ s✐•❡♥❝✐♥❣ Pr❡✈✐♦✉s•② ✐♥✈♦•✈❡❞ ✐♥ ❣❡♥❡ r❡♣r❡ss✐♦♥ ✐♥ ♦t❤❡r s②st❡♠s ✭s❡❡ s❡❝t✐♦♥ ✷✳✺✮✱ ❙P❊◆✬s ❙P❖❈ ❞♦♠❛✐♥ ❛•s♦ ❛♣♣❡❛r❡❞ ❝r✉❝✐❛• ❢♦r ❣❡♥❡ s✐•❡♥❝✐♥❣ ❞✉r✐♥❣ ❳❈■ ✐♥ ♠② ✇♦r❦✳ ■♥❞❡❡❞✱ ❛ ❙P❊◆ tr✉♥❝❛t✐♦♥ •❛❝❦✐♥❣ t❤❡ ❙P❖❈ ❞♦♠❛✐♥ ❢❛✐•❡❞ t♦ r❡s❝✉❡ ❳✲•✐♥❦❡❞ ❣❡♥❡ s✐•❡♥❝✐♥❣ ✉♣♦♥ •♦ss ♦❢ ❡♥❞♦❣❡♥♦✉s ❙P❊◆ ❛♥❞ ❝♦♥s✐st❡♥t•②✱ ❡♥❞♦❣❡♥♦✉s ❞❡•❡t✐♦♥ ♦❢ t❤❡ ❙P❖❈ ❞♦♠❛✐♥ r❡s✉•t❡❞ ✐♥ ❝❤r♦♠♦s♦♠❡✲✇✐❞❡ ❞❡❢❡❝ts ✐♥ ❳✐st✲♠❡❞✐❛t❡❞ s✐•❡♥❝✐♥❣✳ ■♥t❡r❡st✐♥❣•②✱ ❦♥♦❝❦✐♥❣✲♦✉t ❙P❖❈ ❡♥❞♦❣❡♥♦✉s•② r❡s✉•t❡❞ ✐♥ ❛ s•✐❣❤t•② •❡ss s❡✈❡r❡ ♣❤❡✲ ♥♦t②♣❡ t❤❛♥ t❤❡ ♦♥❡ ♦❜s❡r✈❡❞ ✉♣♦♥ ❛✉①✐♥✲♠❡❞✐❛t❡❞ ❞❡♣•❡t✐♦♥ ♦❢ ❡♥❞♦❣❡♥♦✉s ❢✉••✲•❡♥❣t❤ ❙P❊◆✳ ❚❤✐s s♠❛•• ♣❤❡♥♦t②♣✐❝ ❞✐✛❡r❡♥❝❡ ❝♦✉•❞ ♣♦ss✐❜•② ❜❡ ❡①♣•❛✐♥❡❞ ❜② ❝♦♠♣❡♥s❛t♦r② ♠❡❝❤❛♥✐s♠s ❛r✐s✐♥❣ ✐♥ t❤❡ ❝♦♥st✐t✉t✐✈❡ ❙♣❡♥ ∆❙P❖❈ s❝❡♥❛r✐♦✱ ✇❤✐❝❤ ❝❛♥♥♦t t❛❦❡ ♣•❛❝❡ ✐♥ t❤❡ ❝❛s❡ ♦❢ t❤❡ ❛❝✉t❡ ❛♥❞ ❝♦♥❞✐t✐♦♥❛• ❙P❊◆ •♦ss ❛❝❤✐❡✈❡❞ ✐♥ t❤❡ ❞❡❣r♦♥ ❝♦♥t❡①t✳ ❍♦✇✲ ❡✈❡r✱ ✐t ✐s ♠♦r❡ •✐❦❡•② t❤❛t t❤❡ s♠❛•• ❞✐✛❡r❡♥❝❡ ❜❡t✇❡❡♥ t❤❡ ❙♣❡♥ ∆❙P❖❈ ❛♥❞ ❙P❊◆✲❞❡❣r♦♥ ♣❤❡♥♦t②♣❡s ✐s ❡①♣•❛✐♥❡❞ ❜② t❤❡ ❢❛❝t t❤❛t r❡❣✐♦♥s ❞✐st✐♥❝t ❢r♦♠ ❙P❖❈ ❛•s♦ ♣•❛② ❛ r♦•❡ ✐♥ ♠❡❞✐❛t✐♥❣ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❞✉r✐♥❣ ❳❈■✳ •✐✈❡♥ t❤❡ ✈❡r② str♦♥❣ ❙♣❡♥ ∆❙P❖❈ ♣❤❡♥♦t②♣❡✱ t❤❡ r♦•❡ ♦❢ t❤❡s❡ r❡❣✐♦♥s ✐s ♦♥•② ♠✐♥♦r✳ ❚❤❡ r❡❣✐♦♥s ✐♥✈♦•✈❡❞ ❛r❡ ♥❡✐t❤❡r t❤❡ ||▼✶ ✭s❡❡ s❡❝t✐♦♥ ✷✳✹✮ ♥♦r t❤❡ |■❉ ✭♥✉❝•❡❛r r❡✲ ❝❡♣t♦r ✐♥t❡r❛❝t✐♦♥✱ s❡❡ s❡❝t✐♦♥ ✷✳✺✮ ❞♦♠❛✐♥s✱ ❛s ❤✐❣❤•✐❣❤t❡❞ ❜② t❤❡ ❝♦♠♣•❡♠❡♥t❛t✐♦♥ ❛♥❛•②s✐s✳ ■t ✐s ❛•s♦ ❤✐❣❤•② ✉♥•✐❦❡•② t❤❛t t❤❡ |❇P■❉ ❞♦♠❛✐♥ ✕ ✇❤✐❝❤ ♠❡❞✐❛t❡s ✐♥t❡r❛❝t✐♦♥ ✇✐t❤ |❇P✲❏ ✭❛♥❞ ❤❡♥❝❡ ◆♦t❝❤ s✐❣♥❛•✐♥❣✱ s❡❡ s❡❝t✐♦♥ ✷✳✻✮ ✕ ✐s ✐♥✈♦•✈❡❞ ✐♥ ❳❈■✳ ■♥❞❡❡❞✱ ◆♦t❝❤ s✐❣♥❛•✐♥❣ ✐s ❞✐s♣❡♥s❛❜•❡ ❢♦r ❡❛r•② ❡♠❜r②♦♥✐❝ ❞❡✈❡•♦♣♠❡♥t✱ ✇✐t❤ ♠❛t❡r♥❛•✲③②❣♦t✐❝ |❜♣• ✲❑❖ ♠♦✉s❡ ❡♠❜r②♦s ❣✐✈✐♥❣ r✐s❡ t♦ ❛•• t❤r❡❡ ❣❡r♠ •❛②❡rs ❬❙♦✉✐•❤♦• ❡t ❛•✳✱ ✷✵✵✻(✱ ❝♦♥✲ tr❛r② t♦ ✇❤❛t ✇♦✉•❞ ❜❡ ♦❜s❡r✈❡❞ s❤♦✉•❞ ❳❈■ ❜❡ ❝♦♠♣r♦♠✐s❡❞ ❬▼❛r❛❤r❡♥s ❡t ❛•✳✱ ✶✾✾✼(✳ ❚❤❡ ❙P❊◆ r❡❣✐♦♥s ✇❤♦s❡ ❢✉♥❝t✐♦♥ ■ ❞✐❞ ♥♦t ✐♥✈❡st✐❣❛t❡ ✐♥ t❤❡ ❝♦♠♣•❡♠❡♥t❛t✐♦♥ ❛s✲ s❛② ❝♦rr❡s♣♦♥❞ t♦ ✈❡r② •♦♥❣ str❡t❝❤❡s ♦❢ ❛♠✐♥♦ ❛❝✐❞s ♠❛♣♣✐♥❣ t♦ ♣r❡❞✐❝t❡❞ ✐♥tr✐♥s✐❝❛••② ❞✐s♦r❞❡r❡❞ r❡❣✐♦♥s ✭■❉|s✮✳ •✐✈❡♥ t❤❛t ■❉|s ❛r❡ ❦♥♦✇♥ t♦ ❡♥❛❜•❡ •♦✇ ❛✣♥✐t② ♣r♦t❡✐♥✲ ♣r♦t❡✐♥ ✐♥t❡r❛❝t✐♦♥s✱ ✇❤✐❝❤ ❢❛✈♦r ✐♥❝r❡❛s❡s ✐♥ ♣r♦t❡✐♥ ❝♦♥❝❡♥tr❛t✐♦♥ •♦❝❛••② ❬❇❛♥❛♥✐ ❡t ❛•✳✱ ✷✵✶✼❀ ❈❤♦♥❣ ❡t ❛•✳✱ ✷✵✶✽(✱ ✐t ✐s ❝♦♥❝❡✐✈❛❜•❡ t❤❛t t❤❡s❡ r❡❣✐♦♥s ❝♦✉•❞ ❜❡ ✐♥✈♦•✈❡❞ ✐♥ ❤✐❣❤❡r✲ ♦r❞❡r ❛ss❡♠❜•② ✇✐t❤ ❳✐st ❛♥❞ ♦t❤❡r ♣r♦t❡✐♥ ♣❛rt♥❡rs✱ •❡❛❞✐♥❣ t♦ t❤❡ ♠♦•❡❝✉•❛r ❝♦♠♣❛rt✲ ♠❡♥t❛•✐③❛t✐♦♥ ♦❢ t❤❡ ❳✐✱ ✇❤✐❝❤ ❤❛s ❜❡❡♥ ♣r♦♣♦s❡❞ t♦ r❡❣✉•❛t❡ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❞✉r✐♥❣ ❳❈■ ❬❈❡r❛s❡ ❡t ❛•✳✱ ✷✵✶✾(✳ ■♥ ❢❛❝t✱ ❛ r❡❝❡♥t r❡♣♦rt ❢r♦♠ t❤❡ P•❛t❤ •❛❜ s❤♦✇s t❤❛t ❳✐st ♥✉❝•❡❛t❡s t❤❡ ❢♦r♠❛t✐♦♥ ♦❢ ❙P❊◆ ❝•✉st❡rs✱ ✇❤✐❝❤ ❞②♥❛♠✐❝❛••② ✐♥❝r❡❛s❡ ✐♥ ❝♦♥❝❡♥tr❛t✐♦♥ t❤r♦✉❣❤ ✶✼✽ ❉✐s❝✉ss✐♦♥ ❙P❊◆ ♠❡❝❤❛♥✐s♠s ♣r♦t❡✐♥✲♣r♦t❡✐♥ ✐♥t❡r❛❝t✐♦♥s ❬▼❛r❦❛❦✐ ❡t ❛•✳✱ ✷✵✷✵(✳ |❡♠❛r❦❛❜•②✱ t❤❡ ✧❛rt✐✜❝✐❛•✧ r❡❝r✉✐t♠❡♥t ♦❢ ❙P❖❈ ❛•♦♥❡ t♦ ❳✐st ❛♥❞ t❤❡ ❳ ❝❤r♦♠♦✲ s♦♠❡✱ t❤r♦✉❣❤ t❤❡ ❇❣• st❡♠✲•♦♦♣ t❡t❤❡r✐♥❣ s②st❡♠✱ ✐s s✉✣❝✐❡♥t t♦ ❝❛rr② ♦✉t ❛ ♠❛•♦r ❢r❛❝t✐♦♥ ♦❢ ❙P❊◆✲❞❡♣❡♥❞❡♥t ❣❡♥❡ s✐•❡♥❝✐♥❣✱ ❝❤r♦♠♦s♦♠❡ ✇✐❞❡✳ ■♠♣♦rt❛♥t•②✱ t❤❡ ♠❛❣♥✐✲ t✉❞❡ ♦❢ t❤✐s ❳✐st✰❙P❖❈ r❡s❝✉❡ ❡✛❡❝t ✐s ♥♦t ❤♦♠♦❣❡♥❡♦✉s t❤r♦✉❣❤♦✉t t❤❡ ❳ ❝❤r♦♠♦s♦♠❡✱ ❞❡❝r❡❛s✐♥❣ ❛s t❤❡ ❣❡♥♦♠✐❝ ❞✐st❛♥❝❡ t♦ t❤❡ ❳✐st •♦❝✉s ✐♥❝r❡❛s❡s✳ ❚❤✐s ❤✐❣❤•✐❣❤ts t❤❡ •✐♠✲ ✐t❛t✐♦♥ ♦❢ t❤❡ ❇❣•✲t❡t❤❡r✐♥❣ ❛♣♣r♦❛❝❤✱ ✐♥ ✇❤✐❝❤ ❙P❖❈ ✐s ❜❡✐♥❣ ❛rt✐✜❝✐❛••② ❛♥❝❤♦r❡❞ t♦ ❳✐st ❡①♦♥ ✼ ❛♥❞ ♣r❡s✉♠❛❜•② ♥♦t ❜✐♥❞✐♥❣ ✇✐t❤ t❤❡ s❛♠❡ st♦✐❝❤✐♦♠❡tr② ❛s ✐t ✇♦✉•❞ ✐♥ t❤❡ ❝♦♥t❡①t ♦❢ ❙P❊◆ ♦♥ ❳✐st ❆✲r❡♣❡❛t✳ ◆❡✈❡rt❤❡•❡ss✱ ❙P❖❈ ❝•❡❛r•② ❡♠❡r❣❡s ❛s ❙P❊◆✬s ❡s✲ s❡♥t✐❛• ❡✛❡❝t♦r ♦❢ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❞✉r✐♥❣ ❳❈■✱ ❣❛t❤❡r✐♥❣ ❛•• ❡•❡♠❡♥ts r❡q✉✐r❡❞ t♦ ✐♥str✉❝t ❣❡♥❡ s✐•❡♥❝✐♥❣ ❛✉t♦♥♦♠♦✉s•②✳ ✾✳✷✳✷ ❙P❖❈ ✐s ❛ ♠♦•❡❝✉•❛r ✐♥t❡❣r❛t♦r ♦❢ ❡♣✐❣❡♥❡t✐❝ ❛♥❞ tr❛♥s❝r✐♣✲ t✐♦♥❛• ❝♦♥tr♦• ❚❤❡ ❝❤❛r❛❝t❡r✐③❛t✐♦♥ ♦❢ ❙P❖❈✬s ♣r♦t❡✐♥ ✐♥t❡r❛❝t♦♠❡ r❡✈❡❛•❡❞ t❤❛t ✐ts ♣❛rt♥❡rs ✐♥❝•✉❞❡ ◆❈♦|✴❙▼|❚ ✭❡①♣❡❝t❡❞•②✮✱ t❤❡ ◆✉|❉ ❝♦♠♣•❡①✱ t❤❡ ♠ ✻ ❆ |◆❆ ♠❡t❤②•❛t✐♦♥ ♠❛❝❤✐♥❡r② ❛s ✇❡•• ❛s t❤❡ |◆❆P■■ tr❛♥s❝r✐♣t✐♦♥ ♠❛❝❤✐♥❡r②✳ ❍❡♥❝❡✱ ❙P❖❈ ❛❝ts ❛s ❛ ❤✉❜ ❞✉r✐♥❣ ❳❈■✱ ❛••♦✇✐♥❣ ❙P❊◆ t♦ ❜r✐❞❣❡ ❳✐st ✇✐t❤ s❡✈❡r❛• ♣❛r❛••❡• ❢❛❝t♦rs ✐♥✈♦•✈❡❞ ✐♥ tr❛♥s❝r✐♣t✐♦♥ ❛♥❞ ❝❤r♦♠❛t✐♥ r❡❣✉•❛t✐♦♥✳ ■❞❡♥t✐❢②✐♥❣ ♣r♦t❡✐♥ ♣❛rt♥❡rs ♦❢ ❙P❊◆ ❛••♦✇s t♦ ❣♦ ❛ •❛②❡r ❜❡②♦♥❞ t❤❡ ❳✐st✲✐♥t❡r❛❝t♦♠❡ ♦r✐❣✐♥❛••② r❡✈❡❛•❡❞ ✐♥ t❤❡ ♣r♦t❡♦♠✐❝ st✉❞✐❡s ✭s❡❡ s❡❝t✐♦♥ ✶✳✶✸✮✱ ❜r✐♥❣✐♥❣ ♥❡✇ ♠❡❝❤❛♥✐st✐❝ ✐♥s✐❣❤ts ✐♥t♦ t❤❡ ❳❈■ ♣r♦❝❡ss✳ ■t ❝♦✉•❞ ❜❡ ✐♥❢♦r♠❛t✐✈❡ t♦ r❡♣❡❛t t❤❡ ❳✐st✲♣r♦t❡♦♠✐❝ ❡①♣❡r✲ ✐♠❡♥ts ❬❈❤✉ ❡t ❛•✳✱ ✷✵✶✺❀ ▼❝❍✉❣❤ ❡t ❛•✳✱ ✷✵✶✺❀ ▼✐♥❛•✐❣✐ ❡t ❛•✳✱ ✷✵✶✺( ✉♥❞❡r ❙P❊◆✲❞❡♣•❡t❡❞ ❝♦♥❞✐t✐♦♥s✱ s♦ ❛s t♦ r❡✈❡❛• ✇❤❡t❤❡r ❛♥② ♦❢ t❤❡ ♦r✐❣✐♥❛••② ✐❞❡♥t✐✜❡❞ ❳✐st✲✐♥t❡r❛❝t✐♥❣ ♣r♦t❡✐♥s ❛r❡ ✐♥ ❢❛❝t ❜r♦✉❣❤t ✐♥ ✈✐❛ ❙P❊◆✳ ✾✳✷✳✷✳✶ |❡✲❡✈❛•✉❛t✐♥❣ t❤❡ ❡ss❡♥t✐❛•✐t② ♦❢ t❤❡ ❙P❊◆✲◆❈♦|✴❙▼|❚✲❍❉❆❈✸ ❛①✐s ❚❤❡ ◆❈♦|✴❙▼|❚ ❝♦♠♣•❡① ✇❛s ❡①♣❡❝t❡❞•② r❡tr✐❡✈❡❞ ✉♣♦♥ ❙P❖❈ ✐♠♠✉♥♦♣r❡❝✐♣✐t❛✲ t✐♦♥✱ ❛♥❞ s♦ ✇❛s ❍❉❆❈✸✳ ❋✉rt❤❡r♠♦r❡✱ ❍❉❆❈✸ ✇❛s ❢♦✉♥❞ t♦ ❜❡ ♣r❡✲❜♦✉♥❞ t♦ ❳✲•✐♥❦❡❞ ❡♥❤❛♥❝❡rs ✭✐♥st❡❛❞ ♦❢ ❜❡✐♥❣ ❛❝t✐✈❡•② r❡❝r✉✐t❡❞ ❞✉r✐♥❣ ❳❈■✮✱ ✇❤❡r❡ ✐ts ♦❝❝✉♣❛♥❝② str♦♥❣•② ♦✈❡r•❛♣s t❤❛t ♦❢ ❙P❊◆✲t❛r❣❡t❡❞ ❡♥❤❛♥❝❡rs✳ ❍❡♥❝❡✱ t❤✐s ✇♦r❦ r❡✜♥❡s t❤❡ ♠♦❞❡• ❢♦r ❙P❊◆ ❢✉♥❝t✐♦♥ ❞✉r✐♥❣ ❳❈■ ♣r❡✈✐♦✉s•② ♣r♦♣♦s❡❞ ❜② ▼❝❍✉❣❤ ❡t ❛•✳✱ ♣✐♥♣♦✐♥t✐♥❣ t❤❛t ❢♦••♦✇✐♥❣ ❳✐st ❝♦❛t✐♥❣✱ ❙P❊◆ ❛❝❝✉♠✉•❛t❡s ❛t ❡♥❤❛♥❝❡rs ✇❤❡r❡ ✐t ❛❝t✐✈❛t❡s ♣r❡✲❜♦✉♥❞ ❍❉❆❈✸ ❜② ✶✼✾ ❉✐s❝✉ss✐♦♥ ❙P❊◆ ♠❡❝❤❛♥✐s♠s r❡❝r✉✐t✐♥❣ ◆❈♦|✴❙▼|❚ t❤r♦✉❣❤ ✐ts ❙P❖❈ ❞♦♠❛✐♥✳ ❋✉rt❤❡r♠♦r❡✱ ❍❉❆❈✸✲❞❡♣❡♥❞❡♥t ❞❡❛❝❡t②•❛t✐♦♥ ♣•❛②s ❛ r♦•❡ ✐♥ ❳✐st✲♠❡❞✐❛t❡❞ ❣❡♥❡ s✐•❡♥❝✐♥❣✱ ❜✉t t❤✐s r♦•❡ ✐s ♥♦t ❛s ❡ss❡♥t✐❛• ❛s ▼❝❍✉❣❤ ❡t ❛•✳ ♦r✐❣♥✐❛••② ♣r♦♣♦s❡❞✳ ■♥❞❡❡❞✱ ✇❡ r❡♣♦rt t❤❛t ❍❉❆❈✸ •♦ss ♦♥•② ❞❡•❛②s ❳❈■ ✐♥ ♠❊❙❈s ❬➏②•✐❝③ ❡t ❛•✳✱ ✷✵✶✾(✱ ❛♥❞ ✉♥♣✉❜•✐s❤❡❞ ❞❛t❛ ❢r♦♠ ♦✉r •❛❜ s❤♦✇ t❤❛t ✐♠♣r✐♥t❡❞ ❳❈■ ✐s ♥♦t ❛•t❡r❡❞ ✐♥ ❍❞❛❝✸ ❑❖ ❢❡♠❛•❡ ❡♠❜r②♦s✱ ✐♥❞✐❝❛t✐♥❣ t❤❛t ❛•t❤♦✉❣❤ ❍❉❆❈✸ ♠❛② ❢❛❝✐•✐t❛t❡ ❡✣❝✐❡♥t ❣❡♥❡ s✐•❡♥❝✐♥❣ ❞✉r✐♥❣ ❳❈■✱ ✐t ❞♦❡s ♥♦t tr✐❣❣❡r ✐t✳ ❈♦♥✈❡rs❡•②✱ •♦ss ♦❢ ❙P❊◆ ✕ ♦r ❙P❖❈ ♦♥•② ✕ ✐s ❛ss♦❝✐❛t❡❞ ✇✐t❤ s❡✈❡r❡•② ❤✐♥❞❡r❡❞ s✐•❡♥❝✐♥❣ ♦❢ ✈✐rt✉❛••② ❛•• ❳✲•✐♥❦❡❞ ❣❡♥❡s✱ ❞❡♠♦♥str❛t✐♥❣ t❤❛t ❙P❊◆✴❙P❖❈ ♠❡❞✐❛t❡s ❣❡♥❡ s✐•❡♥❝✐♥❣ t❤r♦✉❣❤ ❛❞❞✐t✐♦♥❛• ♠❡❝❤❛♥✐s♠s ✇❤✐❝❤ ❞♦ ♥♦t ✐♥✈♦•✈❡ ❍❉❆❈✸✳ ❈♦♥s✐st❡♥t•②✱ ❍❉❆❈✸ ✐s ♥♦t r❡❛❞✐•② ❜♦✉♥❞ ❛t ❳✲•✐♥❦❡❞ ♣r♦♠♦t❡rs ✕ ❝♦♥tr❛r② t♦ ❙P❊◆ ✕ s✉❣❣❡st✐♥❣ t❤❛t t❤❡ ❢✉♥❝t✐♦♥ ♦❢ ❙P❊◆ ❛t ♣r♦♠♦t❡rs ❤❛s t♦ ❜❡ ❡①♣•❛✐♥❡❞ ✐♥❞❡♣❡♥❞❡♥t•② ♦❢ ❍❉❆❈✸✳ ✾✳✷✳✷✳✷ ❆ ♣♦ss✐❜•❡ r♦•❡ ❢♦r ♥✉❝•❡♦s♦♠❡ r❡♠♦❞❡•✐♥❣ ❞✉r✐♥❣ ❳❈■ ❙P❖❈ ❝♦✲♣✉r✐✜❡s ✇✐t❤ ❛•• ❝♦♠♣♦♥❡♥ts ♦❢ t❤❡ ◆✉|❉ ❝♦♠♣•❡① ❬r❡✈✐❡✇❡❞ ✐♥ ▲❛✐ ❛♥❞ ❲❛❞❡✱ ✷✵✶✶(✱ ✇❤✐❝❤ ❝❛rr✐❡s ♥✉❝•❡♦s♦♠❡ r❡♠♦❞❡•✐♥❣ ✭t❤r♦✉❣❤ ❆❚P✲❞❡♣❡♥❞❡♥t ❤✐st♦♥❡ s•✐❞✲ ✐♥❣ ❜② ❈❍❉✸✴✹✴✺ ❤❡•✐❝❛s❡s✮ ❛♥❞ ❤✐st♦♥❡ ❞❡❛❝❡t②•❛t✐♦♥ ✭t❤r♦✉❣❤ ❍❉❆❈✶✴✷✮ ❛❝t✐✈✐t✐❡s✱ ❜♦t❤ ♦❢ ✇❤✐❝❤ ❛r❡ ✐♥✈♦•✈❡❞ ✐♥ s✐•❡♥❝✐♥❣ tr❛♥s❝r✐♣t✐♦♥ ❬❲❛❞❡ ❡t ❛•✳✱ ✶✾✾✽❀ )❤❛♥❣ ❡t ❛•✳✱ ✶✾✾✽❀ ❳✉❡ ❡t ❛•✳✱ ✶✾✾✽❀ ❚♦♥❣ ❡t ❛•✳✱ ✶✾✾✽❀ ❇♦r♥❡•ö✈ ❡t ❛•✳✱ ✷✵✶✽(✳ |❡♠❛r❦❛❜•②✱ ❜✐♥❞✐♥❣ ❛t ❳✲•✐♥❦❡❞ ♣r♦♠♦t❡rs ❜② t✇♦ ◆✉|❉ s✉❜✉♥✐ts ✭❈❍❉✹ ❛♥❞ ▼❇❉✸✮ str♦♥❣•② ♦✈❡r•❛♣s t❤❛t ♦❢ ❙P❊◆✳ ❋✉rt❤❡r♠♦r❡✱ ❛✉①✐♥✲♠❡❞✐❛t❡❞ ❞❡♣•❡t✐♦♥ ♦❢ ❈❍❉✹ ❞✉r✐♥❣ ❳❈■ ✐♥ ♠❊❙❈s r❡s✉•ts ✐♥ ♠♦❞❡r❛t❡ ❞❡❢❡❝ts ✐♥ ❳✐st✲♠❡❞✐❛t❡❞ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❬❉♦ss✐♥✱ ✉♥✲ ♣✉❜•✐s❤❡❞(✳ •✐✈❡♥ t❤❛t ♥❡✐t❤❡r ❍❉❆❈✶ ♥♦r ❍❉❆❈✷ ✭t❤❡ t✇♦ ◆✉|❉✲❛ss♦❝✐❛t❡❞ ❍❉❆❈s✮ ❛r❡ ✐♥✈♦•✈❡❞ ✐♥ ❳❈■ ❬➏②•✐❝③ ❡t ❛•✳✱ ✷✵✶✾(✱ t❤❡s❡ r❡s✉•ts ❛•t♦❣❡t❤❡r ✐♥❞✐❝❛t❡ t❤❛t t❤❡ ◆✉|❉ ❝♦♠♣•❡① ♣•❛②s ❛ r♦•❡ ✐♥ ❳✐st✲♠❡❞✐❛t❡❞ ❣❡♥❡ s✐•❡♥❝✐♥❣✱ s♣❡❝✐✜❝❛••② t❤r♦✉❣❤ ✐ts ❈❍❉✹✲ ♠❡❞✐❛t❡❞ ♥✉❝•❡♦s♦♠❡ r❡♠♦❞❡•✐♥❣ ❢✉♥❝t✐♦♥✳ ❙✉♣♣♦rt✐♥❣ t❤✐s ❝♦♥❝•✉s✐♦♥✱ ❈❍❉✹✲❞❡♣❡♥❞❡♥t ♥✉❝•❡♦s♦♠❡ r❡♠♦❞❡•✐♥❣ ✇❛s r❡❝❡♥t•② s❤♦✇♥ t♦ ❞✐s♣•❛❝❡ |◆❆P■■ ❢r♦♠ ❚❙❙s✱ t❤✉s ♣❛rt✐❝✐✲ ♣❛t✐♥❣ t♦ tr❛♥s❝r✐♣t✐♦♥❛• s✐•❡♥❝✐♥❣ ❬❇♦r♥❡•ö✈ ❡t ❛•✳✱ ✷✵✶✽(✳ ■♠♣♦rt❛♥t•②✱ t❤❡ ❙P❖❈✴◆✉|❉ ✐♥t❡r❛❝t✐♦♥ s✉❣❣❡sts t❤❛t ❙P❊◆ ♠❛② r❡❣✉•❛t❡ ◆✉|❉✬s ❢✉♥❝t✐♦♥ ❞✉r✐♥❣ ❳❈■✱ ❛•t❤♦✉❣❤ ❛❞✲ ❞✐t✐♦♥❛• ✇♦r❦ ✐s ♥♦✇ r❡q✉✐r❡❞ t♦ ❞❡t❡r♠✐♥❡ ♣r❡❝✐s❡•② ❤♦✇✱ ✇❤❡♥ ❛♥❞ ✇❤❡r❡ t❤✐s ❤❛♣♣❡♥s✳ ✾✳✷✳✷✳✸ ❙P❖❈ ❛s ❛ ❞✐r❡❝t r❡♣r❡ss♦r ♦❢ |◆❆P■■ ❢✉♥❝t✐♦♥ ❙P❖❈ ❝♦✲✐♠♠✉♥♦♣r❡❝✐♣✐t❛t❡s ✇✐t❤ s❡✈❡r❛• ❝♦♠♣♦♥❡♥ts ♦❢ t❤❡ |◆❆P■■ tr❛♥s❝r✐♣t✐♦♥ ♠❛❝❤✐♥❡r②✱ ❛s ✇❡•• ❛s ✇✐t❤ ♠✉•t✐♣•❡ ❣❡♥❡r❛• tr❛♥s❝r✐♣t✐♦♥ ✐♥✐t✐❛t✐♦♥ ❛♥❞ ❡•♦♥❣❛t✐♦♥ ❢❛❝t♦rs✳ ✶✽✵ ❉✐s❝✉ss✐♦♥ ❙P❊◆ ♠❡❝❤❛♥✐s♠s ❲❤❛t ✐s ♠♦r❡✱ ❙P❖❈ ✐♥t❡r❛❝ts ❞✐r❡❝t•② ✇✐t❤ t❤❡ ❈❚❉ ♦❢ t❤❡ P❖▲|✷❆ ✭|P❇✶✮ |◆❆P■■ s✉❜✉♥✐t✱ s♣❡❝✐✜❝❛••② ✇❤❡♥ t❤❡ •❛tt❡r ✐s ♣❤♦s♣❤♦r②•❛t❡❞ ♦♥ ❙❡r✐♥❡ ✺✳ ❙tr✉❝t✉r❛• ❛♥❛•②s✐s r❡✈❡❛•s t❤❛t t❤❡ ❙❡r✺ ♣❤♦s♣❤♦s❡r✐♥❡ ❝♦♠♣r✐s❡❞ ✇✐t❤✐♥ t❤❡ ❈❚❉ (❙P❚❙P❙ ❤❡♣t❛♣❡♣t✐❞❡ r❡♣❡❛t ❡♥❣❛❣❡s ✐♥ s❡✈❡r❛• ✐♥t❡r❛❝t✐♦♥s ✇✐t❤ ❛t •❡❛st ✸ r❡s✐❞✉❡s •♦❝❛t❡❞ ✐♥ t❤❡ ❜❛s✐❝ ♣❛t❝❤ ♦❢ ❙P❖❈✳ ❚❤❡s❡ ♠♦•❡❝✉•❛r ✐♥s✐❣❤ts ♣r♦✈✐❞❡ ❛ ♠❡❝❤❛♥✐st✐❝ ❜❛s✐s ❢♦r t❤❡ ♦❜s❡r✈❛t✐♦♥s t❤❛t✿ ✭✐✮ ❛❝t✐✈❡ tr❛♥s❝r✐♣t✐♦♥ ✐s r❡q✉✐r❡❞ ❢♦r ❙P❊◆ ❜✐♥❞✐♥❣ t♦ ❝❤r♦♠❛t✐♥ ✭✐✐✮ ❙P❊◆ ❝❤r♦♠❛t✐♥ r❡❝r✉✐t♠❡♥t ❞✉r✐♥❣ ❳❈■ ♠❛•♦r•② ❝♦rr❡•❛t❡s ✇✐t❤ ❙❡r✺✲♣❤♦s♣❤♦r②•❛t❡❞ |◆❆P■■✲❜♦✉♥❞ s✐t❡s ✭✐✐✐✮ ❣❡♥❡ s✐•❡♥❝✐♥❣ ✭✐✳❡✳ •♦ss ♦❢ tr❛♥s❝r✐❜✐♥❣ |◆❆P■■✮ r❡s✉•ts ✐♥ ❙P❊◆ ❞✐ss♦❝✐❛t✐♦♥ ❢r♦♠ ❝❤r♦♠❛t✐♥ ❇✐♦•♦❣✐❝❛••②✱ s✉❝❤ ❛ ❞✐r❡❝t ❙P❊◆✴|◆❆P■■ ✐♥t❡r❛❝t✐♦♥ str♦♥❣•② ❤✐♥ts t❤❛t ❙P❊◆ ❝❛♥ r❡♣r❡ss tr❛♥s❝r✐♣t✐♦♥ ❞✐r❡❝t•②✳ ❚❤❡ s♣❡❝✐✜❝✐t② ♦❢ ❙P❖❈ ❢♦r ❙❡r✺✲♣❤♦s♣❤♦r②•❛t❡❞ |◆❆P■■ ✭r❡❣❛r❞•❡ss ♦❢ ✇❤❡t❤❡r ♦r ♥♦t ❙❡r✷ ❛♥❞ ❙❡r✼ ❛r❡ ❛•s♦ ♣❤♦s♣❤♦r②•❛t❡❞✮ s❤♦✇s t❤❛t ❙P❊◆ ❤❛s t❤❡ ♣♦t❡♥t✐❛• t♦ t❛r❣❡t ❛♥② |◆❆ ♣♦•②♠❡r❛s❡ ■■ ♠♦•❡❝✉•❡ ❡♥❣❛❣❡❞ ✐♥ t❤❡ ❡❛r•② st❛❣❡s ♦❢ t❤❡ tr❛♥s❝r✐♣t✐♦♥ ♣r♦❝❡ss ✭✐✳❡✳ ✐♥✐t✐❛t✐♦♥✱ ♣r♦♠♦t❡r ❡s❝❛♣❡ ❛♥❞ t❤❡ s✇✐t❝❤ ✐♥t♦ ♣r♦❞✉❝t✐✈❡ ❡•♦♥❣❛t✐♦♥✮✳ |❛t✐♦♥❛••②✱ t❤✐s s❡❡♠s t♦ ❜❡ t❤❡ ♠♦st str❛✐❣❤t❢♦r✇❛r❞ ✇❛② t♦ ♣❡rt✉r❜ ♦♥❣♦✐♥❣ tr❛♥s❝r✐♣t✐♦♥ ❛❝t✐✈✐t②✱ t❤✉s ❡①♣•❛✐♥✐♥❣ ❤♦✇ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❝❛♥ ♦❝❝✉r s♦ ❡✣❝✐❡♥t•② ❛♥❞ r♦❜✉st•② ❞✉r✐♥❣ ❳❈■✳ ❇✐♦♣❤②s✐❝❛• ❛♥❛•②s✐s r❡✈❡❛•❡❞ t❤❛t t❤❡ ❛✣♥✐t② ♦❢ t❤❡ ❙P❖❈✴❙❡r✺P✲❈❚❉ ✐♥t❡r❛❝t✐♦♥ ✐s r❡•❛t✐✈❡•② ✇❡❛❦ ✭❑ ❞ ≈✶✵✉▼✮✳ ■♥ ❝♦♠♣❛r✐s♦♥✱ t❤❡ ❛✣♥✐t② ❜❡t✇❡❡♥ ❙P❖❈ ❛♥❞ ♣❤♦s♣❤♦r②✲ •❛t❡❞ ◆❈♦|✴❙▼|❚ ✐s r❡♣♦rt❡❞•② ❛t •❡❛st ✶✵ t✐♠❡s ❣r❡❛t❡r ✭❑ ❞ ❁✶✉▼✮ ❬❖s✇❛•❞ ❡t ❛•✳✱ ✷✵✶✻(✳ ❙✉❝❤ ♠♦❞❡st ❜✐♥❞✐♥❣ ❜❡t✇❡❡♥ ❙P❊◆ ❛♥❞ |◆❆P■■ ✐s t♦ ❜❡ ❡①♣❡❝t❡❞✳ ■♥❞❡❡❞✱ ❣✐✈❡♥ t❤❡ ♣♦t❡♥t r❡♣r❡ss✐✈❡ ❛❝t✐✈✐t② ♦❢ ❙P❊◆✱ ❛ ♠✉❝❤ str♦♥❣❡r ❛✣♥✐t② ✇♦✉•❞ s✉❣❣❡st t❤❛t ❙P❊◆ ✐s ✧❝♦♥st✐t✉t✐✈❡•②✧ ❛ss♦❝✐❛t❡❞ ✇✐t❤ |◆❆P■■✱ ❣❡♥♦♠❡ ✇✐❞❡✱ ❤❡♥❝❡ ❤✐♥❞❡r✐♥❣ tr❛♥s❝r✐♣✲ t✐♦♥ ❞r❛♠❛t✐❝❛••②✳ ❋✉rt❤❡r♠♦r❡✱ ✐♥t❡r❛❝t✐♦♥ ❛✣♥✐t✐❡s ❜❡t✇❡❡♥ ❈❚❉✲♣❤♦s♣❤♦♣❡♣t✐❞❡s ❛♥❞ ♦t❤❡r ❈❚❉✲✐♥t❡r❛❝t✐♥❣ ♣r♦t❡✐♥s ❬❊✐❝❦ ❛♥❞ •❡②❡r✱ ✷✵✶✸❀ ❱❛s✐••❡✈❛ ❡t ❛•✳✱ ✷✵✵✽( ❛r❡ ✇✐t❤✐♥ t❤❡ s❛♠❡ ♦r❞❡r ♦❢ ♠❛❣♥✐t✉❞❡ ❛s t❤❡ ❙❡r✺P✲❈❚❉✴❙P❖❈ ✐♥t❡r❛❝t✐♦♥ ❛✣♥✐t②✱ ❝♦♥✜r♠✐♥❣ t❤❡ r❡•❡✈❛♥❝❡ ♦❢ ♦✉r ♠❡❛s✉r❡♠❡♥t✳ ❚❤❡ ✇❡❛❦❡r ❙P❊◆✴|◆❆P■■ ✐♥t❡r❛❝t✐♦♥ ✭❝♦♠♣❛r❡❞ t♦ ❙P❖❈✴◆❈♦|✴❙▼|❚✮ ✐s t♦ ❜❡ ❝♦♥tr❛st❡❞ ✇✐t❤ t❤❡ ❢❛❝t t❤❛t t❤❡ |◆❆P■■✲❈❚❉ ♠♦t✐❢ ✭(❙P❚❙P❙✮ r❡❝♦❣♥✐③❡❞ ❜② ❙P❖❈ ✐s r❡♣❡❛t❡❞ ♠♦r❡ t❤❛♥ ✺✵ t✐♠❡s ✐♥ t❤❡ |◆❆P■■ ♠♦•❡❝✉•❡ ✭s❡❡ s✉❜s❡❝t✐♦♥ ✼✳✶✳✶✮✱ ✇❤✐•❡ t❤❡ ◆❈♦|✴❙▼|❚ ♠♦t✐❢ ✭▲❙❉❙✮ r❡❝♦❣♥✐③❡❞ ❜② ❙P❖❈ ✐s ♣r❡s❡♥t ✐♥ ♦♥•② ♦♥❡ ❝♦♣② ♣❡r ◆❈♦|✴❙▼|❚ ♠♦•❡❝✉•❡ ✭s❡❡ s✉❜s❡❝t✐♦♥ ✼✳✶✳✷✮✳ ❚❤✐s ✇♦✉•❞ ✐♥ t❤❡♦r② ❛••♦✇ ♠✉•t✐♣•❡ ❙P❖❈ ♠♦•❡❝✉•❡s t♦ ❜✐♥❞ t♦ ❛ s✐♥❣•❡ |◆❆P■■ ♠♦•❡❝✉•❡ ✐♥ ✈✐✈♦ ✭❋✐❣✉r❡ ✾✳✶❆✮✱ ❛♥❞ ✇❡ s❤♦✇ t❤❛t t❤✐s ✐s ✐♥❞❡❡❞ t❤❡ ❝❛s❡ ✐♥ ✈✐tr♦✳ ✶✽✶ ❉✐s❝✉ss✐♦♥ ❙P❊◆ ♠❡❝❤❛♥✐s♠s ■♥ •✐❣❤t ♦❢ t❤❡ ♦❜s❡r✈❛t✐♦♥ t❤❛t ❙P❊◆ ❛❝❝✉♠✉•❛t❡s ♠❛ss✐✈❡•② ❛r♦✉♥❞ t❤❡ ❳ ❝❤r♦♠♦✲ s♦♠❡ ❢♦••♦✇✐♥❣ ❳✐st ❝♦❛t✐♥❣✱ ✐t ❢♦••♦✇s t❤❛t t❤❡ •♦❝❛• ❝♦♥❝❡♥tr❛t✐♦♥ ♦❢ ❙P❊◆ ✐♥ t❤❡ ✈✐❝✐♥✐t② ♦❢ ❳✲•✐♥❦❡❞ ❝❤r♦♠❛t✐♥ ✐♥❝r❡❛s❡s ❞r❛st✐❝❛••② ❞✉r✐♥❣ ❳❈■✳ ❙✉❝❤ ❝r♦✇❞✐♥❣ ♦❢ ❙P❊◆ •✐❦❡•② ♦✈❡r❝♦♠❡s t❤❡ ✇❡❛❦ ❜✐♥❞✐♥❣ ❛✣♥✐t② ♦❢ ❙P❊◆ ❢♦r |◆❆P■■✱ s❤✐❢t✐♥❣ t❤❡ ♠♦•❡❝✉•❛r ❜❛•❛♥❝❡ ✐♥ ❢❛✈♦r ♦❢ t❤❡ ❙P❊◆✴|◆❆P■■ ✐♥t❡r❛❝t✐♦♥✱ ✇✐t❤ ♠❛♥② ❙P❊◆✴❙P❖❈ ♠♦•❡❝✉•❡s ✐♥ t❤❡ ✈✐❝✐♥✐t② ♦❢ tr❛♥s❝r✐❜✐♥❣ |◆❆ ♣♦•②♠❡r❛s❡s✳ ❚❤✐s ♠❛❦❡s ❙P❊◆ ❛ ✉♥✐q✉❡ tr❛♥s❝r✐♣t✐♦♥❛• r❡❣✉•❛t♦r ✐♥ t❤❡ s❡♥s❡ t❤❛t ❞❡s♣✐t❡ ❤❛✈✐♥❣ ❛ str♦♥❣ r❡♣r❡ss✐✈❡ ♣♦t❡♥t✐❛•✱ t❤✐s ♣♦t❡♥t✐❛• ✐s ✐♥✐t✐❛••② ✧❞♦r♠❛♥t✧✱ ❛♥❞ ❝❛♥ ♦♥•② ❜❡ ♠♦❜✐•✐③❡❞ t❤r♦✉❣❤ ✐ts ❝♦♥❞✐t✐♦♥❛• ❛❝❝✉♠✉•❛t✐♦♥ ❛t t❛r❣❡t •♦❝✐✱ t❤r♦✉❣❤ ❛♥ |◆❆ ✐♥t❡r♠❡❞✐❛t❡ ✭✐✳❡✳ ❳✐st✮✳ ❲❤❡t❤❡r t❤❡ ❙P❊◆✴|◆❆P■■ ✐♥t❡r❛❝t✐♦♥ ✐s ♦❢ ❢✉♥❝t✐♦♥❛• r❡•❡✈❛♥❝❡ ❢♦r ❣❡♥❡ s✐•❡♥❝✐♥❣ ❞✉r✐♥❣ ❳❈■ ♥❡❡❞s t♦ ❜❡ ✐♥✈❡st✐❣❛t❡❞✳ ❙tr✉❝t✉r❛• ❝♦♠♣❛r✐s♦♥ ♦❢ t❤❡ ❙P❖❈✴❙❡r✺P✲❈❚❉ ❛♥❞ ❙P❖❈✴♣❤♦s♣❤♦✲❙▼|❚ ❝♦♠♣•❡①❡s r❡✈❡❛•s t❤❛t |◆❆P■■ ❛♥❞ ◆❈♦|✴❙▼|❚ ❜✐♥❞ t♦ t❤❡ s❛♠❡ r❡❣✐♦♥ ♦❢ ❙P❖❈ ✭✐✳❡✳ t❤❡ ❜❛s✐❝ ♣❛t❝❤✮✱ ❡♥❣❛❣✐♥❣ ✐♥ ✐♥t❡r♠♦•❡❝✉•❛r ✐♥t❡r❛❝t✐♦♥s ✇✐t❤ t❤❡ s❛♠❡ ❙P❖❈ r❡s✐❞✉❡s✳ •✐✈❡♥ t❤❛t t❤❡ ❙P❖❈✴◆❈♦|✲❙▼|❚✴❍❉❆❈✸ ❛①✐s ♣•❛②s ❛ r♦•❡ ✐♥ ❳✐st✲♠❡❞✐❛t❡❞ ❣❡♥❡ s✐•❡♥❝✐♥❣✱ t❤❡ ❢✉♥❝t✐♦♥ ♦❢ t❤❡ ❙P❖❈✴|◆❆P■■ ✐♥t❡r❛❝t✐♦♥ ❞✉r✲ ✐♥❣ ❳❈■ ❤❛s t♦ ❜❡ ❛❞❞r❡ss❡❞ ✐♥ ❛ ❝♦♥t❡①t ✇❤❡r❡ ✐t ❤❛s ❜❡❡♥ ❣❡♥❡t✐❝❛••② ✉♥❝♦✉♣•❡❞ ❢r♦♠ t❤❡ ❙P❖❈✲◆❈♦|✴❙▼|❚ ✐♥t❡r❛❝t✐♦♥✳ ❚❤❡ ❡①♣❡r✐♠❡♥t ■ ♣r♦♣♦s❡ ✐s t♦ t❡st t❤❡ ❛❜✐•✐t② ♦❢ ❞✐✛❡r❡♥t ❙P❖❈ ♠✉t❛♥ts ✭♠✉t❛t❡❞ ❛t ❦❡② r❡s✐❞✉❡s ✐♠♣•✐❝❛t❡❞ ✐♥ t❤❡ ✐♥t❡r❛❝t✐♦♥ ✇✐t❤ t❤❡ ❙❡r✺P✲❈❚❉✮ t♦ r❡s❝✉❡ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❞❡❢❡❝ts ❛ss♦❝✐❛t❡❞ ✇✐t❤ ❛✉①✐♥✲♠❡❞✐❛t❡❞ ❞❡♣•❡t✐♦♥ ♦❢ ❡♥❞♦❣❡♥♦✉s ❙P❊◆ ✭❋✐❣✉r❡ ✾✳✶❆✱ ❛♥ ❡①♣❡r✐♠❡♥t ♣r✐♥❝✐♣❛••② s✐♠✐•❛r t♦ t❤❡ ♦♥❡ ❞❡s❝r✐❜❡❞ ✐♥ s✉❜s✉❜s❡❝t✐♦♥ ✻✳✸✳✷✳✹✮✱ ✐♥ ❛ ❝❡••✉•❛r ❜❛❝❦❣r♦✉♥❞ ✐♥ ✇❤✐❝❤ t❤❡ ▲❙❉❙✲♠♦t✐❢ ♦❢ ❡♥❞♦❣❡✲ ♥♦
✕ ❙❝❤❡♠❡ ♦❢ t❤❡ ♣r♦♣♦s❡❞ ❡①♣❡r✐♠❡♥t t♦ t❡st t❤❡ ❢✉♥❝t✐♦♥❛•✐t② ♦❢ t❤❡ ❙P❖❈✴|◆❆P■■ ✐♥t❡r❛❝t✐♦♥ ❞✉r✐♥❣ ❳❈■✳ ✭❆✮ ▼✉t❛t✐♥❣ ❙P❖❈ ❛t ❦❡② r❡s✐❞✉❡s ✭s❤♦✇♥ ❛s ♣✉r♣•❡ ❝✐r❝•❡s✮ ❛••♦✇s t❤❡ ❞✐sr✉♣t✐♦♥ ♦❢ ✐ts ✐♥t❡r❛❝t✐♦♥ ✇✐t❤ |◆❆P■■✳ ❇❣•✲t❡t❤❡r✐♥❣ ♦❢ t❤❡ ♠✉t❛t❡❞ ❙P❖❈ t♦ ❳✐st |◆❆ ❡♥❛❜•❡s t❡st✐♥❣ ✇❤❡t❤❡r t❤❡ ❛❜s❡♥❝❡ ♦❢ t❤❡ ❙P❖❈✴|◆❆P■■ ✐♥t❡r❛❝t✐♦♥ ❤❛s ❛♥ ✐♠♣❛❝t ♦♥ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❞✉r✐♥❣ ❳❈■✳ ✭❇✮ ❯♥❝♦✉♣•✐♥❣ ♦❢ t❤❡ ❙P❖❈✴|◆❆P■■ ❛①✐s ❢r♦♠ t❤❡ ◆❈♦|✴❙▼|❚✲❍❉❆❈✸ ❛①✐s ✐s ❛❝❤✐❡✈❡❞ ❜② ♣❡r❢♦r♠✐♥❣ t❤❡ ♣r♦♣♦s❡❞ ❡①♣❡r✐♠❡♥t ✐♥ ❛ ❜❛❝❦❣r♦✉♥❞ ✇❤❡r❡ t❤❡ ▲❙❉❙ ♠♦t✐❢s ♦❢ ◆❈♦| ❛♥❞ ❙▼|❚ ❤❛✈❡ ❜❡❡♥ ❞❡•❡t❡❞ ❤♦♠♦③②❣♦✉s•②✱ t❤❡r❡❜② ❡♥s✉r✐♥❣ t❤❛t ◆❈♦|✴❙▼|❚ ❝❛♥♥♦t ❡♥❣❛❣❡ ✇✐t❤ ❙P❖❈ ❛♥②♠♦r❡✳ ✶✽✷ ❉✐s❝✉ss✐♦♥ ❙P❊◆ ♠❡❝❤❛♥✐s♠s ❆ss✉♠✐♥❣ t❤❛t ❙P❊◆✬s ✐♥t❡r❛❝t✐♦♥ ✇✐t❤ |◆❆P■■ ✐s ✐♠♣♦rt❛♥t ❢♦r ❣❡♥❡ s✐•❡♥❝✐♥❣ ❞✉r✐♥❣ ❳❈■✱ ♦♥❡ ❝♦✉•❞ t❤❡♥ ♣r♦♣♦s❡ t❤❛t ❳✐st✲♠❡❞✐❛t❡❞ tr❛♥s❝r✐♣t✐♦♥❛• s✐•❡♥❝✐♥❣ t❛❦❡s ♣•❛❝❡ t❤r♦✉❣❤ t❤❡ ❢♦••♦✇✐♥❣ st❡♣s✿ ✭✐✮ ❳✐st ❡①t❡♥s✐✈❡•② r❡❝r✉✐ts ❙P❊◆ ✭t❤r♦✉❣❤ t❤❡ ❆✲r❡♣❡❛t✮ t♦ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡✱ •❡❛❞✲ ✐♥❣ t♦ ❛♥ ✐♥❝r❡❛s❡ ✐♥ ❙P❊◆ ❝♦♥❝❡♥tr❛t✐♦♥ •♦❝❛••② ❛r♦✉♥❞ ❳✲•✐♥❦❡❞ ❝❤r♦♠❛t✐♥✳ ✭✐✐✮ ❙P❊◆✬s ✐♥t❡r❛❝t✐♦♥ ✇✐t❤ |◆❆P■■ ✐♥❤✐❜✐ts t❤❡ tr❛♥s❝r✐♣t✐♦♥ ♣r♦❝❡ss ❛♥❞ tr✐❣❣❡rs ❣❡♥❡ s✐•❡♥❝✐♥❣✳ ✭✐✐✐✮ ❙P❊◆✲♠❡❞✐❛t❡❞ ❛❝t✐✈❛t✐♦♥ ♦❢ ❍❉❆❈✸ ❛t ❡♥❤❛♥❝❡rs ❛❝❝❡♥t✉❛t❡s✴❢❛❝✐•✐t❛t❡s ❣❡♥❡ s✐✲ •❡♥❝✐♥❣✳ ✭✐✈✮ ❳✐st✲❞❡♣❡♥❞❡♥t r❡❝r✉✐t♠❡♥t ♦❢ P♦•②❝♦♠❜ ✭t❤r♦✉❣❤ t❤❡ ❇✲r❡♣❡❛t✮ ❢✉rt❤❡r •♦❝❦s ❣❡♥❡ s✐•❡♥❝✐♥❣✱ ✐♥ s②♥❡r❣② ✇✐t❤ ❉◆❆ ♠❡t❤②•❛t✐♦♥✳ ✾✳✷✳✷✳✹ ❙P❖❈ ✐s ❛ ♥♦✈❡• ✐♥t❡r❛❝t✐♦♥ ❞♦♠❛✐♥ ♦❢ t❤❡ ♠ ✻ ❆ ✇r✐t✐♥❣ ♠❛❝❤✐♥❡r② ❳✐st ♠ ✻ ❆ |◆❆ ♠❡t❤②•❛t✐♦♥ ❤❛s ♣r❡✈✐♦✉s•② ❜❡❡♥ s❤♦✇♥ t♦ ❜❡ ✐♠♣♦rt❛♥t ❢♦r ❳❈■✱ ❛•t❤♦✉❣❤ t❤❡ ❡①t❡♥t t♦ ✇❤✐❝❤ t❤✐s ♠❛r❦ ♣•❛②s ❛ r♦•❡ ✐♥ ❳✐st✲♠❡❞✐❛t❡❞ ❣❡♥❡ s✐•❡♥❝✐♥❣ ✐s ❝♦♥tr♦✈❡rs✐❛• ✭s❡❡ s✉❜s❡❝t✐♦♥ ✶✳✶✸✳✸✮✳ ❙♦ ❢❛r✱ P❛t✐• ❡t ❛•✳ ♣r♦♣♦s❡❞ t❤❛t t❤❡ ♠ ✻ ❆ ♠❛✲ ❝❤✐♥❡r② ✐s r❡❝r✉✐t❡❞ t♦ ❳✐st ✈✐❛ t❤❡ |❇▼✶✺ ❝♦❢❛❝t♦r ✕ ✇❤✐❝❤ ❞✐r❡❝t•② ❜✐♥❞s ❳✐st ❆✲r❡♣❡❛t ✕ ✇✐t❤ s✉❜s❡q✉❡♥t (❚❍❉❈✶ ❜✐♥❞✐♥❣ t♦ ♠❡t❤②•❛t❡❞ ❳✐st ❜❡✐♥❣ ♦❜s❝✉r❡•② ✐♠♣•✐❝❛t❡❞ ✐♥ ❳✲•✐♥❦❡❞ ❣❡♥❡ s✐•❡♥❝✐♥❣ ✭s❡❡ s✉❜s❡❝t✐♦♥ ✶✳✶✸✳✸✮✳ ❙✉r♣r✐s✐♥❣•② ❤♦✇❡✈❡r✱ ❛ r❡❝❡♥t st✉❞② r❡♣♦rts t❤❛t |❇▼✶✺ ✐s ❞✐s♣❡♥s❛❜•❡ ❢♦r ❣❡♥❡ s✐•❡♥❝✐♥❣ ❞✉r✐♥❣ ❳❈■ ❬◆❡st❡r♦✈❛ ❡t ❛•✳✱ ✷✵✶✾(✳ Pr❡✈✐♦✉s ✇♦r❦ ❝♦♥❞✉❝t❡❞ ✐♥ ❉r♦s♦♣❤✐•❛ ❛♥❞ ♠♦✉s❡ s❤♦✇❡❞ t❤❛t |❇▼✶✺ ✐♥t❡r❛❝ts ❞✐✲ r❡❝t•② ✇✐t❤ )❈✸❍✶✸✱ ✇❤✐❝❤ ❜r✐❞❣❡s |❇▼✶✺ ✇✐t❤ t❤❡ r❡st ♦❢ t❤❡ ♠ ✻ ❆✲✇r✐t✐♥❣ ❝♦♠♣•❡①✱ ♥♦t❛❜•② ❲❚❆P ❛♥❞ t❤❡ ▼❊❚❚▲✸✴▼❊❚❚▲✶✹ ♠❡t❤②•tr❛♥s❢❡r❛s❡s ❬❑♥✉❝❦•❡s ❡t ❛•✳✱ ✷✵✶✽(✳ ❚❤❡ r❡❣✐♦♥s ♦❢ |❇▼✶✺ ✐♠♣•✐❝❛t❡❞ ✐♥ t❤✐s ✐♥t❡r❛❝t✐♦♥ ✇❡r❡ ❤♦✇❡✈❡r ♥♦t ❡①♣•♦r❡❞✳ |❡♠❛r❦✲ ❛❜•②✱ ❙P❊◆ ❛♥❞ |❇▼✶✺ s❤❛r❡ t✇♦ str✐❦✐♥❣ ❢❡❛t✉r❡s✳ ❋✐rst✱ t❤❡② ❜♦t❤ ✐♥t❡r❛❝t ❞✐r❡❝t•② ✇✐t❤ t❤❡ ❆✲r❡♣❡❛t ♦❢ ❳✐st t❤r♦✉❣❤ t❤❡✐r |◆❆ r❡❝♦❣♥✐t✐♦♥ ♠♦t✐❢s✳ ❙❡❝♦♥❞✱ t❤❡② ❜♦t❤ ❝❛rr② ❛ ❙P❖❈ ❞♦♠❛✐♥ ❛t t❤❡✐r ✈❡r② ❈✲t❡r♠✐♥❛• ❡♥❞✱ ❛•t❤♦✉❣❤ t❤❡s❡ t✇♦ ❙P❖❈s ♦♥•② s❤❛r❡ ✸✺✪ ❤♦♠♦•♦❣②✳ ■♥ t❤✐s ✇♦r❦✱ ■ s❤♦✇ t❤❛t ❙P❊◆✬s ❙P❖❈ ❞♦♠❛✐♥ ❝♦✲♣✉r✐✜❡s ✇✐t❤ ❛•• s✉❜✉♥✐ts ♦❢ t❤❡ ♠ ✻ ❆✲✇r✐t✐♥❣ ♠❛❝❤✐♥❡r②✱ ❛s ✇❡•• ❛s t❤❡ ♥✉❝•❡❛r ♠ ✻ ❆ r❡❛❞❡r (❚❍❉❈✶✳ ❍❡♥❝❡✱ ■ ♣r♦♣♦s❡ t❤❛t ✐♥t❡r❛❝t✐♦♥ ✇✐t❤ t❤❡ ♠ ✻ ❆ |◆❆ ♠❡t❤②•tr❛♥s❢❡r❛s❡ ❝♦♠♣•❡① ✐s ❛ ❣❡♥❡r❛• ❢❡❛t✉r❡ ♦❢ ❙P❖❈ ❞♦♠❛✐♥s✱ s❤❛r❡❞ ❛❝r♦ss ♦t❤❡r ❙P❖❈✲❝♦♥t❛✐♥✐♥❣ ♣r♦t❡✐♥s✳ •✐✈❡♥ t❤❡ r❡s✉•ts ❢r♦♠ ❑♥✉❝❦•❡s ❡t ❛•✳✱ t❤✐s ✐♥t❡r❛❝t✐♦♥ ✐s ♠♦st •✐❦❡•② ♠❡❞✐❛t❡❞ t❤r♦✉❣❤ )❈✸❍✶✸✳ ❈♦♥s✐st❡♥t•②✱ )❈✸❍✶✸ ✐s t❤❡ ♠♦st ❤✐❣❤•② ❡♥r✐❝❤❡❞ ♠ ✻ ❆✲❛ss♦❝✐❛t❡❞ ❢❛❝t♦r ✐♥ ♠② ❙P❖❈ ■P✲♠❛ss s♣❡❝✲ tr♦♠❡tr② ❛♥❛•②s✐s✳ ✶✽✸ ❉✐s❝✉ss✐♦♥ ❙P❊◆ ♠❡❝❤❛♥✐s♠s ❚❤❡ ❞✐s❝♦✈❡r② t❤❛t ❙P❖❈ ✐♥t❡r❛❝ts ✇✐t❤ t❤❡ ♠ ✻ ❆ ♠❛❝❤✐♥❡r② s✉❣❣❡sts t❤❛t ❙P❊◆ ❝❛♥ ❢✉♥❝t✐♦♥❛••② r❡♣•❛❝❡ |❇▼✶✺✱ t❤❡r❡❜② ❡①♣•❛✐♥✐♥❣ ✇❤② ❞❡❢❡❝ts ✐♥ ❳❈■ ❛r❡ ✐♥❝♦♥s✐st❡♥t•② ♦❜s❡r✈❡❞ ✉♣♦♥ |❜♠✶✺ ❦♥♦❝❦✲♦✉t ♦r ❦♥♦❝❦❞♦✇♥ ❬◆❡st❡r♦✈❛ ❡t ❛•✳✱ ✷✵✶✾❀ P❛t✐• ❡t ❛•✳✱ ✷✵✶✻(✳ ❚❛❦❡♥ t♦❣❡t❤❡r✱ ♠② ✇♦r❦ ❤✐❣❤•✐❣❤ts ❙P❊◆ ❛s ❛ ✈❡r② ♣r♦❜❛❜•❡ ❝❛♥❞✐❞❛t❡ ❢♦r •✐♥❦✐♥❣ ❳✐st ✇✐t❤ ♠ ✻ ❆ |◆❆ ♠❡t❤②•❛t✐♦♥✳ ■t ✐s ♥♦✇ ✐♠♣♦rt❛♥t t♦ ♣r♦✜•❡ ❳✐st ♠ ✻ ❆ ♠❡t❤②•❛t✐♦♥ ✉♣♦♥ ❙P❊◆ •♦ss✱ s♦ ❛s t♦ ❝♦♥✜r♠ t❤❛t ❙P❊◆ ♣•❛②s ❛ r♦•❡ ✐♥ ❳✐st ♠❡t❤②•❛t✐♦♥✳ ◆❡✈❡rt❤❡•❡ss✱ ✐t s❡❡♠s ✉♥•✐❦❡•② t❤❛t s✉❝❤ ❙P❊◆✲❞❡♣❡♥❞❡♥t |◆❆ ♠❡t❤②•❛t✐♦♥ ✐s ✐♠✲ ♣♦rt❛♥t ❢♦r ❳✐st✲♠❡❞✐❛t❡❞ ❣❡♥❡ s✐•❡♥❝✐♥❣✱ ❣✐✈❡♥ t❤❛t ♠❊❙❈s s❤♦✇ ✉♥♣❡rt✉r❜❡❞ ❳❈■ ✉♣♦♥ ❛✉①✐♥✲♠❡❞✐❛t❡❞ ❞❡♣•❡t✐♦♥ ♦❢ )❈✸❍✶✸ ❬❉♦ss✐♥✱ ✉♥♣✉❜•✐s❤❡❞(✳ ❍♦✇❡✈❡r✱ ❙P❊◆✲❞❡♣❡♥❞❡♥t |◆❆ ♠❡t❤②•❛t✐♦♥ ❝♦✉•❞ r❡❣✉•❛t❡ ❳✐st ♣♦st✲tr❛♥s❝r✐♣t✐♦♥❛••② ✭❡✳❣✳ ♣r♦♠♦t✐♥❣ ✐ts st❛❜✐•✐t②✮✱ ❛♥❞ ❝♦✉•❞ t❤✉s ❡①♣•❛✐♥ t❤❡ r❡❞✉❝❡❞ ❳✐st •❡✈❡•s ♦❜s❡r✈❡❞ ✉♥❞❡r ❙P❊◆✲❞❡♣•❡t❡❞ ❝♦♥❞✐t✐♦♥s ✭s❡❡ s✉❜s❡❝t✐♦♥ ✽✳✻✳✷✮✳ ■♥ ♦♥❡ ♦❢ t❤❡ ❳✐st ♣r♦t❡♦♠✐❝ st✉❞✐❡s✱ ❍♦✇❛r❞ ❈❤❛♥❣✬s •❛❜ r❡♣♦rt❡❞ t❤❛t t❤❡ ❲❚❆P ♠ ✻ ❆ ❝♦❢❛❝t♦r ❝♦✲♣✉r✐✜❡s ✇✐t❤ ❢✉••✲•❡♥❣t❤ ❳✐st |◆❆ ✕ ❜✉t ♥♦t ❳✐st ∆❆✲r❡♣❡❛t ✕ ❛♥❞ ❤❡♥❝❡ ❝♦♥❝•✉❞❡❞ t❤❛t ❲❚❆P ✐♥t❡r❛❝ts ✇✐t❤ t❤❡ ❆✲r❡♣❡❛t ♦❢ ❳✐st ❬❈❤✉ ❡t ❛•✳✱ ✷✵✶✺(✳ ❍♦✇❡✈❡r✱ ❛•t❤♦✉❣❤ s❡✈❡r❛• st✉❞✐❡s ❤❛✈❡ ♥♦✇ s❤♦✇♥ t❤❛t ❙P❊◆ ❞✐r❡❝t•② ❜✐♥❞s ❳✐st ❆✲r❡♣❡❛t ❬▼♦♥✲ ❢♦rt ❡t ❛•✳✱ ✷✵✶✺❀ ▲✉ ❡t ❛•✳✱ ✷✵✶✻❀ ❈❛rt❡r ❡t ❛•✳✱ ✷✵✷✵(✱ ♥♦ s✉❝❤ ❞✐r❡❝t ✐♥t❡r❛❝t✐♦♥ ❤❛s ❜❡❡♥ r❡♣♦rt❡❞ ❢♦r ❲❚❆P✳ ■♥ •✐❣❤t ♦❢ ♠② ✇♦r❦ r❡✈❡❛•✐♥❣ t❤❛t ❙P❊◆ ✐s ❛ ❜♦♥❛ ✜❞❡ ✐♥t❡r❛❝t♦r ♦❢ t❤❡ ♠ ✻ ❆ |◆❆ ♠❡t❤②•❛t✐♦♥ ♠❛❝❤✐♥❡r② ✭✇✐t❤ ❲❚❆P s❝♦r✐♥❣ ❛s ❛ ❤✐❣❤•② ❡♥r✐❝❤❡❞ ❝❛♥❞✐✲ ❞❛t❡ ✐♥ t❤❡ ❙P❖❈ ♣r♦t❡✐♥ ✐♥t❡r❛❝t♦♠❡✮✱ ■ ♣r♦♣♦s❡ t❤❛t ❲❚❆P ✐s ✐♥ ❢❛❝t ❜r✐❞❣❡❞ t♦ ❳✐st ✐♥❞✐r❡❝t•② ✈✐❛ ❙P❊◆✱ ❤❡♥❝❡ ❝♦✲♣✉r✐❢②✐♥❣ ✇✐t❤ ❳✐st ✐♥ ❛♥ ❆✲r❡♣❡❛t ❞❡♣❡♥❞❡♥t ♠❛♥♥❡r✳ ✾✳✸ ❙P❊◆✲❢❛♠✐•② ♣r♦t❡✐♥s ❛s |◆❆✲❣✉✐❞❡❞ tr❛♥s❝r✐♣✲ t✐♦♥❛• r❡♣r❡ss♦rs ❙P❊◆ ✐s ❝♦♥s❡r✈❡❞ ❛❝r♦ss t❤❡ ❛♥✐♠❛• ❦✐♥❣❞♦♠ ✭s❡❡ s❡❝t✐♦♥ ✷✳✷✮✱ ❛♥❞ ✐ts ||▼✷✲✹ ❛♥❞ ❙P❖❈ ❞♦♠❛✐♥s ❛r❡ r❡♠❛r❦❛❜•② ✇❡•• ❝♦♥s❡r✈❡❞ ✐♥ ✈❡rt❡❜r❛t❡s✳ ❋✉rt❤❡r♠♦r❡✱ s❡✈❡r❛• ♣r♦t❡✐♥s ❝♦♥t❛✐♥✐♥❣ ❜♦t❤ ||▼ ❛♥❞ ❙P❖❈ ❞♦♠❛✐♥s ❛r❡ ❢♦✉♥❞ ❛❝r♦ss ❢✉♥❣✐✱ ♣•❛♥t ❛♥❞ ❛♥✐♠❛• ❦✐♥❣❞♦♠s✳ ❚❤❡r❡❢♦r❡✱ ✐t ✐s t❡♠♣t✐♥❣ t♦ s♣❡❝✉•❛t❡ t❤❛t t❤❡ ♣r♦t❡✐♥ ✐♥t❡r❛❝t♦♠❡ ♦❢ ❙P❊◆✬s ❙P❖❈ ❞♦♠❛✐♥ ✐s ✈❡r② s✐♠✐•❛r ❛❝r♦ss ✈❡rt❡❜r❛t❡s✱ ❛♥❞ t❤❛t s♦♠❡ ♦❢ t❤❡ ♠❡❝❤❛♥✐s♠s ✐❞❡♥t✐✜❡❞ ✐♥ t❤✐s ✇♦r❦ ♠❛② ❛•s♦ ❜❡ ❜r✐❞❣❡❞ ❜② ❙P❖❈ ✐♥ ♦t❤❡r ❙P❖❈✲❝♦♥t❛✐♥✐♥❣ ♣r♦t❡✐♥s✳ ■❢ t❤❛t ✇❡r❡ t❤❡ ❝❛s❡✱ ❙P❊◆ ❛♥❞ ♦t❤❡r ❙P❊◆✲r❡•❛t❡❞ ♣r♦t❡✐♥s ✇♦✉•❞ ❡♠❡r❣❡ ❛s |◆❆✲ ❣✉✐❞❡❞ tr❛♥s❝r✐♣t✐♦♥❛• r❡♣r❡ss♦rs✱ ❜r✐❞❣✐♥❣ s❡✈❡r❛• •❛②❡rs ♦❢ ❝❤r♦♠❛t✐♥ ❛♥❞ tr❛♥s❝r✐♣t✐♦♥❛• ❝♦♥tr♦• t♦ ❡♥s✉r❡ ♣♦t❡♥t ❣❡♥❡ r❡♣r❡ss✐♦♥✳ ■t ✐s ♥♦✇ ❝r✉❝✐❛• t♦ ❝❤❛r❛❝t❡r✐③❡ t❤❡ r❡♣❡rt♦✐r❡ ♦❢ |◆❆s t❤❛t ✐♥t❡r❛❝t s♣❡❝✐✜❝❛••② ✇✐t❤ ❙P❊◆✱ ❛❝r♦ss t✐ss✉❡s ❛♥❞ ❛♥✐♠❛•s✳ ❆ r❡❝❡♥t st✉❞② ✶✽✹ ❉✐s❝✉ss✐♦♥ ❙P❊◆ ♠❡❝❤❛♥✐s♠s ❢♦✉♥❞ t❤❛t ✐♥ ❛❞❞✐t✐♦♥ t♦ ❳✐st✱ ❙P❊◆✬s ||▼✷✲✹ ❞♦♠❛✐♥s ❜✐♥❞ t♦ ❡♥❞♦❣❡♥♦✉s r❡tr♦✈✐r✉s ❑ ❡•❡♠❡♥ts ✭❊|❱❑s✮✱ ❛ ❢❛♠✐•② ♦❢ ✐♥t❡❣r❛t❡❞ r❡tr♦✈✐r✉s❡s s❡q✉❡♥❝❡s ❬❈❛rt❡r ❡t ❛•✳✱ ✷✵✷✵(✳ ❚❤r♦✉❣❤ ❜✐♥❞✐♥❣ ❛❝t✐✈❡•② tr❛♥s❝r✐❜❡❞ ❊|❱❑s✱ ❙P❊◆ r❡♣r❡ss❡s ❊|❱❑ tr❛♥s❝r✐♣t✐♦♥ ❛♥❞ ❡♥s✉r❡s ❣❡♥♦♠✐❝ s✉r✈❡✐•❛♥❝❡ ❬❈❛rt❡r ❡t ❛•✳✱ ✷✵✷✵(✳ ❚❤❡ s✐•❡♥❝✐♥❣ ♠❡❝❤❛♥✐s♠s ✐♥✈♦•✈❡❞ ✇❡r❡ ♥♦t ❛❞❞r❡ss❡❞✱ ❜✉t t❤❡s❡ ♠❡❝❤❛♥✐s♠s ❛r❡ •✐❦❡•② t♦ ❜❡ s✐♠✐•❛r t♦ t❤♦s❡ ✐♥✈♦•✈❡❞ ✐♥ ❳❈■✳ ❍✐❣❤•✐❣❤t✐♥❣ t❤❛t ❙P❖❈ ❞♦♠❛✐♥s ❣❡♥❡r❛••② ♠❛② ❜❡ ✐♥✈♦•✈❡❞ ✐♥ s✐♠✐•❛r ♠❡❝❤❛♥✐s♠s✱ ❛ r❡❝❡♥t st✉❞② ❝♦♥❞✉❝t❡❞ ✐♥ ♣•❛♥ts ✭❆r❛❜✐❞♦♣s✐s✮ ❢♦✉♥❞ t❤❛t ❇❖|❉❊| ♣r♦t❡✐♥s ✕ ✇❤✐❝❤ ❝❛rr② ❛ ❙P❖❈ ❞♦♠❛✐♥ ✕ ❛❝t ❛s tr❛♥s❝r✐♣t✐♦♥❛• ✐♥s✉•❛t♦rs ❜② ♣r♦♠♦t✐♥❣ ✸✬ |◆❆P■■✲♣❛✉s✐♥❣ ❬(✉ ❡t ❛•✳✱ ✷✵✶✾(✳ ❚❤❡ ♣r❡❝✐s❡ ♠❡❝❤❛♥✐s♠ ❜② ✇❤✐❝❤ t❤✐s ❤❛♣♣❡♥s ✇❛s ♥♦t ❛❞❞r❡ss❡❞✱ ❜✉t ✐t ✐s t❡♠♣t✐♥❣ t♦ ❤②♣♦t❤❡s✐③❡ t❤❛t t❤❡ ❙P❖❈ ❞♦♠❛✐♥ ♦❢ ❇❖|❉❊| ♣r♦t❡✐♥s ♠❛② ❜❡ ✐♥t❡r❛❝t✐♥❣ ✇✐t❤ t❤❡ ❈❚❉ ❞♦♠❛✐♥ ♦❢ ❆t|◆❆P■■ ❛❝❝♦r❞✐♥❣ t♦ ❈❚❉ ♣❤♦s♣❤♦r②•❛t✐♦♥ st❛t✉s✱ ✇✐t❤ ❛ s♣❡❝✐✜❝✐t② t♦✇❛r❞s ♣❤♦s♣❤♦r②•❛t✐♦♥ ❛t ❙❡r✐♥❡ ✷✱ ✇❤✐❝❤ ✐s ♠♦st ❡♥r✐❝❤❡❞ ❛t t❤❡ ✸✬ ❡♥❞ ♦❢ ❣❡♥❡s ✭s❡❡ s✉❜s❡❝t✐♦♥ ✼✳✶✳✶✮✳ ❋✉rt❤❡r♠♦r❡✱ t❤❡ ♣•❛♥t ♣r♦t❡✐♥ ❋P❆ ✕ ✇❤✐❝❤ r❡❣✉•❛t❡s ✢♦✇❡r✐♥❣ t✐♠❡ ❛♥❞ ❝❛rr✐❡s ||▼ ❛♥❞ ❙P❖❈ ❞♦♠❛✐♥s ✕ ❤❛s ❜❡❡♥ s❤♦✇♥ t♦ s✐•❡♥❝❡ ❝❤r♦♠❛t✐♥ ✐♥ ❛♥ |◆❆✲❞❡♣❡♥❞❡♥t ♠❛♥♥❡r ❬❇ä✉r•❡ ❡t ❛•✳✱ ✷✵✵✼( ❛♥❞ r❡❣✉•❛t❡ ✸✬✲❡♥❞ ♣r♦❝❡ss✐♥❣ ❬❍♦r♥②✐❦ ❡t ❛•✳✱ ✷✵✶✵❀ ❙♦♥♠❡③ ❡t ❛•✳✱ ✷✵✶✶(✳ ■♠♣♦rt❛♥t•②✱ ❋P❆✬s ❙P❖❈ ❞♦♠❛✐♥ s❤❛r❡s str✉❝t✉r❛• s✐♠✐•❛r✐t② ✇✐t❤ ❙P❊◆✬s ❙P❖❈ ❞♦♠❛✐♥✱ ♥♦t❛❜•② ❛t t❤❡ •❡✈❡• ♦❢ t❤❡ ❜❛s✐❝ ♣❛t❝❤ ❬)❤❛♥❣ ❡t ❛•✳✱ ✷✵✶✻(✳ Iws1/
C R R M s S P O C PAF1 complex Ser5 P SPOC ❋✐❣✉r❡ ✾✳✷ ✕ ❲♦r❦✐♥❣ ♠♦❞❡• ❢♦r ❙P❊◆ ❢✉♥❝t✐♦♥ ❞✉r✐♥❣ ❳❈■✳ ✭❆✮ ❆t t❤❡ ✈❡r② ❜❡❣✐♥♥✐♥❣ ♦❢ ❳❈■✱ ❙P❊◆ ✐s r❡❝r✉✐t❡❞ ❜② ❳✐st |◆❆ ✇❤✐•❡ ✐t ✐s ❜❡✐♥❣ tr❛♥s❝r✐❜❡❞✳ ❚❤❡ ✐♥t❡r❛❝t✐♦♥ ❜❡t✇❡❡♥ t❤❡ ❙P❖❈ ❞♦♠❛✐♥ ❛♥❞ t❤❡ ♠ ✻ ❆ ✇r✐t✐♥❣ ♠❛❝❤✐♥❡r② ♠❛② ❜❡ ✐♥✈♦•✈❡❞ ✐♥ ❳✐st |◆❆ ♠❡t❤②•❛t✐♦♥✳ ✭❇✮ ❳✐st t❤❡♥ ❣✉✐❞❡s ❙P❊◆ t♦ ❳✲•✐♥❦❡❞ ❡♥❤❛♥❝❡rs ❛♥❞ ♣r♦♠♦t❡rs✱ ✇❤❡r❡ t❤❡ ❙P❖❈ ❞♦♠❛✐♥ tr✐❣❣❡rs ❣❡♥❡ s✐•❡♥❝✐♥❣ ❜② ✐♥t❡r❛❝t✐♥❣ ✇✐t❤ t❤❡ ◆❈♦|✴❙▼|❚ ❝♦♠♣•❡①✱ t❤❡ ◆✉|❉ ❝♦♠♣•❡① ❛s ✇❡•• ❛s t❤❡ tr❛♥s❝r✐♣t✐♦♥ ♠❛❝❤✐♥❡r②✳ ✭❈✮ ❋♦••♦✇✐♥❣ ❣❡♥❡ s✐•❡♥❝✐♥❣✱ t❤❡ ✧❛❝t✐✈❡ ❝❤r♦♠❛t✐♥✧ ❝♦♥t❡①t ❢❛✈♦r❛❜•❡ ❢♦r ❙P❊◆ ❜✐♥❞✐♥❣ ✐s •♦st✱ ❛♥❞ ❙P❊◆ ❤❡♥❝❡ ❞✐s❡♥❣❛❣❡s ❢r♦♠ ❳✲•✐♥❦❡❞ ❝❤r♦♠❛t✐♥✳ ❉✐r❡❝t ❙P❖❈✲✐♥t❡r❛❝t♦rs ❛r❡ •❛❜❡•❡❞ ✐♥ ❜♦•❞✳ ❚❛❦❡♥ t♦❣❡t❤❡r✱ ♠② ✇♦r❦ ❛♥❞ t❤❡ ❡①✐st✐♥❣ •✐t❡r❛t✉r❡ s✉❣❣❡st t❤❛t |◆❆✲♠❡❞✐❛t❡❞ r❡✲ ❝r✉✐t♠❡♥t ♦❢ ❙P❊◆✲❢❛♠✐•② ♣r♦t❡✐♥s ✐s ❛ ✇✐❞❡s♣r❡❛❞ ♠❡❛♥s t♦ r❡♣r❡ss tr❛♥s❝r✐♣t✐♦♥ ❛❝✉t❡•② ❛t t❛r❣❡t •♦❝✐✱ t❤r♦✉❣❤ s✐♠✉•t❛♥❡♦✉s•② ❡♥❣❛❣✐♥❣ ✇✐t❤ ♠✉•t✐♣•❡ •❛②❡rs ♦❢ ❡♣✐❣❡♥❡t✐❝ ❛♥❞ tr❛♥s❝r✐♣t✐♦♥❛• r❡❣✉•❛t✐♦♥✳ ■♥ t❤❡ ❝♦♥t❡①t ♦❢ ❳❈■✱ ■ ♣r♦♣♦s❡ t❤❛t ❙P❊◆ ✐s ❛ ♠♦•❡❝✉•❛r ✐♥t❡❣r❛t♦r ✇❤✐❝❤ •✐♥❦s ❳✐st |◆❆ ✇✐t❤ t❤❡ tr❛♥s❝r✐♣t✐♦♥ ♠❛❝❤✐♥❡r②✱ ❤✐st♦♥❡ ❞❡❛❝❡t②•❛s❡s✱ ✶✽✺ ❉✐s❝✉ss✐♦♥ ❙P❊◆ ♠❡❝❤❛♥✐s♠s ♥✉❝•❡♦s♦♠❡ r❡♠♦❞❡••❡rs ❛♥❞ |◆❆ ♠ ✻ ❆✲tr❛♥s❢❡r❛s❡s t♦ ♣r♦♠♦t❡ ❡✣❝✐❡♥t ❛♥❞ r♦❜✉st ❣❡♥❡ s✐•❡♥❝✐♥❣ ✭❋✐❣✉r❡ ✾✳✷✮✳ ❆s ❛ ✇❤♦•❡✱ t❤✐s ✇♦r❦ ❞❡♠♦♥str❛t❡s t❤❛t t❤❡ st✉❞② ♦❢ ❳ ❝❤r♦♠♦✲ s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥ ♣r♦✈✐❞❡s ❛ ♣♦✇❡r❢✉• ♠♦❞❡• t♦ ❞✐s❝♦✈❡r ❢✉♥❞❛♠❡♥t❛• ❛s♣❡❝ts ♦❢ ♣r♦t❡✐♥ ❢✉♥❝t✐♦♥ ❛♥❞ ❣❡♥❡ r❡❣✉•❛t✐♦♥✳ ✶✽✻ P❛rt ❱ |❡❢❡r❡♥❝❡s |❡❢❡r❡♥❝❡s |❊❋❊|❊◆❈❊❙ |❡❢❡r❡♥❝❡s ❆❞❛♠s✱ P✳ ❉✳✱ ❆❢♦♥✐♥❡✱ P✳ ❱✳✱ ❇✉♥❦ó❝③✐✱ •✳✱ ❈❤❡♥✱ ❱✳ ❇✳✱ ❉❛✈✐s✱ ■✳ ❲✳✱ ❊❝❤♦•s✱ ◆✳✱ ❍❡❛❞❞✱ ❏✳ ❏✳✱ ❍✉♥❣✱ ▲✳ ❲✳✱ ❑❛♣r❛•✱ •✳ ❏✳✱ •r♦ss❡✲❑✉♥st•❡✈❡✱ |✳ ❲✳✱ ▼❝❈♦②✱ ❆✳ ❏✳✱ ▼♦r✐❛rt②✱ ◆✳ ❲✳✱ ❖❡✛♥❡r✱ |✳✱ |❡❛❞✱ |✳ ❏✳✱ |✐❝❤❛r❞s♦♥✱ ❉✳ ❈✳✱ |✐❝❤❛r❞s♦♥✱ ❏✳ ❙✳✱ ❚❡r✇✐••✐❣❡r✱ ❚✳ ❈✳✱ ❛♥❞ )✇❛rt✱ P✳ ❍✳ ✭✷✵✶✵✮✳ P❍❊◆■❳✿ ❆ ❝♦♠♣r❡❤❡♥s✐✈❡ P②t❤♦♥✲❜❛s❡❞ s②st❡♠ ❢♦r ♠❛❝r♦♠♦•❡❝✉•❛r str✉❝t✉r❡ s♦•✉t✐♦♥✳ ❆❝t❛ ❈r②st❛••♦❣r❛♣❤✐❝❛ ❙❡❝t✐♦♥ ❉✿ ❇✐♦•♦❣✐❝❛• ❈r②st❛••♦❣r❛♣❤②✱ ✻✻✭✷✮✿✷✶✸✕✷✷✶✳ ❆•♠❡✐❞❛✱ ▼✳✱ P✐♥t❛❝✉❞❛✱ •✳✱ ▼❛s✉✐✱ ❖✳✱ ❑♦s❡❦✐✱ (✳✱ •❞✉•❛✱ ▼✳✱ ❈❡r❛s❡✱ ❆✳✱ ❇r♦✇♥✱ ❉✳✱ ▼♦✉•❞✱ ❆✳✱ ■♥♥♦❝❡♥t✱ ❈✳✱ ◆❛❦❛②❛♠❛✱ ▼✳✱ ❙❝❤❡r♠❡••❡❤✱ ▲✳✱ ◆❡st❡r♦✈❛✱ ❚✳ ❇✳✱ ❑♦s❡❦✐✱ ❍✳✱ ❛♥❞ ❇r♦❝❦❞♦r✛✱ ◆✳ ✭✷✵✶✼✮✳ P❈•❋✸✴✺✲P|❈✶ ✐♥✐t✐❛t❡s P♦•②❝♦♠❜ r❡❝r✉✐t♠❡♥t ✐♥ ❳ ❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥✳ ❙❝✐❡♥❝❡✱ ✸✺✻✭✻✸✹✷✮✿✶✵✽✶✕✶✵✽✹✳ ❆♥❞r✐❛ts✐•❛✈♦✱ ▼✳✱ ❙t❡❢❛♥✉tt✐✱ ▼✳✱ ❙✐✉❞❡•❛✱ ❑✳✱ P❡r❞✐❣♦t♦✱ ❈✳ ◆✳✱ ❇♦✉♠❛r❞✱ ❇✳✱ •❡r✈❛✐s✱ ▲✳✱ •✐••❡t✲▼❛r❦♦✇s❦❛✱ ❆✳✱ ❆• )♦✉❛❜✐✱ ▲✳✱ ❙❝❤✇❡✐s❣✉t❤✱ ❋✳✱ ❛♥❞ ❇❛r❞✐♥✱ ❆✳ ❏✳ ✭✷✵✶✽✮✳ ❙♣❡♥ •✐♠✐ts ✐♥t❡st✐♥❛• st❡♠ ❝❡•• s❡•❢✲r❡♥❡✇❛•✳ P▲♦❙ •❡♥❡t✐❝s✱ ✶✹✭✶✶✮✿❡✶✵✵✼✼✼✸✳ ❆r✐❡t✐✱ ❋✳✱ •❛❜✉s✱ ❈✳✱ ❚❛♠❜❛•♦✱ ▼✳✱ ❍✉❡t✱ ❚✳✱ |♦✉♥❞✱ ❆✳✱ ❛♥❞ ❚❤♦r❡✱ ❙✳ ✭✷✵✶✹✮✳ ❚❤❡ ❝r②st❛• str✉❝t✉r❡ ♦❢ t❤❡ s♣•✐t ❡♥❞ ♣r♦t❡✐♥ ❙❍❆|P ❛❞❞s ❛ ♥❡✇ •❛②❡r ♦❢ ❝♦♠♣•❡①✐t② t♦ ♣r♦t❡✐♥s ❝♦♥t❛✐♥✐♥❣ |◆❆ r❡❝♦❣♥✐t✐♦♥ ♠♦t✐❢s✳ ◆✉❝•❡✐❝ ❆❝✐❞s |❡s❡❛r❝❤✱ ✹✷✭✶✵✮✿✻✼✹✷✕✻✼✺✷✳ ❆r✐②♦s❤✐✱ ▼✳ ❛♥❞ ❙❝❤✇❛❜❡✱ ❏✳ ❲✳ |✳ ✭✷✵✵✸✮✳ ❆ ❝♦♥s❡r✈❡❞ str✉❝t✉r❛• ♠♦t✐❢ r❡✈❡❛•s t❤❡ ❡ss❡♥t✐❛• tr❛♥s❝r✐♣t✐♦♥❛• r❡♣r❡ss✐♦♥ ❢✉♥❝t✐♦♥ ♦❢ ❙♣❡♥ ♣r♦t❡✐♥s ❛♥❞ t❤❡✐r r♦•❡ ✐♥ ❞❡✈❡•♦♣✲ ♠❡♥t❛• s✐❣♥❛•✐♥❣✳ •❡♥❡s ✫ ❞❡✈❡•♦♣♠❡♥t✱ ✶✼✭✶✺✮✿✶✾✵✾✕✷✵✳ ❆r♠❡♥✐❛✱ ❏✳✱ ❲❛♥❦♦✇✐❝③✱ ❙✳ ❆✳✱ ▲✐✉✱ ❉✳✱ •❛♦✱ ❏✳✱ ❑✉♥❞r❛✱ |✳✱ |❡③♥✐❦✱ ❊✳✱ ❈❤❛t✐•❛✱ ❲✳ ❑✳✱ ❈❤❛❦r❛✈❛rt②✱ ❉✳✱ ❍❛♥✱ •✳ ❈✳✱ ❈♦•❡♠❛♥✱ ■✳✱ ▼♦♥t❣♦♠❡r②✱ ❇✳✱ Pr✐t❝❤❛r❞✱ ❈✳✱ ▼♦rr✐ss❡②✱ ❈✳✱ ❇❛r❜✐❡r✐✱ ❈✳ ❊✳✱ ❇❡•tr❛♥✱ ❍✳✱ ❙❜♦♥❡r✱ ❆✳✱ )❛❢❡✐r✐♦✉✱ )✳✱ ▼✐r❛♥❞❛✱ ❙✳✱ ❇✐❡•s❦✐✱ ❈✳ ▼✳✱ P❡♥s♦♥✱ ❆✳ ❱✳✱ ❚♦•♦♥❡♥✱ ❈✳✱ ❍✉❛♥❣✱ ❋✳ ❲✳✱ |♦❜✐♥s♦♥✱ ❉✳✱ ❲✉✱ (✳ ▼✳✱ ▲♦♥✐❣r♦✱ |✳✱ •❛rr❛✇❛②✱ ▲✳ ❆✳✱ ❉❡♠✐❝❤❡•✐s✱ ❋✳✱ ❑❛♥t♦✛✱ P✳ ❲✳✱ ❚❛♣•✐♥✱ ▼✳ ❊✳✱ ❆❜✐❞❛✱ ❲✳✱ ❚❛②•♦r✱ ❇✳ ❙✳✱ ❙❝❤❡r✱ ❍✳ ■✳✱ ◆❡•s♦♥✱ P✳ ❙✳✱ ❉❡ ❇♦♥♦✱ ❏✳ ❙✳✱ |✉❜✐♥✱ ▼✳ ❆✳✱ ❙❛✇②❡rs✱ ❈✳ ▲✳✱ ❈❤✐♥♥❛✐②❛♥✱ ❆✳ ▼✳✱ ❙❝❤✉•t③✱ ◆✳✱ ❛♥❞ ❱❛♥ ❆••❡♥✱ ❊✳ ▼✳ ✭✷✵✶✽✮✳ ❚❤❡ •♦♥❣ t❛✐• ♦❢ ♦♥❝♦❣❡♥✐❝ ❞r✐✈❡rs ✐♥ ♣r♦st❛t❡ ❝❛♥❝❡r✳ ◆❛t✉r❡ •❡♥❡t✐❝s✱ ✺✵✭✺✮✿✻✹✺✕✻✺✶✳ ✶✽✽ |❡❢❡r❡♥❝❡s |❊❋❊|❊◆❈❊❙ ❆st❡r✱ ❏✳ ❈✳✱ P❡❛r✱ ❲✳ ❙✳✱ ❛♥❞ ❇•❛❝❦•♦✇✱ ❙✳ ❈✳ ✭✷✵✶✼✮✳ ❚❤❡ ❱❛r✐❡❞ |♦•❡s ♦❢ ◆♦t❝❤ ✐♥ ❈❛♥❝❡r✳ ❆♥♥✉❛• |❡✈✐❡✇ ♦❢ P❛t❤♦•♦❣②✿ ▼❡❝❤❛♥✐s♠s ♦❢ ❉✐s❡❛s❡✱ ✶✷✭✶✮✿✷✹✺✕✷✼✺✳ ❆✉❣✉✐✱ ❙✳✱ ◆♦r❛✱ ❊✳ P✳✱ ❛♥❞ ❍❡❛r❞✱ ❊✳ ✭✷✵✶✶✮✳ |❡❣✉•❛t✐♦♥ ♦❢ ❳✲❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥ ❜② t❤❡ ❳✲✐♥❛❝t✐✈❛t✐♦♥ ❝❡♥tr❡✳ ◆❛t✉r❡ |❡✈✐❡✇s •❡♥❡t✐❝s✱ ✶✷✭✻✮✿✹✷✾✕✹✹✷✳ ❇❛❝❤tr♦❣✱ ❉✳ ✭✷✵✶✸✮✳ (✲❝❤r♦♠♦s♦♠❡ ❡✈♦•✉t✐♦♥✿ ❡♠❡r❣✐♥❣ ✐♥s✐❣❤ts ✐♥t♦ ♣r♦❝❡ss❡s ♦❢ (✲ ❝❤r♦♠♦s♦♠❡ ❞❡❣❡♥❡r❛t✐♦♥✳ ◆❛t✉r❡ |❡✈✐❡✇s •❡♥❡t✐❝s✱ ✶✹✭✷✮✿✶✶✸✕✶✷✹✳ ❇❛♥❛♥✐✱ ❙✳ ❋✳✱ ▲❡❡✱ ❍✳ ❖✳✱ ❍②♠❛♥✱ ❆✳ ❆✳✱ ❛♥❞ |♦s❡♥✱ ▼✳ ❑✳ ✭✷✵✶✼✮✳ ❇✐♦♠♦•❡❝✉•❛r ❝♦♥❞❡♥s❛t❡s✿ ❖r❣❛♥✐③❡rs ♦❢ ❝❡••✉•❛r ❜✐♦❝❤❡♠✐str②✳ ❇❛r❛✉✱ ❏✳✱ ❚❡✐ss❛♥❞✐❡r✱ ❆✳✱ )❛♠✉❞✐♦✱ ◆✳✱ |♦②✱ ❙✳✱ ◆❛•❡ss♦✱ ❱✳✱ ❍ér❛✉•t✱ (✳✱ •✉✐••♦✉✱ ❋✳✱ ❛♥❞ ❇♦✉r❝✬❤✐s✱ ❉✳ ✭✷✵✶✻✮✳ ❚❤❡ ❉◆❆ ♠❡t❤②•tr❛♥s❢❡r❛s❡ ❉◆▼❚✸❈ ♣r♦t❡❝ts ♠❛•❡ ❣❡r♠ ❝❡••s ❢r♦♠ tr❛♥s♣♦s♦♥ ❛❝t✐✈✐t②✳ ❙❝✐❡♥❝❡✱ ✸✺✹✭✻✸✶✹✮✿✾✵✾✕✾✶✷✳ ❇❛rr✱ ▼✳ ▲✳ ❛♥❞ ❇❡rtr❛♠✱ ❊✳ •✳ ✭✶✾✹✾✮✳ ❆ ♠♦r♣❤♦•♦❣✐❝❛• ❞✐st✐♥❝t✐♦♥ ❜❡t✇❡❡♥ ♥❡✉r♦♥❡s ♦❢ t❤❡ ♠❛•❡ ❛♥❞ ❢❡♠❛•❡✱ ❛♥❞ t❤❡ ❜❡❤❛✈✐♦✉r ♦❢ t❤❡ ♥✉❝•❡♦•❛r s❛t❡••✐t❡ ❞✉r✐♥❣ ❛❝❝❡•❡r❛t❡❞ ♥✉❝•❡♦♣r♦t❡✐♥ s②♥t❤❡s✐s ❬✷(✳ ◆❛t✉r❡✱ ✶✻✸✭✹✶✹✽✮✿✻✼✻✕✻✼✼✳ ❇❛rt♦♥✱ ❉✳ ❊✳✱ ❉❛✈✐❞✱ ❋✳ ◆✳✱ ❛♥❞ ▼❡rr✐♥❣t♦♥✱ ▼✳ ✭✶✾✻✹✮✳ ❚❤❡ ♣♦s✐t✐♦♥s ♦❢ t❤❡ s❡① ❝❤r♦♠♦s♦♠❡s ✐♥ t❤❡ ❤✉♠❛♥ ❝❡•• ✐♥ ♠✐t♦s✐s✳ ❆♥♥❛•s ♦❢ ❍✉♠❛♥ •❡♥❡t✐❝s✱ ✷✽✭✶✲✸✮✿✶✷✸✕ ✶✷✽✳ ❇ä✉r•❡✱ ■✳✱ ❙♠✐t❤✱ ▲✳✱ ❇❛✉•❝♦♠❜❡✱ ❉✳ ❈✳✱ ❛♥❞ ❉❡❛♥✱ ❈✳ ✭✷✵✵✼✮✳ ❲✐❞❡s♣r❡❛❞ r♦•❡ ❢♦r t❤❡ ✢♦✇❡r✐♥❣✲t✐♠❡ r❡❣✉•❛t♦rs ❋❈❆ ❛♥❞ ❋P❆ ✐♥ |◆❆✲♠❡❞✐❛t❡❞ ❝❤r♦♠❛t✐♥ s✐•❡♥❝✐♥❣✳ ❙❝✐❡♥❝❡✱ ✸✶✽✭✺✽✹✼✮✿✶✵✾✕✶✶✷✳ ❇❡•❛❣❤③❛•✱ ❍✳✱ ❉❡❦❦❡r✱ ❏✳✱ ❛♥❞ •✐❜❝✉s✱ ❏✳ ❍✳ ✭✷✵✶✼✮✳ ❍✐✲❈ ✷✳✵✿ ❆♥ ♦♣t✐♠✐③❡❞ ❍✐✲❈ ♣r♦❝❡✲ ❞✉r❡ ❢♦r ❤✐❣❤✲r❡s♦•✉t✐♦♥ ❣❡♥♦♠❡✲✇✐❞❡ ♠❛♣♣✐♥❣ ♦❢ ❝❤r♦♠♦s♦♠❡ ❝♦♥❢♦r♠❛t✐♦♥✳ ▼❡t❤♦❞s✱ ✶✷✸✿✺✻✕✻✺✳ ❇❡r❣✱ ❙✳✱ ❑✉tr❛✱ ❉✳✱ ❑r♦❡❣❡r✱ ❚✳✱ ❙tr❛❡❤•❡✱ ❈✳ ◆✳✱ ❑❛✉s•❡r✱ ❇✳ ❳✳✱ ❍❛✉❜♦•❞✱ ❈✳✱ ❙❝❤✐❡❣❣✱ ▼✳✱ ❆•❡s✱ ❏✳✱ ❇❡✐❡r✱ ❚✳✱ |✉❞②✱ ▼✳✱ ❊r❡♥✱ ❑✳✱ ❈❡r✈❛♥t❡s✱ ❏✳ ■✳✱ ❳✉✱ ❇✳✱ ❇❡✉tt❡♥♠✉❡••❡r✱ ❋✳✱ ❲♦•♥②✱ ❆✳✱ )❤❛♥❣✱ ❈✳✱ ❑♦❡t❤❡✱ ❯✳✱ ❍❛♠♣r❡❝❤t✱ ❋✳ ❆✳✱ ❛♥❞ ❑r❡s❤✉❦✱ ❆✳ ✭✷✵✶✾✮✳ ✐•❛st✐❦✿ ✐♥t❡r❛❝t✐✈❡ ♠❛❝❤✐♥❡ •❡❛r♥✐♥❣ ❢♦r ✭❜✐♦✮✐♠❛❣❡ ❛♥❛•②s✐s✳ ◆❛t✉r❡ ▼❡t❤♦❞s✱ ✶✻✭✶✷✮✿✶✷✷✻✕ ✶✷✸✷✳ ❇❡r•❡t❝❤✱ ❏✳ ❇✳✱ ▼❛✱ ❲✳✱ (❛♥❣✱ ❋✳✱ ❙❤❡♥❞✉r❡✱ ❏✳✱ ◆♦❜•❡✱ ❲✳ ❙✳✱ ❉✐st❡❝❤❡✱ ❈✳ ▼✳✱ ❛♥❞ ❉❡♥❣✱ ❳✳ ✭✷✵✶✺✮✳ ❊s❝❛♣❡ ❢r♦♠ ❳ ■♥❛❝t✐✈❛t✐♦♥ ❱❛r✐❡s ✐♥ ▼♦✉s❡ ❚✐ss✉❡s✳ P▲♦❙ •❡♥❡t✐❝s✱ ✶✶✭✸✮✿❡✶✵✵✺✵✼✾✳ ❇❡r•❡t❝❤✱ ❏✳ ❇✳✱ (❛♥❣✱ ❋✳✱ ❳✉✱ ❏✳✱ ❈❛rr❡•✱ ▲✳✱ ❛♥❞ ❉✐st❡❝❤❡✱ ❈✳ ▼✳ ✭✷✵✶✶✮✳ •❡♥❡s t❤❛t ❡s❝❛♣❡ ❢r♦♠ ❳ ✐♥❛❝t✐✈❛t✐♦♥✳ ❍✉♠❛♥ ❣❡♥❡t✐❝s✱ ✶✸✵✭✷✮✿✷✸✼✕✹✺✳ ✶✽✾ |❡❢❡r❡♥❝❡s |❊❋❊|❊◆❈❊❙ ❇•❛❝❦❜✉r♥✱ •✳ ▼✳✱ ❈❤❡r✜•s✱ ❏✳✱ ▼♦ss✱ •✳ P✳✱ |✐❝❤❛r❞s✱ ◆✳ •✳ ❏✳✱ ❲❛•t❤♦✱ ❏✳ P✳✱ ❲✐••✐❛♠s✱ ◆✳ ❍✳✱ ❛♥❞ ❲✐tt✐♥❣❤♦❢❡r✱ ❆✳ ✭✷✵✶✼✮✳ ■❯P❆❈ |❡❝♦♠♠❡♥❞❛t✐♦♥s ❍♦✇ t♦ ♥❛♠❡ ❛t♦♠s ✐♥ ♣❤♦s♣❤❛t❡s✱ ♣♦•②♣❤♦s♣❤❛t❡s✱ t❤❡✐r ❞❡r✐✈❛t✐✈❡s ❛♥❞ ♠✐♠✐❝s✱ ❛♥❞ tr❛♥s✐t✐♦♥ st❛t❡ ❛♥❛✲ •♦❣✉❡s ❢♦r ❡♥③②♠❡✲❝❛t❛•②s❡❞ ♣❤♦s♣❤♦r②• tr❛♥s❢❡r r❡❛❝t✐♦♥s ✭■❯P❆❈ |❡❝♦♠♠❡♥❞❛t✐♦♥s ✷✵✶✻✮✳ P✉r❡ ❆♣♣•✳ ❈❤❡♠✱ ✽✾✭✺✮✿✻✺✸✕✻✼✺✳ ❇•❛❝❦•❡❞❣❡✱ ◆✳ P✳✱ ❋❛r❝❛s✱ ❆✳ ▼✳✱ ❑♦♥❞♦✱ ❚✳✱ ❑✐♥❣✱ ❍✳ ❲✳✱ ▼❝•♦✉r❛♥✱ ❏✳ ❋✳✱ ❍❛♥ss❡♥✱ ▲✳ ▲✳✱ ■t♦✱ ❙✳✱ ❈♦♦♣❡r✱ ❙✳✱ ❑♦♥❞♦✱ ❑✳✱ ❑♦s❡❦✐✱ (✳✱ ■s❤✐❦✉r❛✱ ❚✳✱ ▲♦♥❣✱ ❍✳ ❑✳✱ ❙❤❡❛❤❛♥✱ ❚✳ ❲✳✱ ❇r♦❝❦❞♦r✛✱ ◆✳✱ ❑❡ss•❡r✱ ❇✳ ▼✳✱ ❑♦s❡❦✐✱ ❍✳✱ ❛♥❞ ❑•♦s❡✱ |✳ ❏✳ ✭✷✵✶✹✮✳ ❱❛r✐❛♥t P|❈✶ ❝♦♠♣•❡①✲❞❡♣❡♥❞❡♥t ❍✷❆ ✉❜✐q✉✐t②•❛t✐♦♥ ❞r✐✈❡s P|❈✷ r❡❝r✉✐t♠❡♥t ❛♥❞ ♣♦•②❝♦♠❜ ❞♦♠❛✐♥ ❢♦r♠❛t✐♦♥✳ ❈❡••✱ ✶✺✼✭✻✮✿✶✹✹✺✕✶✹✺✾✳ ❇•❡✇✐tt✱ ▼✳ ❊✳✱ •❡♥❞r❡•✱ ❆✳ ❱✳✱ P❛♥❣✱ )✳✱ ❙♣❛rr♦✇✱ ❉✳ ❇✳✱ ❲❤✐t❡•❛✇✱ ◆✳✱ ❈r❛✐❣✱ ❏✳ ▼✳✱ ❆♣❡❞❛✐•❡✱ ❆✳✱ ❍✐•t♦♥✱ ❉✳ ❏✳✱ ❉✉♥✇♦♦❞✐❡✱ ❙✳ ▲✳✱ ❇r♦❝❦❞♦r✛✱ ◆✳✱ ❑❛②✱ •✳ ❋✳✱ ❛♥❞ ❲❤✐t❡•❛✇✱ ❊✳ ✭✷✵✵✽✮✳ ❙♠❝❍❉✶✱ ❝♦♥t❛✐♥✐♥❣ ❛ str✉❝t✉r❛•✲♠❛✐♥t❡♥❛♥❝❡✲♦❢✲❝❤r♦♠♦s♦♠❡s ❤✐♥❣❡ ❞♦✲ ♠❛✐♥✱ ❤❛s ❛ ❝r✐t✐❝❛• r♦•❡ ✐♥ ❳ ✐♥❛❝t✐✈❛t✐♦♥✳ ◆❛t✉r❡ •❡♥❡t✐❝s✱ ✹✵✭✺✮✿✻✻✸✕✻✻✾✳ ❇•❡✇✐tt✱ ▼✳ ❊✳✱ ❱✐❝❦❛r②♦✉s✱ ◆✳ ❑✳✱ ❍❡♠•❡②✱ ❙✳ ❏✳✱ ❆s❤❡✱ ❆✳✱ ❇r✉①♥❡r✱ ❚✳ ❏✳✱ Pr❡✐s✱ ❏✳ ■✳✱ ❆r❦❡••✱ |✳✱ ❛♥❞ ❲❤✐t❡•❛✇✱ ❊✳ ✭✷✵✵✺✮✳ ❆♥ ◆✲❡t❤②•✲◆✲♥✐tr♦s♦✉r❡❛ s❝r❡❡♥ ❢♦r ❣❡♥❡s ✐♥✈♦•✈❡❞ ✐♥ ✈❛r✐❡❣❛t✐♦♥ ✐♥ t❤❡ ♠♦✉s❡✳ Pr♦❝❡❡❞✐♥❣s ♦❢ t❤❡ ◆❛t✐♦♥❛• ❆❝❛❞❡♠② ♦❢ ❙❝✐❡♥❝❡s ♦❢ t❤❡ ❯♥✐t❡❞ ❙t❛t❡s ♦❢ ❆♠❡r✐❝❛✱ ✶✵✷✭✷✶✮✿✼✻✷✾✕✼✻✸✹✳ ❇♦❣❣s✱ ❇✳ ❆✳✱ ❈❤❡✉♥❣✱ P✳✱ ❍❡❛r❞✱ ❊✳✱ ❙♣❡❝t♦r✱ ❉✳ ▲✳✱ ❈❤✐♥❛✉•t✱ ❆✳ ❈✳✱ ❛♥❞ ❆••✐s✱ ❈✳ ❉✳ ✭✷✵✵✷✮✳ ❉✐✛❡r❡♥t✐❛••② ♠❡t❤②•❛t❡❞ ❢♦r♠s ♦❢ ❤✐st♦♥❡ ❍✸ s❤♦✇ ✉♥✐q✉❡ ❛ss♦❝✐❛t✐♦♥ ♣❛tt❡r♥s ✇✐t❤ ✐♥❛❝t✐✈❡ ❤✉♠❛♥ ❳ ❝❤r♦♠♦s♦♠❡s✳ ◆❛t✉r❡ •❡♥❡t✐❝s✱ ✸✵✭✶✮✿✼✸✕✼✻✳ ❇♦r❡♥s③t❡✐♥✱ ▼✳✱ ❖❦❛♠♦t♦✱ ■✳✱ ❙②①✱ ▲✳✱ •✉✐•❜❛✉❞✱ •✳✱ P✐❝❛r❞✱ ❈✳✱ ❆♥❝❡•✐♥✱ ❑✳✱ •❛•✉♣❛✱ |✳✱ ❉✐❛❜❛♥❣♦✉❛②❛✱ P✳✱ ❙❡r✈❛♥t✱ ◆✳✱ ❇❛r✐••♦t✱ ❊✳✱ ❙✉r❛♥✐✱ ❆✳✱ ❙❛✐t♦✉✱ ▼✳✱ ❈❤❡♥✱ ❈✳ ❏✳✱ ❆♥❛st❛ss✐❛❞✐s✱ ❑✳✱ ❛♥❞ ❍❡❛r❞✱ ❊✳ ✭✷✵✶✼❛✮✳ ❈♦♥tr✐❜✉t✐♦♥ ♦❢ ❡♣✐❣❡♥❡t✐❝ •❛♥❞s❝❛♣❡s ❛♥❞ tr❛♥s❝r✐♣t✐♦♥ ❢❛❝t♦rs t♦ ❳✲❝❤r♦♠♦s♦♠❡ r❡❛❝t✐✈❛t✐♦♥ ✐♥ t❤❡ ✐♥♥❡r ❝❡•• ♠❛ss✳ ◆❛t✉r❡ ❈♦♠♠✉♥✐❝❛t✐♦♥s✱ ✽✭✶✮✿✶✕✶✹✳ ❇♦r❡♥s③t❡✐♥✱ ▼✳✱ ❙②①✱ ▲✳✱ ❆♥❝❡•✐♥✱ ❑✳✱ ❉✐❛❜❛♥❣♦✉❛②❛✱ P✳✱ P✐❝❛r❞✱ ❈✳✱ ▲✐✉✱ ❚✳✱ ▲✐❛♥❣✱ ❏✳✲❇✳✱ ❱❛ss✐•❡✈✱ ■✳✱ •❛•✉♣❛✱ |✳✱ ❙❡r✈❛♥t✱ ◆✳✱ ❇❛r✐••♦t✱ ❊✳✱ ❙✉r❛♥✐✱ ❆✳✱ ❈❤❡♥✱ ❈✳✲❏✳✱ ❛♥❞ ❍❡❛r❞✱ ❊✳ ✭✷✵✶✼❜✮✳ ❳✐st✲❞❡♣❡♥❞❡♥t ✐♠♣r✐♥t❡❞ ❳ ✐♥❛❝t✐✈❛t✐♦♥ ❛♥❞ t❤❡ ❡❛r•② ❞❡✈❡•♦♣♠❡♥t❛• ❝♦♥s❡q✉❡♥❝❡s ♦❢ ✐ts ❢❛✐•✉r❡✳ ◆❛t✉r❡ ❙tr✉❝t✉r❛• ✫ ▼♦•❡❝✉•❛r ❇✐♦•♦❣②✳ ❇♦r♥❡•ö✈✱ ❙✳✱ |❡②♥♦•❞s✱ ◆✳✱ ❳❡♥♦♣❤♦♥t♦s✱ ▼✳✱ •❤❛r❜✐✱ ❙✳✱ ❏♦❤♥st♦♥❡✱ ❊✳✱ ❋•♦②❞✱ |✳✱ |❛•s❡r✱ ▼✳✱ ❙✐❣♥♦•❡t✱ ❏✳✱ ▲♦♦s✱ |✳✱ ❉✐❡t♠❛♥♥✱ ❙✳✱ ❇❡rt♦♥❡✱ P✳✱ ❛♥❞ ❍❡♥❞r✐❝❤✱ ❇✳ ✭✷✵✶✽✮✳ ❚❤❡ ◆✉❝•❡♦s♦♠❡ |❡♠♦❞❡•✐♥❣ ❛♥❞ ❉❡❛❝❡t②•❛t✐♦♥ ❈♦♠♣•❡① ▼♦❞✉•❛t❡s ❈❤r♦♠❛t✐♥ ❙tr✉❝✲ t✉r❡ ❛t ❙✐t❡s ♦❢ ❆❝t✐✈❡ ❚r❛♥s❝r✐♣t✐♦♥ t♦ ❋✐♥❡✲❚✉♥❡ •❡♥❡ ❊①♣r❡ss✐♦♥✳ ▼♦•❡❝✉•❛r ❈❡••✱ ✼✶✭✶✮✿✺✻✕✼✷✳❡✹✳ ✶✾✵ |❡❢❡r❡♥❝❡s |❊❋❊|❊◆❈❊❙ ❇♦✉r❣❡♦✐s✱ ❈✳ ❆✳✱ ▲❛q✉❡rr✐❡r❡✱ ❋✳✱ ❍❡♠♦♥✱ ❉✳✱ ❍✉❜❡rt✱ ❏✳✱ ❛♥❞ ❇♦✉t❡✐••❡✱ ▼✳ ✭✶✾✽✺✮✳ ◆❡✇ ❞❛t❛ ♦♥ t❤❡ ✐♥ s✐t✉ ♣♦s✐t✐♦♥ ♦❢ t❤❡ ✐♥❛❝t✐✈❡ ❳ ❝❤r♦♠♦s♦♠❡ ✐♥ t❤❡ ✐♥t❡r♣❤❛s❡ ♥✉❝•❡✉s ♦❢ ❤✉♠❛♥ ✜❜r♦❜•❛sts✳ ❍✉♠❛♥ •❡♥❡t✐❝s✱ ✻✾✭✷✮✿✶✷✷✕✶✷✾✳ ❇♦✉s❛r❞✱ ❆✳✱ |❛♣♦s♦✱ ❆✳ ❈✳✱ ➏②•✐❝③✱ ❏✳ ❏✳✱ P✐❝❛r❞✱ ❈✳✱ P✐r❡s✱ ❱✳ ❇✳✱ ◗✐✱ (✳✱ •✐•✱ ❈✳✱ ❙②①✱ ▲✳✱ ❈❤❛♥❣✱ ❍✳ (✳✱ ❍❡❛r❞✱ ❊✳✱ ❛♥❞ ❞❛ |♦❝❤❛✱ ❙✳ ❚✳ ✭✷✵✶✾✮✳ ❚❤❡ r♦•❡ ♦❢ ❳✐st✲♠❡❞✐❛t❡❞ P♦•②❝♦♠❜ r❡❝r✉✐t♠❡♥t ✐♥ t❤❡ ✐♥✐t✐❛t✐♦♥ ♦❢ ❳✲❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥✳ ❊▼❇❖ r❡♣♦rts✱ ✷✵✭✶✵✮✳ ❇r♦❝❦❞♦r✛✱ ◆✳ ✭✷✵✶✾✮✳ ▲♦❝❛•✐③❡❞ ❛❝❝✉♠✉•❛t✐♦♥ ♦❢ ❳✐st |◆❆ ✐♥ ❳ ❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛✲ t✐♦♥✳ ❖♣❡♥ ❇✐♦•♦❣②✱ ✾✭✶✷✮✳ ❇r♦❝❦❞♦r✛✱ ◆✳✱ ❆s❤✇♦rt❤✱ ❆✳✱ ❑❛②✱ •✳ ❋✳✱ ❈♦♦♣❡r✱ P✳✱ ❙♠✐t❤✱ ❙✳✱ ▼❝❈❛❜❡✱ ❱✳ ▼✳✱ ◆♦r✲ r✐s✱ ❉✳ P✳✱ P❡♥♥②✱ •✳ ❉✳✱ P❛t❡•✱ ❉✳✱ ❛♥❞ |❛st❛♥✱ ❙✳ ✭✶✾✾✶✮✳ ❈♦♥s❡r✈❛t✐♦♥ ♦❢ ♣♦s✐t✐♦♥ ❛♥❞ ❡①❝•✉s✐✈❡ ❡①♣r❡ss✐♦♥ ♦❢ ♠♦✉s❡ ❳✐st ❢r♦♠ t❤❡ ✐♥❛❝t✐✈❡ ❳ ❝❤r♦♠♦s♦♠❡✳ ◆❛t✉r❡✱ ✸✺✶✭✻✸✷✹✮✿✸✷✾✕✸✸✶✳ ❇r♦❝❦❞♦r✛✱ ◆✳✱ ❆s❤✇♦rt❤✱ ❆✳✱ ❑❛②✱ •✳ ❋✳✱ ▼❝❈❛❜❡✱ ❱✳ ▼✳✱ ◆♦rr✐s✱ ❉✳ P✳✱ ❈♦♦♣❡r✱ P✳ ❏✳✱ ❙✇✐❢t✱ ❙✳✱ ❛♥❞ |❛st❛♥✱ ❙✳ ✭✶✾✾✷✮✳ ❚❤❡ ♣r♦❞✉❝t ♦❢ t❤❡ ♠♦✉s❡ ❳✐st ❣❡♥❡ ✐s ❛ ✶✺ ❦❜ ✐♥❛❝t✐✈❡ ❳✲s♣❡❝✐✜❝ tr❛♥s❝r✐♣t ❝♦♥t❛✐♥✐♥❣ ♥♦ ❝♦♥s❡r✈❡❞ ❖|❋ ❛♥❞ •♦❝❛t❡❞ ✐♥ t❤❡ ♥✉❝•❡✉s✳ ❈❡••✱ ✼✶✭✸✮✿✺✶✺✕✷✻✳ ❇r♦✇♥✱ ❈✳ ❏✳✱ ❇❛••❛❜✐♦✱ ❆✳✱ |✉♣❡rt✱ ❏✳ ▲✳✱ ▲❛❢r❡♥✐❡r❡✱ |✳ •✳✱ •r♦♠♣❡✱ ▼✳✱ ❚♦♥•♦r❡♥③✐✱ |✳✱ ❛♥❞ ❲✐••❛r❞✱ ❍✳ ❋✳ ✭✶✾✾✶✮✳ ❆ ❣❡♥❡ ❢r♦♠ t❤❡ r❡❣✐♦♥ ♦❢ t❤❡ ❤✉♠❛♥ ❳ ✐♥❛❝t✐✈❛t✐♦♥ ❝❡♥tr❡ ✐s ❡①♣r❡ss❡❞ ❡①❝•✉s✐✈❡•② ❢r♦♠ t❤❡ ✐♥❛❝t✐✈❡ ❳ ❝❤r♦♠♦s♦♠❡✳ ◆❛t✉r❡✱ ✸✹✾✭✻✸✵✹✮✿✸✽✕✹✹✳ ❇r♦✇♥✱ ❈✳ ❏✳✱ ❍❡♥❞r✐❝❤✱ ❇✳ ❉✳✱ |✉♣❡rt✱ ❏✳ ▲✳✱ ▲❛❢r❡♥✐èr❡✱ |✳ •✳✱ ❳✐♥❣✱ (✳✱ ▲❛✇r❡♥❝❡✱ ❏✳✱ ❛♥❞ ❲✐••❛r❞✱ ❍✳ ❋✳ ✭✶✾✾✷✮✳ ❚❤❡ ❤✉♠❛♥ ❳■❙❚ ❣❡♥❡✿ ❆♥❛•②s✐s ♦❢ ❛ ✶✼ ❦❜ ✐♥❛❝t✐✈❡ ❳✲ s♣❡❝✐✜❝ |◆❆ t❤❛t ❝♦♥t❛✐♥s ❝♦♥s❡r✈❡❞ r❡♣❡❛ts ❛♥❞ ✐s ❤✐❣❤•② •♦❝❛•✐③❡❞ ✇✐t❤✐♥ t❤❡ ♥✉❝•❡✉s✳ ❈❡••✱ ✼✶✭✸✮✿✺✷✼✕✺✹✷✳ ❇r♦✇♥✱ ❈✳ ❏✳ ❛♥❞ ❲✐••❛r❞✱ ❍✳ ❋✳ ✭✶✾✾✹✮✳ ❚❤❡ ❤✉♠❛♥ ❳✲✐♥❛❝t✐✈❛t✐♦♥ ❝❡♥tr❡ ✐s ♥♦t r❡q✉✐r❡❞ ❢♦r ♠❛✐♥t❡♥❛♥❝❡ ♦❢ ❳✲❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥✳ ◆❛t✉r❡✱ ✸✻✽✭✻✹✻✼✮✿✶✺✹✕✶✺✻✳ ❇✉r❛t♦✇s❦✐✱ ❙✳ ✭✷✵✵✸✮✳ ❚❤❡ ❈❚❉ ❝♦❞❡✳ ❈❛r❞♦③♦✱ ❚✳ ❛♥❞ P❛❣❛♥♦✱ ▼✳ ✭✷✵✵✹✮✳ ❚❤❡ ❙❈❋ ✉❜✐q✉✐t✐♥ •✐❣❛s❡✿ ■♥s✐❣❤ts ✐♥t♦ ❛ ♠♦•❡❝✉•❛r ♠❛❝❤✐♥❡✳ ❈❛rr❡•✱ ▲✳ ❛♥❞ ❇r♦✇♥✱ ❈✳ ❏✳ ✭✷✵✶✼✮✳ ❲❤❡♥ t❤❡ •②♦♥✭■③❡❞ ❝❤r♦♠♦s♦♠❡✮ r♦❛rs✿ ❖♥❣♦✐♥❣ ❡①♣r❡ss✐♦♥ ❢r♦♠ ❛♥ ✐♥❛❝t✐✈❡ ❳ ❝❤r♦♠♦s♦♠❡✳ P❤✐•♦s♦♣❤✐❝❛• ❚r❛♥s❛❝t✐♦♥s ♦❢ t❤❡ |♦②❛• ❙♦❝✐❡t② ❇✿ ❇✐♦•♦❣✐❝❛• ❙❝✐❡♥❝❡s✱ ✸✼✷✭✶✼✸✸✮✳ ✶✾✶ |❡❢❡r❡♥❝❡s |❊❋❊|❊◆❈❊❙ ❈❛rr❡•✱ ▲✳✱ ❈♦tt•❡✱ ❆✳ ❆✳✱ •♦❣•✐♥✱ ❑✳ ❈✳✱ ❛♥❞ ❲✐••❛r❞✱ ❍✳ ❋✳ ✭✶✾✾✾✮✳ ❆ ✜rst✲❣❡♥❡r❛t✐♦♥ ❳✲ ✐♥❛❝t✐✈❛t✐♦♥ ♣r♦✜•❡ ♦❢ t❤❡ ❤✉♠❛♥ ❳ ❝❤r♦♠♦s♦♠❡✳ Pr♦❝❡❡❞✐♥❣s ♦❢ t❤❡ ◆❛t✐♦♥❛• ❆❝❛❞❡♠② ♦❢ ❙❝✐❡♥❝❡s ♦❢ t❤❡ ❯♥✐t❡❞ ❙t❛t❡s ♦❢ ❆♠❡r✐❝❛✱ ✾✻✭✷✺✮✿✶✹✹✹✵✕✶✹✹✹✹✳ ❈❛rt❡r✱ ❆✳ ❈✳✱ ❳✉✱ ❏✳✱ ◆❛❦❛♠♦t♦✱ ▼✳ (✳✱ ❲❡✐✱ (✳✱ )❛r♥❡❣❛r✱ ❇✳ ❏✳✱ ❙❤✐✱ ◗✳✱ ❇r♦✉❣❤t♦♥✱ ❏✳ P✳✱ |❛♥s♦♠✱ |✳ ❈✳✱ ❙❛•❤♦tr❛✱ ❆✳✱ ◆❛❣❛r❛•❛✱ ❙✳ ❉✳✱ ▲✐✱ |✳✱ ❉♦✉✱ ❉✳ |✳✱ (♦st✱ ❑✳ ❊✳✱ ❈❤♦✱ ❙✳ ❲✳✱ ▼✐str②✱ ❆✳✱ ▲♦♥❣❛❦❡r✱ ▼✳ ❚✳✱ ❑❤❛✈❛r✐✱ P✳ ❆✳✱ ❇❛t❡②✱ |✳ ❚✳✱ ❲✉tt❦❡✱ ❉✳ ❙✳✱ ❛♥❞ ❈❤❛♥❣✱ ❍✳ (✳ ✭✷✵✷✵✮✳ ❙♣❡♥ •✐♥❦s r♥❛✲♠❡❞✐❛t❡❞ ❡♥❞♦❣❡♥♦✉s r❡tr♦✈✐r✉s s✐•❡♥❝✐♥❣ ❛♥❞ ① ❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥✳ ❡▲✐❢❡✱ ✾✿✶✕✺✽✳ ❈❡r❛s❡✱ ❆✳✱ ❆r♠❛♦s✱ ❆✳✱ ◆❡✉♠❛②❡r✱ ❈✳✱ ❆✈♥❡r✱ P✳✱ •✉tt♠❛♥✱ ▼✳✱ ❛♥❞ ❚❛rt❛❣•✐❛✱ •✳ •✳ ✭✷✵✶✾✮✳ P❤❛s❡ s❡♣❛r❛t✐♦♥ ❞r✐✈❡s ❳✲❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥✿ ❛ ❤②♣♦t❤❡s✐s✳ ❈❡r❛s❡✱ ❆✳✱ ❙♠❡❡ts✱ ❉✳✱ ❚❛♥❣✱ (✳ ❆✳✱ •❞✉•❛✱ ▼✳✱ ❑r❛✉s✱ ❋✳✱ ❙♣✐✈❛❦♦✈✱ ▼✳✱ ▼♦✐♥❞r♦t✱ ❇✳✱ ▲❡•❡✉✱ ▼✳✱ ❚❛tt❡r♠✉s❝❤✱ ❆✳✱ ❉❡♠♠❡r•❡✱ ❏✳✱ ◆❡st❡r♦✈❛✱ ❚✳ ❇✳✱ •r❡❡♥✱ ❈✳✱ ❖tt❡✱ ❆✳ P✳✱ ❙❝❤❡r♠❡••❡❤✱ ▲✳✱ ❛♥❞ ❇r♦❝❦❞♦r✛✱ ◆✳ ✭✷✵✶✹✮✳ ❙♣❛t✐❛• s❡♣❛r❛t✐♦♥ ♦❢ ❳✐st |◆❆ ❛♥❞ ♣♦•②❝♦♠❜ ♣r♦t❡✐♥s r❡✈❡❛•❡❞ ❜② s✉♣❡rr❡s♦•✉t✐♦♥ ♠✐❝r♦s❝♦♣②✳ Pr♦❝❡❡❞✐♥❣s ♦❢ t❤❡ ◆❛t✐♦♥❛• ❆❝❛❞❡♠② ♦❢ ❙❝✐❡♥❝❡s ♦❢ t❤❡ ❯♥✐t❡❞ ❙t❛t❡s ♦❢ ❆♠❡r✐❝❛✱ ✶✶✶✭✻✮✿✷✷✸✺✕✷✷✹✵✳ ❈❤❛♣♠❛♥✱ |✳ ❉✳✱ ❍❡✐❞❡♠❛♥♥✱ ▼✳✱ ❍✐♥t❡r♠❛✐r✱ ❈✳✱ ❛♥❞ ❊✐❝❦✱ ❉✳ ✭✷✵✵✽✮✳ ▼♦•❡❝✉•❛r ❡✈♦•✉✲ t✐♦♥ ♦❢ t❤❡ |◆❆ ♣♦•②♠❡r❛s❡
♥ ❍✉♠♠❡•❡♥✱ P✳✱ ❋❡•❞♠❛♥✱ ❆✳ ▲✳✱ ▲✐♥❦✱ ❇✳ ❑✳✱ ◆♦✈❛❦✱ ❆✳ ❏✳✱ ❈❡r❤❛♥✱ ❏✳ |✳✱ ❍❛❜❡r✲ ♠❛♥♥✱ ❚✳ ▼✳✱ ❙✐❡❜❡rt✱ |✳✱ |♦s❡♥✇❛•❞✱ ❆✳✱ ❚❤♦r♥❡r✱ ❆✳ |✳✱ ▼❡②❡rs♦♥✱ ▼✳ ▲✳✱ •♦•✉❜✱ ❚✳ |✳✱ ❇❡r♦✉❦❤✐♠✱ |✳✱ ❲✉•❢✱ •✳ •✳✱ ❖tt✱ •✳✱ |♦❞✐❣✱ ❙✳ ❏✳✱ ▼♦♥t✐✱ ❙✳✱ ◆❡✉❜❡r❣✱ ❉✳ ❙✳✱ ▲♦❡✤❡r✱ ▼✳✱ P❢r❡✉♥❞s❝❤✉❤✱ ▼✳✱ ❚rü♠♣❡r✱ ▲✳✱ •❡t③✱ •✳✱ ❛♥❞ ❙❤✐♣♣✱ ▼✳ ❆✳ ✭✷✵✶✽✮✳ ▼♦•❡❝✲ ✉•❛r s✉❜t②♣❡s ♦❢ ❞✐✛✉s❡ •❛r❣❡ ❇ ❝❡•• •②♠♣❤♦♠❛ ❛r❡ ❛ss♦❝✐❛t❡❞ ✇✐t❤ ❞✐st✐♥❝t ♣❛t❤♦❣❡♥✐❝ ♠❡❝❤❛♥✐s♠s ❛♥❞ ♦✉t❝♦♠❡s✳ ◆❛t✉r❡ ▼❡❞✐❝✐♥❡✱ ✷✹✭✺✮✿✻✼✾✕✻✾✵✳ ❈❤❛r•❡s✇♦rt❤✱ ❇✳ ✭✶✾✾✻✮✳ ❚❤❡ ❡✈♦•✉t✐♦♥ ♦❢ ❝❤r♦♠♦s♦♠❛• s❡① ❞❡t❡r♠✐♥❛t✐♦♥ ❛♥❞ ❞♦s❛❣❡ ❝♦♠♣❡♥s❛t✐♦♥✳ ❈✉rr❡♥t ❜✐♦•♦❣② ✿ ❈❇✱ ✻✭✷✮✿✶✹✾✕✻✷✳ ❈❤❛r•❡s✇♦rt❤✱ ❉✳✱ ❈❤❛r•❡s✇♦rt❤✱ ❇✳✱ ❛♥❞ ▼❛r❛✐s✱ •✳ ✭✷✵✵✺✮✳ ❙t❡♣s ✐♥ t❤❡ ❡✈♦•✉t✐♦♥ ♦❢ ❤❡t❡r♦♠♦r♣❤✐❝ s❡① ❝❤r♦♠♦s♦♠❡s✳ ❍❡r❡❞✐t②✱ ✾✺✭✷✮✿✶✶✽✕✶✷✽✳ ❈❤❛✉♠❡✐•✱ ❏✳✱ ▲❡ ❇❛❝❝♦♥✱ P✳✱ ❲✉t③✱ ❆✳✱ ❛♥❞ ❍❡❛r❞✱ ❊✳ ✭✷✵✵✻✮✳ ❆ ♥♦✈❡• r♦•❡ ❢♦r ❳✐st |◆❆ ✐♥ t❤❡ ❢♦r♠❛t✐♦♥ ♦❢ ❛ r❡♣r❡ss✐✈❡ ♥✉❝•❡❛r ❝♦♠♣❛rt♠❡♥t ✐♥t♦ ✇❤✐❝❤ ❣❡♥❡s ❛r❡ r❡❝r✉✐t❡❞ ✇❤❡♥ s✐•❡♥❝❡❞✳ •❡♥❡s ✫ ❞❡✈❡•♦♣♠❡♥t✱ ✷✵✭✶✻✮✿✷✷✷✸✕✸✼✳ ✶✾✷ |❡❢❡r❡♥❝❡s |❊❋❊|❊◆❈❊❙ ❈❤❡•♠✐❝❦✐✱ ❚✳✱ |♦❣❡r✱ ❊✳✱ ❚❡✐ss❛♥❞✐❡r✱ ❆✳✱ ❉✉r❛✱ ▼✳✱ ❇♦♥♥❡✈✐••❡✱ ▲✳✱ |✉❝•✐✱ ❙✳✱ ❉♦ss✐♥✱ ❋✳✱ ❋♦✉❛ss✐❡r✱ ❈✳✱ ▲❛♠❡✐r❛s✱ ❙✳✱ ❛♥❞ ❇♦✉r❝✬❤✐s✱ ❉✳ ✭✷✵✷✶✮✳ ♠✻❆ |◆❆ ♠❡t❤②•❛t✐♦♥ r❡❣✉•❛t❡s t❤❡ ❢❛t❡ ♦❢ ❡♥❞♦❣❡♥♦✉s r❡tr♦✈✐r✉s❡s✳ ◆❛t✉r❡✱ ✺✾✶✭✼✽✹✾✮✿✸✶✷✕✸✶✻✳ ❈❤❡♥✱ ❈✳ ❑✳✱ ❇•❛♥❝♦✱ ▼✳✱ ❏❛❝❦s♦♥✱ ❈✳✱ ❆③♥❛✉r②❛♥✱ ❊✳✱ ❖••✐❦❛✐♥❡♥✱ ◆✳✱ ❙✉r❦❛✱ ❈✳✱ ❈❤♦✇✱ ❆✳✱ ❈❡r❛s❡✱ ❆✳✱ ▼❝❉♦♥❡•✱ P✳✱ ❛♥❞ •✉tt♠❛♥✱ ▼✳ ✭✷✵✶✻✮✳ ❳✐st r❡❝r✉✐ts t❤❡ ❳ ❝❤r♦♠♦s♦♠❡ t♦ t❤❡ ♥✉❝•❡❛r •❛♠✐♥❛ t♦ ❡♥❛❜•❡ ❝❤r♦♠♦s♦♠❡✲✇✐❞❡ s✐•❡♥❝✐♥❣✳ ❙❝✐❡♥❝❡✱ ✸✺✹✭✻✸✶✶✮✿✹✻✽✕✹✼✷✳ ❈❤❡♥✱ ❋✳ ❛♥❞ |❡❜❛②✱ ■✳ ✭✷✵✵✵✮✳ s♣•✐t ❡♥❞s✱ ❛ ♥❡✇ ❝♦♠♣♦♥❡♥t ♦❢ t❤❡ ❉r♦s♦♣❤✐•❛ ❊•❋ r❡❝❡♣✲ t♦r ♣❛t❤✇❛②✱ r❡❣✉•❛t❡s ❞❡✈❡•♦♣♠❡♥t ♦❢ ♠✐❞•✐♥❡ ❣•✐❛• ❝❡••s✳ ❈✉rr❡♥t ❇✐♦•♦❣②✱ ✶✵✭✶✺✮✿✾✹✸✕ ✾✹✻✳ ❈❤❡♥✱ ❏✳✱ ◆✐❦♦•❛✐t❝❤✐❦✱ ❖✳✱ ❙✐♥❣❤✱ ❏✳✱ ❲r✐❣❤t✱ ❆✳✱ ❇❡♥❝s✐❝s✱ ❈✳ ❊✳✱ ❈♦✣♥✱ ❏✳ ▼✳✱ ◆✐✱ ◆✳✱ ▲♦❝❦❡tt✱ ❙✳✱ P❛t❤❛❦✱ ❱✳ ❑✳✱ ❛♥❞ ❍✉✱ ❲✳ ❙✳ ✭✷✵✵✾✮✳ ❍✐❣❤ ❡✣❝✐❡♥❝② ♦❢ ❍■❱✲✶ ❣❡✲ ♥♦♠✐❝ |◆❆ ♣❛❝❦❛❣✐♥❣ ❛♥❞ ❤❡t❡r♦③②❣♦t❡ ❢♦r♠❛t✐♦♥ r❡✈❡❛•❡❞ ❜② s✐♥❣•❡ ✈✐r✐♦♥ ❛♥❛•②s✐s✳ Pr♦❝❡❡❞✐♥❣s ♦❢ t❤❡ ◆❛t✐♦♥❛• ❆❝❛❞❡♠② ♦❢ ❙❝✐❡♥❝❡s ♦❢ t❤❡ ❯♥✐t❡❞ ❙t❛t❡s ♦❢ ❆♠❡r✐❝❛✱ ✶✵✻✭✸✷✮✿✶✸✺✸✺✕✶✸✺✹✵✳ ❈❤♦✱ ❊✳ ❏✳✱ ❚❛❦❛❣✐✱ ❚✳✱ ▼♦♦r❡✱ ❈✳ |✳✱ ❛♥❞ ❇✉r❛t♦✇s❦✐✱ ❙✳ ✭✶✾✾✼✮✳ ♠|◆❆ ❝❛♣♣✐♥❣ ❡♥③②♠❡ ✐s r❡❝r✉✐t❡❞ t♦ t❤❡ tr❛♥s❝r✐♣t✐♦♥ ❝♦♠♣•❡① ❜② ♣❤♦s♣❤♦r②•❛t✐♦♥ ♦❢ t❤❡ |◆❆ ♣♦•②♠❡r❛s❡ ■■ ❝❛r❜♦①②✲t❡r♠✐♥❛• ❞♦♠❛✐♥✳ •❡♥❡s ❛♥❞ ❉❡✈❡•♦♣♠❡♥t✱ ✶✶✭✷✹✮✿✸✸✶✾✕✸✸✷✻✳ ❈❤♦♥❣✱ ❙✳✱ ❉✉❣❛st✲❉❛r③❛❝q✱ ❈✳✱ ▲✐✉✱ )✳✱ ❉♦♥❣✱ P✳✱ ❉❛✐•❡②✱ •✳ ▼✳✱ ❈❛tt♦❣•✐♦✱ ❈✳✱ ❍❡❝❦❡rt✱ ❆✳✱ ❇❛♥❛•❛✱ ❙✳✱ ▲❛✈✐s✱ ▲✳✱ ❉❛r③❛❝q✱ ❳✳✱ ❛♥❞ ❚•✐❛♥✱ |✳ ✭✷✵✶✽✮✳ ■♠❛❣✐♥❣ ❞②♥❛♠✐❝ ❛♥❞ s❡•❡❝t✐✈❡ •♦✇✲❝♦♠♣•❡①✐t② ❞♦♠❛✐♥ ✐♥t❡r❛❝t✐♦♥s t❤❛t ❝♦♥tr♦• ❣❡♥❡ tr❛♥s❝r✐♣t✐♦♥✳ ❙❝✐❡♥❝❡✱ ✸✻✶✭✻✹✵✵✮✳ ❈❤✉✱ ❈✳✱ )❤❛♥❣✱ ◗✳ ❈✳✱ ❉❛ |♦❝❤❛✱ ❙✳ ❚✳✱ ❋•②♥♥✱ |✳ ❆✳✱ ❇❤❛r❛❞✇❛•✱ ▼✳✱ ❈❛•❛❜r❡s❡✱ ❏✳ ▼✳✱ ▼❛❣♥✉s♦♥✱ ❚✳✱ ❍❡❛r❞✱ ❊✳✱ ❛♥❞ ❈❤❛♥❣✱ ❍✳ (✳ ✭✷✵✶✺✮✳ ❙②st❡♠❛t✐❝ ❞✐s❝♦✈❡r② ♦❢ ❳✐st |◆❆ ❜✐♥❞✐♥❣ ♣r♦t❡✐♥s✳ ❈❡••✱ ✶✻✶✭✷✮✿✹✵✹✕✹✶✻✳ ❈❤✉r❡❛✉✱ ❈✳✱ ❈❤❛♥t❛•❛t✱ ❙✳✱ |♦♠✐t♦✱ ❆✳✱ •❛•✈❛♥✐✱ ❆✳✱ ❉✉r❡t✱ ▲✳✱ ❆✈♥❡r✱ P✳✱ ❛♥❞ |♦✉❣❡✉••❡✱ ❈✳ ✭✷✵✶✶✮✳ ❋t① ✐s ❛ ♥♦♥✲❝♦❞✐♥❣ |◆❆ ✇❤✐❝❤ ❛✛❡❝ts ❳✐st ❡①♣r❡ss✐♦♥ ❛♥❞ ❝❤r♦♠❛t✐♥ str✉❝✲ t✉r❡ ✇✐t❤✐♥ t❤❡ ❳✲✐♥❛❝t✐✈❛t✐♦♥ ❝❡♥t❡r r❡❣✐♦♥✳ ❍✉♠❛♥ ▼♦•❡❝✉•❛r •❡♥❡t✐❝s✱ ✷✵✭✹✮✿✼✵✺✕ ✼✶✽✳ ❈•❡♠s♦♥✱ ❈✳ ▼✳✱ ▼❝◆❡✐•✱ ❏✳ ❆✳✱ ❲✐••❛r❞✱ ❍✳ ❋✳✱ ❛♥❞ ▲❛✇r❡♥❝❡✱ ❏✳ ❇✳ ✭✶✾✾✻✮✳ ❳■❙❚ |◆❆ ♣❛✐♥ts t❤❡ ✐♥❛❝t✐✈❡ ❳ ❝❤r♦♠♦s♦♠❡ ❛t ✐♥t❡r♣❤❛s❡✿ ❊✈✐❞❡♥❝❡ ❢♦r ❛ ♥♦✈❡• |◆❆ ✐♥✈♦•✈❡❞ ✐♥ ♥✉❝•❡❛r✴❝❤r♦♠♦s♦♠❡ str✉❝t✉r❡✳ ❏♦✉r♥❛• ♦❢ ❈❡•• ❇✐♦•♦❣②✱ ✶✸✷✭✸✮✿✷✺✾✕✷✼✺✳ ❈♦❦❡r✱ ❍✳✱ ❲❡✐✱ •✳✱ ▼♦✐♥❞r♦t✱ ❇✳✱ ▼♦❤❛♠♠❡❞✱ ❙✳✱ ◆❡st❡r♦✈❛✱ ❚✳✱ ❛♥❞ ❇r♦❝❦❞♦r✛✱ ◆✳ ✭✷✵✷✵✮✳ ❚❤❡ r♦•❡ ♦❢ t❤❡ ❳✐st ✺✬ ♠✻❆ r❡❣✐♦♥ ❛♥❞ |❇▼✶✺ ✐♥ ❳ ❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥✳ ❲❡••❝♦♠❡ ❖♣❡♥ |❡s❡❛r❝❤✱ ✺✳ ✶✾✸ |❡❢❡r❡♥❝❡s |❊❋❊|❊◆❈❊❙ ❈♦•♦❣♥♦r✐✱ ❉✳✱ ❙✉♥✇♦♦✱ ❍✳✱ ❑r✐③✱ ❆✳ ❏✳✱ ❲❛♥❣✱ ❈✳ (✳✱ ❛♥❞ ▲❡❡✱ ❏✳ ❚✳ ✭✷✵✶✾✮✳ ❳✐st ❉❡•❡t✐♦♥❛• ❆♥❛•②s✐s |❡✈❡❛•s ❛♥ ■♥t❡r❞❡♣❡♥❞❡♥❝② ❜❡t✇❡❡♥ ❳✐st |◆❆ ❛♥❞ P♦•②❝♦♠❜ ❈♦♠♣•❡①❡s ❢♦r ❙♣r❡❛❞✐♥❣ ❛•♦♥❣ t❤❡ ■♥❛❝t✐✈❡ ❳✳ ▼♦•❡❝✉•❛r ❈❡••✱ ✼✹✭✶✮✿✶✵✶✕✶✶✼✳❡✶✵✳ ❈♦♦♣❡r✱ ❙✳✱ •r✐•③❡♥❤♦✉t✱ ❆✳✱ ❯♥❞❡r✇♦♦❞✱ ❊✳✱ ❆♥❝❡•✐♥✱ ❑✳✱ )❤❛♥❣✱ ❚✳✱ ◆❡st❡r♦✈❛✱ ❚✳ ❇✳✱ ❆♥✐•✲❑✐r♠✐③✐t❛s✱ ❇✳✱ ❇❛ss❡tt✱ ❆✳✱ ❑♦♦✐str❛✱ ❙✳ ▼✳✱ ❆❣❣❡r✱ ❑✳✱ ❍❡•✐♥✱ ❑✳✱ ❍❡❛r❞✱ ❊✳✱ ❛♥❞ ❇r♦❝❦❞♦r✛✱ ◆✳ ✭✷✵✶✻✮✳ ❏❛r✐❞✷ ❜✐♥❞s ♠♦♥♦✲✉❜✐q✉✐t②•❛t❡❞ ❍✷❆ •②s✐♥❡ ✶✶✾ t♦ ♠❡❞✐❛t❡ ❝r♦sst❛•❦ ❜❡t✇❡❡♥ P♦•②❝♦♠❜ ❝♦♠♣•❡①❡s P|❈✶ ❛♥❞ P|❈✷✳ ◆❛t✉r❡ ❈♦♠♠✉♥✐❝❛t✐♦♥s✱ ✼✭✶✮✿✶✕✽✳ ❈♦st❛♥③✐✱ ❈✳ ❛♥❞ P❡❤rs♦♥✱ ❏✳ |✳ ✭✶✾✾✽✮✳ ❍✐st♦♥❡ ♠❛❝r♦❍✷❆✶ ✐s ❝♦♥❝❡♥tr❛t❡❞ ✐♥ t❤❡ ✐♥❛❝t✐✈❡ ❳ ❝❤r♦♠♦s♦♠❡ ♦❢ ❢❡♠❛•❡ ♠❛♠♠❛•s✳ ◆❛t✉r❡✱ ✸✾✸✭✻✻✽✺✮✿✺✾✾✕✻✵✶✳ ❈s❛♥❦♦✈s③❦✐✱ •✳✱ ◆❛❣②✱ ❆✳✱ ❛♥❞ ❏❛❡♥✐s❝❤✱ |✳ ✭✷✵✵✶✮✳ ❙②♥❡r❣✐s♠ ♦❢ ❳✐st |◆❆✱ ❉◆❆ ♠❡t❤②•❛t✐♦♥✱ ❛♥❞ ❤✐st♦♥❡ ❤②♣♦❛❝❡t②•❛t✐♦♥ ✐♥ ♠❛✐♥t❛✐♥✐♥❣ ❳ ❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥✳ ❏♦✉r♥❛• ♦❢ ❈❡•• ❇✐♦•♦❣②✱ ✶✺✸✭✹✮✿✼✼✸✕✼✽✸✳ ❉❛ |♦❝❤❛✱ ❙✳ ❚✳ ❛♥❞ ❍❡❛r❞✱ ❊✳ ✭✷✵✶✼✮✳ ◆♦✈❡• ♣•❛②❡rs ✐♥ ❳ ✐♥❛❝t✐✈❛t✐♦♥✿ ■♥s✐❣❤ts ✐♥t♦ ❳✐st✲♠❡❞✐❛t❡❞ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❛♥❞ ❝❤r♦♠♦s♦♠❡ ❝♦♥❢♦r♠❛t✐♦♥✳ ❞❛ |♦❝❤❛✱ ❙✳✱ ❇♦❡✈❛✱ ❱✳✱ ❊s❝❛♠✐••❛✲❉❡•✲❆r❡♥❛•✱ ▼✳✱ ❆♥❝❡•✐♥✱ ❑✳✱ •r❛♥✐❡r✱ ❈✳✱ ▼❛t✐❛s✱ ◆✳✱ ❙❛♥✉••✐✱ ❙✳✱ ❈❤♦✇✱ ❏✳✱ ❙❝❤✉•③✱ ❊✳✱ P✐❝❛r❞✱ ❈✳✱ ❑❛♥❡❦♦✱ ❙✳✱ ❍❡•✐♥✱ ❑✳✱ |❡✐♥❜❡r❣✱ ❉✳✱ ❙t❡✇❛rt✱ ❆✳✱ ❲✉t③✱ ❆✳✱ ▼❛r❣✉❡r♦♥✱ |✳✱ ❛♥❞ ❍❡❛r❞✱ ❊✳ ✭✷✵✶✹✮✳ ❏❛r✐❞✷ ■s ■♠♣•✐❝❛t❡❞ ✐♥ t❤❡ ■♥✐t✐❛• ❳✐st✲■♥❞✉❝❡❞ ❚❛r❣❡t✐♥❣ ♦❢ P|❈✷ t♦ t❤❡ ■♥❛❝t✐✈❡ ❳ ❈❤r♦♠♦s♦♠❡✳ ▼♦•❡❝✉•❛r ❈❡••✱ ✺✸✭✷✮✿✸✵✶✕✸✶✻✳ ❉❛rr♦✇✱ ❊✳ ▼✳✱ ❍✉♥t•❡②✱ ▼✳ ❍✳✱ ❉✉❞❝❤❡♥❦♦✱ ❖✳✱ ❙t❛♠❡♥♦✈❛✱ ❊✳ ❑✳✱ ❉✉r❛♥❞✱ ◆✳ ❈✳✱ ❙✉♥✱ )✳✱ ❍✉❛♥❣✱ ❙✳ ❈✳✱ ❙❛♥❜♦r♥✱ ❆✳ ▲✳✱ ▼❛❝❤♦•✱ ■✳✱ ❙❤❛♠✐♠✱ ▼✳✱ ❙❡❜❡r❣✱ ❆✳ P✳✱ ▲❛♥❞❡r✱ ❊✳ ❙✳✱ ❈❤❛❞✇✐❝❦✱ ❇✳ P✳✱ ❛♥❞ ❆✐❞❡♥✱ ❊✳ ▲✳ ✭✷✵✶✻✮✳ ❉❡•❡t✐♦♥ ♦❢ ❉❳)✹ ♦♥ t❤❡ ❤✉♠❛♥ ✐♥❛❝t✐✈❡ ❳ ❝❤r♦♠♦s♦♠❡ ❛•t❡rs ❤✐❣❤❡r✲♦r❞❡r ❣❡♥♦♠❡ ❛r❝❤✐t❡❝t✉r❡✳ Pr♦❝❡❡❞✐♥❣s ♦❢ t❤❡ ◆❛t✐♦♥❛• ❆❝❛❞❡♠② ♦❢ ❙❝✐❡♥❝❡s ♦❢ t❤❡ ❯♥✐t❡❞ ❙t❛t❡s ♦❢ ❆♠❡r✐❝❛✱ ✶✶✸✭✸✶✮✿❊✹✺✵✹✕❊✹✺✶✷✳ ❉❛✈✐❞✱ ❈✳ ❏✳✱ ❇♦②♥❡✱ ❆✳ |✳✱ ▼✐••❤♦✉s❡✱ ❙✳ |✳✱ ❛♥❞ ▼❛♥•❡②✱ ❏✳ ▲✳ ✭✷✵✶✶✮✳ ❚❤❡ |◆❆ ♣♦•②♠❡r❛s❡ ■■ ❈✲t❡r♠✐♥❛• ❞♦♠❛✐♥ ♣r♦♠♦t❡s s♣•✐❝✐♥❣ ❛❝t✐✈❛t✐♦♥ t❤r♦✉❣❤ r❡❝r✉✐t♠❡♥t ♦❢ ❛ ❯✷❆❋✻✺✲Pr♣✶✾ ❝♦♠♣•❡①✳ •❡♥❡s ❛♥❞ ❉❡✈❡•♦♣♠❡♥t✱ ✷✺✭✾✮✿✾✼✷✕✾✽✷✳ ❉❛✈✐❞♦✈✐❝❤✱ ❈✳✱ ❲❛♥❣✱ ❳✳✱ ❈✐❢✉❡♥t❡s✲|♦•❛s✱ ❈✳✱ •♦♦❞r✐❝❤✱ ❑✳ ❏✳✱ •♦♦❞✐♥❣✱ ❆✳ |✳✱ ▲❡❡✱ ❏✳ ❚✳✱ ❛♥❞ ❈❡❝❤✱ ❚✳ |✳ ✭✷✵✶✺✮✳ ❚♦✇❛r❞ ❛ ❝♦♥s❡♥s✉s ♦♥ t❤❡ ❜✐♥❞✐♥❣ s♣❡❝✐✜❝✐t② ❛♥❞ ♣r♦♠✐s❝✉✐t② ♦❢ P|❈✷ ❢♦r |◆❆✳ ▼♦•❡❝✉•❛r ❈❡••✱ ✺✼✭✸✮✿✺✺✷✕✺✺✽✳ ❉❛✈✐❞♦✈✐❝❤✱ ❈✳✱ )❤❡♥❣✱ ▲✳✱ •♦♦❞r✐❝❤✱ ❑✳ ❏✳✱ ❛♥❞ ❈❡❝❤✱ ❚✳ |✳ ✭✷✵✶✸✮✳ Pr♦♠✐s❝✉♦✉s |◆❆ ❜✐♥❞✐♥❣ ❜② P♦•②❝♦♠❜ r❡♣r❡ss✐✈❡ ❝♦♠♣•❡① ✷✳ ◆❛t✉r❡ ❙tr✉❝t✉r❛• ❛♥❞ ▼♦•❡❝✉•❛r ❇✐♦•♦❣②✱ ✷✵✭✶✶✮✿✶✷✺✵✕✶✷✺✼✳ ✶✾✹ |❡❢❡r❡♥❝❡s |❊❋❊|❊◆❈❊❙ ❞❡ ◆❛♣♦•❡s✱ ▼✳✱ ▼❡r♠♦✉❞✱ ❏✳ ❊✳✱ ❲❛❦❛♦✱ |✳✱ ❚❛♥❣✱ (✳ ❆✳✱ ❊♥❞♦❤✱ ▼✳✱ ❆♣♣❛♥❛❤✱ |✳✱ ◆❡st❡r♦✈❛✱ ❚✳ ❇✳✱ ❙✐•✈❛✱ ❏✳✱ ❖tt❡✱ ❆✳ P✳✱ ❱✐❞❛•✱ ▼✳✱ ❑♦s❡❦✐✱ ❍✳✱ ❛♥❞ ❇r♦❝❦❞♦r✛✱ ◆✳ ✭✷✵✵✹✮✳ P♦•②❝♦♠❜ ❣r♦✉♣ ♣r♦t❡✐♥s r✐♥❣✶❆✴❇ •✐♥❦ ✉❜✐q✉✐t②•❛t✐♦♥ ♦❢ ❤✐st♦♥❡ ❍✷❆ t♦ ❤❡r✐t❛❜•❡ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❛♥❞ ❳ ✐♥❛❝t✐✈❛t✐♦♥✳ ❉❡✈❡•♦♣♠❡♥t❛• ❈❡••✱ ✼✭✺✮✿✻✻✸✕✻✼✻✳ ❉❡ ❱r✐❡s✱ ❲✳ ◆✳✱ ❇✐♥♥s✱ ▲✳ ❚✳✱ ❋❛♥❝❤❡r✱ ❑✳ ❙✳✱ ❉❡❛♥✱ ❏✳✱ ▼♦♦r❡✱ |✳✱ ❑❡♠•❡r✱ |✳✱ ❛♥❞ ❑♥♦✇•❡s✱ ❇✳ ❇✳ ✭✷✵✵✵✮✳ ❊①♣r❡ss✐♦♥ ♦❢ ❈r❡ r❡❝♦♠❜✐♥❛s❡ ✐♥ ♠♦✉s❡ ♦♦❝②t❡s✿ ❆ ♠❡❛♥s t♦ st✉❞② ♠❛t❡r♥❛• ❡✛❡❝t ❣❡♥❡s✳ •❡♥❡s✐s✱ ✷✻✭✷✮✿✶✶✵✕✶✶✷✳ ❉❡♥❣✱ ❳✳✱ ❍✐❛tt✱ ❏✳ ❇✳✱ ◆❣✉②❡♥✱ ❉✳ ❑✳✱ ❊r❝❛♥✱ ❙✳✱ ❙t✉r❣✐••✱ ❉✳✱ ❍✐••✐❡r✱ ▲✳ ❲✳✱ ❙❝❤•❡s✐♥❣❡r✱ ❋✳✱ ❉❛✈✐s✱ ❈✳ ❆✳✱ |❡✐♥❦❡✱ ❱✳ ❏✳✱ •✐♥❣❡r❛s✱ ❚✳ |✳✱ ❙❤❡♥❞✉r❡✱ ❏✳✱ ❲❛t❡rst♦♥✱ |✳ ❍✳✱ ❖•✐✈❡r✱ ❇✳✱ ▲✐❡❜✱ ❏✳ ❉✳✱ ❛♥❞ ❉✐st❡❝❤❡✱ ❈✳ ▼✳ ✭✷✵✶✶✮✳ ❊✈✐❞❡♥❝❡ ❢♦r ❝♦♠♣❡♥s❛t♦r② ✉♣r❡❣✉•❛✲ t✐♦♥ ♦❢ ❡①♣r❡ss❡❞ ❳✲•✐♥❦❡❞ ❣❡♥❡s ✐♥ ♠❛♠♠❛•s✱ ❈❛❡♥♦r❤❛❜❞✐t✐s ❡•❡❣❛♥s ❛♥❞ ❉r♦s♦♣❤✐•❛ ♠❡•❛♥♦❣❛st❡r✳ ◆❛t✉r❡ •❡♥❡t✐❝s✱ ✹✸✭✶✷✮✿✶✶✼✾✕✶✶✽✺✳ ❉❡♥❣✱ ❳✳✱ ▼❛✱ ❲✳✱ |❛♠❛♥✐✱ ❱✳✱ ❍✐••✱ ❆✳✱ (❛♥❣✱ ❋✳✱ ❆②✱ ❋✳✱ ❇❡r•❡t❝❤✱ ❏✳ ❇✳✱ ❇•❛✉✱ ❈✳ ❆✳✱ ❙❤❡♥❞✉r❡✱ ❏✳✱ ❉✉❛♥✱ )✳✱ ◆♦❜•❡✱ ❲✳ ❙✳✱ ❛♥❞ ❉✐st❡❝❤❡✱ ❈✳ ▼✳ ✭✷✵✶✺✮✳ ❇✐♣❛rt✐t❡ str✉❝t✉r❡ ♦❢ t❤❡ ✐♥❛❝t✐✈❡ ♠♦✉s❡ ❳ ❝❤r♦♠♦s♦♠❡✳ •❡♥♦♠❡ ❇✐♦•♦❣②✱ ✶✻✭✶✮✿✶✺✷✳ ❉❤❛r♠❛s✐r✐✱ ◆✳✱ ❉❤❛r♠❛s✐r✐✱ ❙✳✱ ❛♥❞ ❊st❡••❡✱ ▼✳ ✭✷✵✵✺✮✳ ❚❤❡ ❋✲❜♦① ♣r♦t❡✐♥ ❚■|✶ ✐s ❛♥ ❛✉①✐♥ r❡❝❡♣t♦r✳ ◆❛t✉r❡✱ ✹✸✺✭✼✵✹✶✮✿✹✹✶✕✹✹✺✳ ❉❤✐♠❛♥✱ ❱✳ ❑✳✱ ❇♦•t✱ ▼✳ ❏✳✱ ❛♥❞ ❲❤✐t❡✱ ❑✳ P✳ ✭✷✵✶✽✮✳ ◆✉❝•❡❛r r❡❝❡♣t♦rs ✐♥ ❝❛♥❝❡r ✲ ❯♥❝♦✈❡r✐♥❣ ♥❡✇ ❛♥❞ ❡✈♦•✈✐♥❣ r♦•❡s t❤r♦✉❣❤ ❣❡♥♦♠✐❝ ❛♥❛•②s✐s✳ ◆❛t✉r❡ |❡✈✐❡✇s •❡♥❡t✐❝s✱ ✶✾✭✸✮✿✶✻✵✕✶✼✹✳ ❉✐ ❈r♦❝❡✱ ▲✳ ❛♥❞ ❍❡•✐♥✱ ❑✳ ✭✷✵✶✸✮✳ ❚r❛♥s❝r✐♣t✐♦♥❛• r❡❣✉•❛t✐♦♥ ❜② P♦•②❝♦♠❜ ❣r♦✉♣ ♣r♦t❡✐♥s✳ ❉✐st❡❝❤❡✱ ❈✳ ▼✳ ❛♥❞ ❇❡r•❡t❝❤✱ ❏✳ ❇✳ ✭✷✵✶✺✮✳ ❳✲❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥ ❛♥❞ ❡s❝❛♣❡✳ ❏♦✉r♥❛• ♦❢ •❡♥❡t✐❝s✱ ✾✹✭✹✮✿✺✾✶✕✺✾✾✳ ❉✐①♦♥✱ ❏✳ |✳✱ ❙❡•✈❛r❛•✱ ❙✳✱ (✉❡✱ ❋✳✱ ❑✐♠✱ ❆✳✱ ▲✐✱ (✳✱ ❙❤❡♥✱ (✳✱ ❍✉✱ ▼✳✱ ▲✐✉✱ ❏✳ ❙✳✱ ❛♥❞ |❡♥✱ ❇✳ ✭✷✵✶✷✮✳ ❚♦♣♦•♦❣✐❝❛• ❞♦♠❛✐♥s ✐♥ ♠❛♠♠❛•✐❛♥ ❣❡♥♦♠❡s ✐❞❡♥t✐✜❡❞ ❜② ❛♥❛•②s✐s ♦❢ ❝❤r♦♠❛t✐♥ ✐♥t❡r❛❝t✐♦♥s✳ ◆❛t✉r❡✱ ✹✽✺✭✼✸✾✽✮✿✸✼✻✕✸✽✵✳ ❉♦♥♦❤♦❡✱ ▼✳ ❊✳✱ ❙✐•✈❛✱ ❙✳ ❙✳✱ P✐♥t❡r✱ ❙✳ ❋✳✱ ❳✉✱ ◆✳✱ ❛♥❞ ▲❡❡✱ ❏✳ ❚✳ ✭✷✵✵✾✮✳ ❚❤❡ ♣•✉r✐♣♦t❡♥❝② ❢❛❝t♦r ❖❝t✹ ✐♥t❡r❛❝ts ✇✐t❤ ❈t❝❢ ❛♥❞ ❛•s♦ ❝♦♥tr♦•s ❳✲❝❤r♦♠♦s♦♠❡ ♣❛✐r✐♥❣ ❛♥❞ ❝♦✉♥t✐♥❣✳ ◆❛t✉r❡✱ ✹✻✵✭✼✷✺✶✮✿✶✷✽✕✶✸✷✳ ❉♦r♦q✉❡③✱ ❉✳ ❇✳✱ ❖rr✲❲❡❛✈❡r✱ ❚✳ ▲✳✱ ❛♥❞ |❡❜❛②✱ ■✳ ✭✷✵✵✼✮✳ ❙♣•✐t ❡♥❞s ❛♥t❛❣♦♥✐③❡s t❤❡ ◆♦t❝❤ ❛♥❞ ♣♦t❡♥t✐❛t❡s t❤❡ ❊•❋| s✐❣♥❛•✐♥❣ ♣❛t❤✇❛②s ❞✉r✐♥❣ ❉r♦s♦♣❤✐•❛ ❡②❡ ❞❡✈❡•♦♣✲ ♠❡♥t✳ ▼❡❝❤❛♥✐s♠s ♦❢ ❉❡✈❡•♦♣♠❡♥t✱ ✶✷✹✭✾✲✶✵✮✿✼✾✷✕✽✵✻✳ ✶✾✺ |❡❢❡r❡♥❝❡s |❊❋❊|❊◆❈❊❙ ❉♦ss✐♥✱ ❋✳✱ P✐♥❤❡✐r♦✱ ■✳✱ ➏②•✐❝③✱ ❏✳ ❏✳✱ |♦❡♥s❝❤✱ ❏✳✱ ❈♦••♦♠❜❡t✱ ❙✳✱ ▲❡ ❙❛✉①✱ ❆✳✱ ❈❤❡•♠✐❝❦✐✱ ❚✳✱ ❆tt✐❛✱ ▼✳✱ ❑❛♣♦♦r✱ ❱✳✱ )❤❛♥✱ (✳✱ ❉✐♥❣•✐✱ ❋✳✱ ▲♦❡✇✱ ❉✳✱ ▼❡r❝❤❡r✱ ❚✳✱ ❉❡❦❦❡r✱ ❏✳✱ ❛♥❞ ❍❡❛r❞✱ ❊✳ ✭✷✵✷✵✮✳ ❙P❊◆ ✐♥t❡❣r❛t❡s tr❛♥s❝r✐♣t✐♦♥❛• ❛♥❞ ❡♣✐❣❡♥❡t✐❝ ❝♦♥tr♦• ♦❢ ❳✲ ✐♥❛❝t✐✈❛t✐♦♥✳ ◆❛t✉r❡✱ ✺✼✽✭✼✼✾✺✮✿✹✺✺✕✹✻✵✳ ❉②❡r✱ ❑✳ ❆✳✱ ❈❛♥•✜❡•❞✱ ❚✳ ❑✳✱ ❛♥❞ •❛rt•❡r✱ ❙✳ ▼✳ ✭✶✾✽✾✮✳ ▼♦•❡❝✉•❛r ❝②t♦•♦❣✐❝❛• ❞✐✛❡r❡♥t✐✲ ❛t✐♦♥ ♦❢ ❛❝t✐✈❡ ❢r♦♠ ✐♥❛❝t✐✈❡ ① ❞♦♠❛✐♥s ✐♥ ✐♥t❡r♣❤❛s❡✿ ■♠♣•✐❝❛t✐♦♥s ❢♦r ① ❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥✳ ❈②t♦❣❡♥❡t✐❝ ❛♥❞ •❡♥♦♠❡ |❡s❡❛r❝❤✱ ✺✵✭✷✲✸✮✿✶✶✻✕✶✷✵✳ ❊❞❡•♠❛♥♥✱ ❏✳✱ ❍♦•③♠❛♥♥✱ ❑✳✱ ❚❛✉s❝❤✱ ❊✳✱ ❙❛✉♥❞❡rs♦♥✱ ❊✳ ❆✳✱ ❏❡❜❛r❛•✱ ❇✳ ▼✳✱ ❙t❡✐♥❜r❡❝❤❡r✱ ❉✳✱ ❉♦•♥✐❦✱ ❆✳✱ ❇•ätt❡✱ ❚✳ ❏✳✱ ▲❛♥❞❛✉✱ ❉✳ ❆✳✱ ❙❛✉❜✱ ❏✳✱ ❊st❡♥❢❡•❞❡r✱ ❙✳✱ ■❜❛❝❤✱ ❙✳✱ ❈②♠✲ ❜❛•✐st❛✱ ❋✳✱ ▲❡❜•♦♥❞✱ ❱✳✱ ❉❡•♠❡r✱ ❆✳✱ ❇❛❤•♦✱ ❏✳✱ |♦❜r❡❝❤t✱ ❙✳✱ ❋✐s❝❤❡r✱ ❑✳✱ •♦❡❞❡✱ ❱✳✱ ❇✉••✐♥❣❡r✱ ▲✳✱ ❲✉✱ ❈✳ ❏✳✱ ▼❡rt❡♥s✱ ❉✳✱ ❋✐❝③✱ •✳✱ •r✐❜❜❡♥✱ ❏✳ •✳✱ ❍❛••❡❦✱ ▼✳✱ ❉ö❤♥❡r✱ ❍✳✱ ❛♥❞ ❙t✐•❣❡♥❜❛✉❡r✱ ❙✳ ✭✷✵✷✵✮✳ •❡♥♦♠✐❝ ❛•t❡r❛t✐♦♥s ✐♥ ❤✐❣❤✲r✐s❦ ❝❤r♦♥✐❝ •②♠♣❤♦❝②t✐❝ •❡✉❦❡♠✐❛ ❢r❡q✉❡♥t•② ❛✛❡❝t ❝❡•• ❝②❝•❡ ❦❡② r❡❣✉•❛t♦rs ❛♥❞ ◆❖❚❈❍✶✲r❡❣✉•❛t❡❞ tr❛♥s❝r✐♣✲ t✐♦♥✳ ❍❛❡♠❛t♦•♦❣✐❝❛✱ ✶✵✺✭✺✮✿✶✸✼✾✕✶✸✾✵✳ ❊❣•♦✛✱ ❙✳✱ ❖✬|❡✐••②✱ ❉✳✱ ❈❤❛♣♠❛♥✱ |✳ ❉✳✱ ❚❛②•♦r✱ ❆✳✱ ❚❛♥③❤❛✉s✱ ❑✳✱ P✐tts✱ ▲✳✱ ❊✐❝❦✱ ❉✳✱ ❛♥❞ ▼✉r♣❤②✱ ❙✳ ✭✷✵✵✼✮✳ ❙❡r✐♥❡✲✼ ♦❢ t❤❡ |◆❆ ♣♦•②♠❡r❛s❡ ■■ ❈❚❉ ✐s s♣❡❝✐✜❝❛••② r❡q✉✐r❡❞ ❢♦r s♥|◆❆ ❣❡♥❡ ❡①♣r❡ss✐♦♥✳ ❙❝✐❡♥❝❡✱ ✸✶✽✭✺✽✺✼✮✿✶✼✼✼✕✶✼✼✾✳ ❊✐❝❦✱ ❉✳ ❛♥❞ •❡②❡r✱ ▼✳ ✭✷✵✶✸✮✳ ❚❤❡ |◆❆ ♣♦•②♠❡r❛s❡ ■■ ❝❛r❜♦①②✲t❡r♠✐♥❛• ❞♦♠❛✐♥ ✭❈❚❉✮ ❝♦❞❡✳ ❊✐•s✱ |✳✱ ❉✐❡t③❡•✱ ❙✳✱ ❇❡rt✐♥✱ ❊✳✱ ❙❝❤rö❝❦✱ ❊✳✱ ❙♣❡✐❝❤❡r✱ ▼✳ |✳✱ |✐❡❞✱ ❚✳✱ |♦❜❡rt✲◆✐❝♦✉❞✱ ▼✳✱ ❈r❡♠❡r✱ ❈✳✱ ❛♥❞ ❈r❡♠❡r✱ ❚✳ ✭✶✾✾✻✮✳ ❚❤r❡❡✲❞✐♠❡♥s✐♦♥❛• r❡❝♦♥str✉❝t✐♦♥ ♦❢ ♣❛✐♥t❡❞ ❤✉♠❛♥ ✐♥t❡r♣❤❛s❡ ❝❤r♦♠♦s♦♠❡s✿ ❆❝t✐✈❡ ❛♥❞ ✐♥❛❝t✐✈❡ ❳ ❝❤r♦♠♦s♦♠❡ t❡rr✐t♦r✐❡s ❤❛✈❡ s✐♠✐•❛r ✈♦•✉♠❡s ❜✉t ❞✐✛❡r ✐♥ s❤❛♣❡ ❛♥❞ s✉r❢❛❝❡ str✉❝t✉r❡✳ ❏♦✉r♥❛• ♦❢ ❈❡•• ❇✐♦•♦❣②✱ ✶✸✺✭✻✮✿✶✹✷✼✕✶✹✹✵✳ ❊♠s•❡②✱ P✳ ❛♥❞ ❈♦✇t❛♥✱ ❑✳ ✭✷✵✵✹✮✳ ❈♦♦t✿ ▼♦❞❡•✲❜✉✐•❞✐♥❣ t♦♦•s ❢♦r ♠♦•❡❝✉•❛r ❣r❛♣❤✐❝s✳ ❆❝t❛ ❈r②st❛••♦❣r❛♣❤✐❝❛ ❙❡❝t✐♦♥ ❉✿ ❇✐♦•♦❣✐❝❛• ❈r②st❛••♦❣r❛♣❤②✱ ✻✵✭✶✷ ■✮✿✷✶✷✻✕✷✶✸✷✳ ❊♥❣r❡✐t③✱ ❏✳ ▼✳✱ P❛♥❞②❛✲❏♦♥❡s✱ ❆✳✱ ▼❝❉♦♥❡•✱ P✳✱ ❙❤✐s❤❦✐♥✱ ❆✳✱ ❙✐r♦❦♠❛♥✱ ❑✳✱ ❙✉r❦❛✱ ❈✳✱ ❑❛❞r✐✱ ❙✳✱ ❳✐♥❣✱ ❏✳✱ •♦r❡♥✱ ❆✳✱ ▲❛♥❞❡r✱ ❊✳ ❙✳✱ P•❛t❤✱ ❑✳✱ ❛♥❞ •✉tt♠❛♥✱ ▼✳ ✭✷✵✶✸✮✳ ❚❤❡ ❳✐st •♥❝|◆❆ ❡①♣•♦✐ts t❤r❡❡✲❞✐♠❡♥s✐♦♥❛• ❣❡♥♦♠❡ ❛r❝❤✐t❡❝t✉r❡ t♦ s♣r❡❛❞ ❛❝r♦ss t❤❡ ❳ ❝❤r♦♠♦s♦♠❡✳ ❙❝✐❡♥❝❡ ✭◆❡✇ (♦r❦✱ ◆✳(✳✮✱ ✸✹✶✭✻✶✹✼✮✿✶✷✸✼✾✼✸✳ ❊s❝❛♠✐••❛✲❉❡•✲❆r❡♥❛•✱ ▼✳✱ ❉❛ |♦❝❤❛✱ ❙✳ ❚✳✱ ❛♥❞ ❍❡❛r❞✱ ❊✳ ✭✷✵✶✶✮✳ ❊✈♦•✉t✐♦♥❛r② ❞✐✈❡r✲ s✐t② ❛♥❞ ❞❡✈❡•♦♣♠❡♥t❛• r❡❣✉•❛t✐♦♥ ♦❢ ❳✲❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥✳ ❍✉♠❛♥ •❡♥❡t✐❝s✱ ✶✸✵✭✷✮✿✸✵✼✕✸✷✼✳ ✶✾✻ |❡❢❡r❡♥❝❡s |❊❋❊|❊◆❈❊❙ ❋❡♥❣✱ (✳✱ ❇♦♠♠❡r✱ •✳ ❚✳✱ )❤❛✐✱ (✳✱ ❆❦②♦•✱ ❆✳✱ ❍✐♥♦✐✱ ❚✳✱ ❲✐♥❡r✱ ■✳✱ ▲✐♥✱ ❍✳ ❱✳✱ ❈❛❞✐❣❛♥✱ ❑✳ ▼✳✱ ❈❤♦✱ ❑✳ |✳✱ ❛♥❞ ❋❡❛r♦♥✱ ❊✳ |✳ ✭✷✵✵✼✮✳ ❉r♦s♦♣❤✐•❛ s♣•✐t ❡♥❞s ❤♦♠♦•♦❣✉❡ ❙❍❆|P ❢✉♥❝t✐♦♥s ❛s ❛ ♣♦s✐t✐✈❡ r❡❣✉•❛t♦r ♦❢ ❲♥t✴β✲❝❛t❡♥✐♥✴❚✲❝❡•• ❢❛❝t♦r s✐❣♥❛•✐♥❣ ✐♥ ♥❡♦♣•❛st✐❝ tr❛♥s❢♦r♠❛t✐♦♥✳ ❈❛♥❝❡r |❡s❡❛r❝❤✱ ✻✼✭✷✮✿✹✽✷✕✹✾✶✳ ❋✉r•❛♥✱ •✳✱ •✉t✐❡rr❡③ ❍❡r♥❛♥❞❡③✱ ◆✳✱ ❍✉r❡t✱ ❈✳✱ •❛•✉♣❛✱ |✳✱ ✈❛♥ ❇❡♠♠❡•✱ ❏✳ •✳✱ |♦♠✐t♦✱ ❆✳✱ ❍❡❛r❞✱ ❊✳✱ ▼♦r❡②✱ ❈✳✱ ❛♥❞ |♦✉❣❡✉••❡✱ ❈✳ ✭✷✵✶✽✮✳ ❚❤❡ ❋t① ◆♦♥❝♦❞✐♥❣ ▲♦❝✉s ❈♦♥✲ tr♦•s ❳ ❈❤r♦♠♦s♦♠❡ ■♥❛❝t✐✈❛t✐♦♥ ■♥❞❡♣❡♥❞❡♥t•② ♦❢ ■ts |◆❆ Pr♦❞✉❝ts✳ ▼♦•❡❝✉•❛r ❈❡••✱ ✼✵✭✸✮✿✹✻✷✕✹✼✷✳❡✽✳ •❛•✉♣❛✱ |✳ ❛♥❞ ❍❡❛r❞✱ ❊✳ ✭✷✵✶✺✮✳ ❳✲❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥✿ ◆❡✇ ✐♥s✐❣❤ts ✐♥t♦ ❝✐s ❛♥❞ tr❛♥s r❡❣✉•❛t✐♦♥✳ ❈✉rr❡♥t ❖♣✐♥✐♦♥ ✐♥ •❡♥❡t✐❝s ❛♥❞ ❉❡✈❡•♦♣♠❡♥t✱ ✸✶✿✺✼✕✻✻✳ •✐❛✐♠♦✱ ❇✳ ❉✳✱ ❖s✇❛•❞✱ ❋✳✱ ❛♥❞ ❇♦r❣❣r❡❢❡✱ ❚✳ ✭✷✵✶✼✮✳ ❉②♥❛♠✐❝ ❝❤r♦♠❛t✐♥ r❡❣✉•❛t✐♦♥ ❛t ◆♦t❝❤ t❛r❣❡t ❣❡♥❡s✳ ❚r❛♥s❝r✐♣t✐♦♥✱ ✽✭✶✮✿✻✶✕✻✻✳ •✐♦r❣❡tt✐✱ ▲✳✱ ▲❛•♦✐❡✱ ❇✳ |✳✱ ❈❛rt❡r✱ ❆✳ ❈✳✱ ❆tt✐❛✱ ▼✳✱ )❤❛♥✱ (✳✱ ❳✉✱ ❏✳✱ ❈❤❡♥✱ ❈✳ ❏✳✱ ❑❛♣•❛♥✱ ◆✳✱ ❈❤❛♥❣✱ ❍✳ (✳✱ ❍❡❛r❞✱ ❊✳✱ ❛♥❞ ❉❡❦❦❡r✱ ❏✳ ✭✷✵✶✻✮✳ ❙tr✉❝t✉r❛• ♦r❣❛♥✐③❛t✐♦♥ ♦❢ t❤❡ ✐♥❛❝t✐✈❡ ❳ ❝❤r♦♠♦s♦♠❡ ✐♥ t❤❡ ♠♦✉s❡✳ ◆❛t✉r❡✱ ✺✸✺✭✼✻✶✸✮✿✺✼✺✕✺✼✾✳ •♦♥t❛♥✱ ❈✳✱ ❆❝❤❛♠❡✱ ❊✳ ▼✳✱ ❉❡♠♠❡rs✱ ❏✳✱ ❇❛r❛❦❛t✱ ❚✳ ❙✳✱ |❡♥t♠❡❡st❡r✱ ❊✳✱ ❱❛♥ ■•❝❦❡♥✱ ❲✳✱ •r♦♦t❡❣♦❡❞✱ ❏✳ ❆✳✱ ❛♥❞ •r✐❜♥❛✉✱ ❏✳ ✭✷✵✶✷✮✳ |◆❋✶✷ ✐♥✐t✐❛t❡s ❳✲❝❤r♦♠♦s♦♠❡ ✐♥❛❝✲ t✐✈❛t✐♦♥ ❜② t❛r❣❡t✐♥❣ |❊❳✶ ❢♦r ❞❡❣r❛❞❛t✐♦♥✳ ◆❛t✉r❡✱ ✹✽✺✭✼✸✾✽✮✿✸✽✻✕✸✾✵✳ •r❛✈❡s✱ ❏✳ ❆✳✱ ❑♦✐♥❛✱ ❊✳✱ ❛♥❞ ❙❛♥❦♦✈✐❝✱ ◆✳ ✭✷✵✵✻✮✳ ❍♦✇ t❤❡ ❣❡♥❡ ❝♦♥t❡♥t ♦❢ ❤✉♠❛♥ s❡① ❝❤r♦♠♦s♦♠❡s ❡✈♦•✈❡❞✳ ❈✉rr❡♥t ❖♣✐♥✐♦♥ ✐♥ •❡♥❡t✐❝s ❛♥❞ ❉❡✈❡•♦♣♠❡♥t✱ ✶✻✭✸✮✿✷✶✾✕✷✷✹✳ •r❛✈❡s✱ ❏✳ ❆✳ ▼✳ ✭✷✵✵✻✮✳ ❙❡① ❝❤r♦♠♦s♦♠❡ s♣❡❝✐❛•✐③❛t✐♦♥ ❛♥❞ ❞❡❣❡♥❡r❛t✐♦♥ ✐♥ ♠❛♠♠❛•s✳ ❈❡••✱ ✶✷✹✭✺✮✿✾✵✶✕✾✶✹✳ •r❛②✱ ❲✳ ▼✳✱ ❑❡♣✐♥s❦✐✱ ❙✳✱ |♦✉s❡✱ ❉✳✱ ▲❡②s❡r✱ ❖✳✱ ❛♥❞ ❊st❡••❡✱ ▼✳ ✭✷✵✵✶✮✳ ❆✉①✐♥ r❡❣✉•❛t❡s ❙❈❋✭❚■|✶✮✲❞❡♣❡♥❞❡♥t ❞❡❣r❛❞❛t✐♦♥ ♦❢ ❆❯❳✴■❆❆ ♣r♦t❡✐♥s✳ ◆❛t✉r❡✱ ✹✶✹✭✻✽✻✶✮✿✷✼✶✕✷✼✻✳ •r✐♠♠✱ ❏✳ ❇✳✱ ❊♥❣•✐s❤✱ ❇✳ P✳✱ ❈❤❡♥✱ ❏✳✱ ❙•❛✉❣❤t❡r✱ ❏✳ P✳✱ )❤❛♥❣✱ )✳✱ |❡✈②❛❦✐♥✱ ❆✳✱ P❛t❡•✱ |✳✱ ▼❛❝❦•✐♥✱ ❏✳ ❏✳✱ ◆♦r♠❛♥♥♦✱ ❉✳✱ ❙✐♥❣❡r✱ |✳ ❍✳✱ ▲✐♦♥♥❡t✱ ❚✳✱ ❛♥❞ ▲❛✈✐s✱ ▲✳ ❉✳ ✭✷✵✶✺✮✳ ❆ ❣❡♥❡r❛• ♠❡t❤♦❞ t♦ ✐♠♣r♦✈❡ ✢✉♦r♦♣❤♦r❡s ❢♦r •✐✈❡✲❝❡•• ❛♥❞ s✐♥❣•❡✲♠♦•❡❝✉•❡ ♠✐❝r♦s❝♦♣②✳ ◆❛t✉r❡ ▼❡t❤♦❞s✱ ✶✷✭✸✮✿✷✹✹✕✷✺✵✳ •✉❡♥t❤❡r✱ ▼✳ •✳✱ ❇❛r❛❦✱ ❖✳✱ ❛♥❞ ▲❛③❛r✱ ▼✳ ❆✳ ✭✷✵✵✶✮✳ ❚❤❡ ❙▼|❚ ❛♥❞ ◆✲❈♦| ❈♦r❡✲ ♣r❡ss♦rs ❆r❡ ❆❝t✐✈❛t✐♥❣ ❈♦❢❛❝t♦rs ❢♦r ❍✐st♦♥❡ ❉❡❛❝❡t②•❛s❡ ✸✳ ▼♦•❡❝✉•❛r ❛♥❞ ❈❡••✉•❛r ❇✐♦•♦❣②✱ ✷✶✭✶✽✮✿✻✵✾✶✕✻✶✵✶✳ •✉❡♥t❤❡r✱ ▼✳ •✳✱ (✉✱ ❏✳✱ ❑❛♦✱ •✳ ❉✳✱ (❡♥✱ ❚✳ ❏✳✱ ❛♥❞ ▲❛③❛r✱ ▼✳ ❆✳ ✭✷✵✵✷✮✳ ❆ss❡♠❜•② ♦❢ t❤❡ ❙▼|❚✲❤✐st♦♥❡ ❞❡❛❝❡t②•❛s❡ ✸ r❡♣r❡ss✐♦♥ ❝♦♠♣•❡① r❡q✉✐r❡s t❤❡ ❚❈P✲✶ r✐♥❣ ❝♦♠♣•❡①✳ •❡♥❡s ❛♥❞ ❉❡✈❡•♦♣♠❡♥t✱ ✶✻✭✷✹✮✿✸✶✸✵✕✸✶✸✺✳ ✶✾✼ |❡❢❡r❡♥❝❡s |❊❋❊|❊◆❈❊❙ ❍❛♥s❡♥✱ ❆✳ ❙✳✱ P✉st♦✈❛✱ ■✳✱ ❈❛tt♦❣•✐♦✱ ❈✳✱ ❚•✐❛♥✱ |✳✱ ❛♥❞ ❉❛r③❛❝q✱ ❳✳ ✭✷✵✶✼✮✳ ❈❚❈❋ ❛♥❞ ❝♦❤❡s✐♥ r❡❣✉•❛t❡ ❝❤r♦♠❛t✐♥ •♦♦♣ st❛❜✐•✐t② ✇✐t❤ ❞✐st✐♥❝t ❞②♥❛♠✐❝s✳ ❡▲✐❢❡✱ ✻✳ ❍❛s❡❣❛✇❛✱ (✳✱ ❇r♦❝❦❞♦r✛✱ ◆✳✱ ❑❛✇❛♥♦✱ ❙✳✱ ❚s✉t✉✐✱ ❑✳✱ ❚s✉t✉✐✱ ❑✳✱ ❛♥❞ ◆❛❦❛❣❛✇❛✱ ❙✳ ✭✷✵✶✵✮✳ ❚❤❡ ♠❛tr✐① ♣r♦t❡✐♥ ❤♥|◆P ❯ ✐s r❡q✉✐r❡❞ ❢♦r ❝❤r♦♠♦s♦♠❛• •♦❝❛•✐③❛t✐♦♥ ♦❢ ①✐st |◆❆✳ ❉❡✈❡•♦♣♠❡♥t❛• ❈❡••✱ ✶✾✭✸✮✿✹✻✾✕✹✼✻✳ ❍❛③❡❣❤✱ ❑✳ ❊✳✱ ◆❡♠❦♦✈✱ ❚✳✱ ❉✬❆•❡ss❛♥❞r♦✱ ❆✳✱ ❉✐••❡r✱ ❏✳ ❉✳✱ ▼♦♥❦s✱ ❏✳✱ ▼❝▼❛♥❛♠❛♥✱ ❏✳ ▲✳✱ ❏♦♥❡s✱ ❑✳ ▲✳✱ ❍❛♥s❡♥✱ ❑✳ ❈✳✱ ❛♥❞ |❡✐s✱ ❚✳ ✭✷✵✶✼✮✳ ❆♥ ❛✉t♦♥♦♠♦✉s ♠❡t❛❜♦•✐❝ r♦•❡ ❢♦r ❙♣❡♥✳ P▲♦❙ •❡♥❡t✐❝s✱ ✶✸✭✻✮✿❡✶✵✵✻✽✺✾✳ ❍❡❛r❞✱ ❊✳✱ |♦✉❣❡✉••❡✱ ❈✳✱ ❆r♥❛✉❞✱ ❉✳✱ ❆✈♥❡r✱ P✳✱ ❆••✐s✱ ❈✳ ❉✳✱ ❛♥❞ ❙♣❡❝t♦r✱ ❉✳ ▲✳ ✭✷✵✵✶✮✳ ▼❡t❤②•❛t✐♦♥ ♦❢ ❤✐st♦♥❡ ❍✸ ❛t ▲②s✲✾ ✐s ❛♥ ❡❛r•② ♠❛r❦ ♦♥ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡ ❞✉r✐♥❣ ❳ ✐♥❛❝t✐✈❛t✐♦♥✳ ❈❡••✱ ✶✵✼✭✻✮✿✼✷✼✕✸✽✳ ❍❡✐❞❡♠❛♥♥✱ ▼✳✱ ❍✐♥t❡r♠❛✐r✱ ❈✳✱ ❱♦ÿ✱ ❑✳✱ ❛♥❞ ❊✐❝❦✱ ❉✳ ✭✷✵✶✸✮✳ ❉②♥❛♠✐❝ ♣❤♦s♣❤♦r②•❛t✐♦♥ ♣❛tt❡r♥s ♦❢ |◆❆ ♣♦•②♠❡r❛s❡ ■■ ❈❚❉ ❞✉r✐♥❣ tr❛♥s❝r✐♣t✐♦♥✳ ❍♦r♥②✐❦✱ ❈✳✱ ❚❡r③✐✱ ▲✳ ❈✳✱ ❛♥❞ ❙✐♠♣s♦♥✱ •✳ •✳ ✭✷✵✶✵✮✳ ❚❤❡ ❙♣❡♥ ❋❛♠✐•② Pr♦t❡✐♥ ❋P❆ ❈♦♥tr♦•s ❆•t❡r♥❛t✐✈❡ ❈•❡❛✈❛❣❡ ❛♥❞ P♦•②❛❞❡♥②•❛t✐♦♥ ♦❢ |◆❆✳ ❉❡✈❡•♦♣♠❡♥t❛• ❈❡••✱ ✶✽✭✷✮✿✷✵✸✕✷✶✸✳ ❍♦s♦❣❛♥❡✱ ▼✳✱ ❋✉♥❛②❛♠❛✱ |✳✱ ❙❤✐r♦t❛✱ ▼✳✱ ❛♥❞ ◆❛❦❛②❛♠❛✱ ❑✳ ✭✷✵✶✻✮✳ ▲❛❝❦ ♦❢ ❚r❛♥s❝r✐♣✲ t✐♦♥ ❚r✐❣❣❡rs ❍✸❑✷✼♠❡✸ ❆❝❝✉♠✉•❛t✐♦♥ ✐♥ t❤❡ •❡♥❡ ❇♦❞②✳ ❈❡•• |❡♣♦rts✱ ✶✻✭✸✮✿✻✾✻✕✼✵✻✳ ❍s✐♥✱ ❏✳ P✳ ❛♥❞ ▼❛♥•❡②✱ ❏✳ ▲✳ ✭✷✵✶✷✮✳ ❚❤❡ |◆❆ ♣♦•②♠❡r❛s❡ ■■ ❈❚❉ ❝♦♦r❞✐♥❛t❡s tr❛♥✲ s❝r✐♣t✐♦♥ ❛♥❞ |◆❆ ♣r♦❝❡ss✐♥❣✳ ❍✉❛♥❣✱ ❊✳ (✳✱ )❤❛♥❣✱ ❏✳✱ ▼✐s❦❛✱ ❊✳ ❆✳✱ •✉❡♥t❤❡r✱ ▼✳ •✳✱ ❑♦✉③❛r✐❞❡s✱ ❚✳✱ ❛♥❞ ▲❛③❛r✱ ▼✳ ❆✳ ✭✷✵✵✵✮✳ ◆✉❝•❡❛r r❡❝❡♣t♦r ❝♦r❡♣r❡ss♦rs ♣❛rt♥❡r ✇✐t❤ ❝•❛ss ■■ ❤✐st♦♥❡ ❞❡❛❝❡t②•❛s❡s ✐♥ ❛ ❙✐♥✸✲✐♥❞❡♣❡♥❞❡♥t r❡♣r❡ss✐♦♥ ♣❛t❤✇❛②✳ •❡♥❡s ❛♥❞ ❉❡✈❡•♦♣♠❡♥t✱ ✶✹✭✶✮✿✹✺✕✺✹✳ ❍✉❛♥❣✱ (✳✱ ❑✐♠✱ ❏✳ ❑✳✱ ❉♦✱ ❉✳ ❱✳✱ ▲❡❡✱ ❈✳✱ P❡♥❢♦•❞✱ ❈✳ ❆✳✱ )②•✐❝③✱ ❏✳ ❏✳✱ ▼❛r✐♦♥✐✱ ❏✳ ❈✳✱ ❍❛❝❦❡tt✱ ❏✳ ❆✳✱ ❛♥❞ ❙✉r❛♥✐✱ ▼✳ ❆✳ ✭✷✵✶✼✮✳ ❙t❡••❛ ♠♦❞✉•❛t❡s tr❛♥s❝r✐♣t✐♦♥❛• ❛♥❞ ❡♥❞♦❣❡✲ ♥♦✉s r❡tr♦✈✐r✉s ♣r♦❣r❛♠s ❞✉r✐♥❣ ♠❛t❡r♥❛•✲t♦✲③②❣♦t✐❝ tr❛♥s✐t✐♦♥✳ ❡▲✐❢❡✱ ✻✳ ❏❛s♥♦✈✐❞♦✈❛✱ ❖✳ ❛♥❞ ❙t❡✢✱ |✳ ✭✷✵✶✸✮✳ ❚❤❡ ❈❚❉ ❝♦❞❡ ♦❢ |◆❆ ♣♦•②♠❡r❛s❡ ■■✿ ❆ str✉❝t✉r❛• ✈✐❡✇✳ ❏❡♦♥✱ (✳ ❛♥❞ ▲❡❡✱ ❏✳ ❚✳ ✭✷✵✶✶✮✳ ((✶ ❚❡t❤❡rs ❳✐st |◆❆ t♦ t❤❡ ✐♥❛❝t✐✈❡ ❳ ♥✉❝•❡❛t✐♦♥ ❝❡♥t❡r✳ ❈❡••✱ ✶✹✻✭✶✮✿✶✶✾✕✶✸✸✳ ❏❡♣♣❡s❡♥✱ P✳ ❛♥❞ ❚✉r♥❡r✱ ❇✳ ▼✳ ✭✶✾✾✸✮✳ ❚❤❡ ✐♥❛❝t✐✈❡ ❳ ❝❤r♦♠♦s♦♠❡ ✐♥ ❢❡♠❛•❡ ♠❛♠♠❛•s ✐s ❞✐st✐♥❣✉✐s❤❡❞ ❜② ❛ •❛❝❦ ♦❢ ❤✐st♦♥❡ ❍✹ ❛❝❡t②•❛t✐♦♥✱ ❛ ❝②t♦❣❡♥❡t✐❝ ♠❛r❦❡r ❢♦r ❣❡♥❡ ❡①♣r❡ss✐♦♥✳ ❈❡••✱ ✼✹✭✷✮✿✷✽✶✕✾✳ ✶✾✽ |❡❢❡r❡♥❝❡s |❊❋❊|❊◆❈❊❙ ❏❡r♦♥✐♠♦✱ ❈✳✱ ❇❛t❛✐••❡✱ ❆✳ |✳✱ ❛♥❞ |♦❜❡rt✱ ❋✳ ✭✷✵✶✸✮✳ ❚❤❡ ✇r✐t❡rs✱ r❡❛❞❡rs✱ ❛♥❞ ❢✉♥❝t✐♦♥s ♦❢ t❤❡ |◆❆ ♣♦•②♠❡r❛s❡ ■■ ❈✲t❡r♠✐♥❛• ❞♦♠❛✐♥ ❝♦❞❡✳ ❑❛❜s❝❤✱ ❲✳ ✭✷✵✶✵❛✮✳ ■♥t❡❣r❛t✐♦♥✱ s❝❛•✐♥❣✱ s♣❛❝❡✲❣r♦✉♣ ❛ss✐❣♥♠❡♥t ❛♥❞ ♣♦st✲r❡✜♥❡♠❡♥t✳ ❆❝t❛ ❈r②st❛••♦❣r❛♣❤✐❝❛ ❙❡❝t✐♦♥ ❉✿ ❇✐♦•♦❣✐❝❛• ❈r②st❛••♦❣r❛♣❤②✱ ✻✻✭✷✮✿✶✸✸✕✶✹✹✳ ❑❛❜s❝❤✱ ❲✳ ✭✷✵✶✵❜✮✳ ❳❉❙✳ ❆❝t❛ ❝r②st❛••♦❣r❛♣❤✐❝❛✳ ❙❡❝t✐♦♥ ❉✱ ❇✐♦•♦❣✐❝❛• ❝r②st❛••♦❣r❛♣❤②✱ ✻✻✭Pt ✷✮✿✶✷✺✕✸✷✳ ❑❛•❛♥tr②✱ ❙✳ ❛♥❞ ▼❛❣♥✉s♦♥✱ ❚✳ ✭✷✵✵✻✮✳ ❚❤❡ P♦•②❝♦♠❜ •r♦✉♣ Pr♦t❡✐♥ ❊❊❉ ■s ❉✐s♣❡♥s❛❜•❡ ❢♦r t❤❡ ■♥✐t✐❛t✐♦♥ ♦❢ |❛♥❞♦♠ ❳✲❈❤r♦♠♦s♦♠❡ ■♥❛❝t✐✈❛t✐♦♥✳ P▲♦❙ •❡♥❡t✐❝s✱ ✷✭✺✮✿❡✻✻✳ ❑❛•❜✱ |✳✱ ▲❛t✇✐❡•✱ ❙✳✱ ❇❛②♠❛③✱ ❍✳ ■✳✱ ❏❛♥s❡♥✱ P✳ ❲✳✱ ▼ü••❡r✱ ❈✳ ❲✳✱ ❱❡r♠❡✉•❡♥✱ ▼✳✱ ❛♥❞ ▼ü••❡r✱ ❏✳ ✭✷✵✶✹✮✳ ❍✐st♦♥❡ ❍✷❆ ♠♦♥♦✉❜✐q✉✐t✐♥❛t✐♦♥ ♣r♦♠♦t❡s ❤✐st♦♥❡ ❍✸ ♠❡t❤②•❛t✐♦♥ ✐♥ P♦•②❝♦♠❜ r❡♣r❡ss✐♦♥✳ ◆❛t✉r❡ ❙tr✉❝t✉r❛• ❛♥❞ ▼♦•❡❝✉•❛r ❇✐♦•♦❣②✱ ✷✶✭✻✮✿✺✻✾✕✺✼✶✳ ❑❛♥❡❦♦✱ ❙✳✱ ❙♦♥✱ ❏✳✱ ❇♦♥❛s✐♦✱ |✳✱ ❙❤❡♥✱ ❙✳ ❙✳✱ ❛♥❞ |❡✐♥❜❡r❣✱ ❉✳ ✭✷✵✶✹✮✳ ◆❛s❝❡♥t |◆❆ ✐♥✲ t❡r❛❝t✐♦♥ ❦❡❡♣s P|❈✷ ❛❝t✐✈✐t② ♣♦✐s❡❞ ❛♥❞ ✐♥ ❝❤❡❝❦✳ •❡♥❡s ✫ ❉❡✈❡•♦♣♠❡♥t✱ ✷✽✭✶✽✮✿✶✾✽✸✕ ✶✾✽✽✳ ❑❛♥♥♦✱ ❚✳✱ ❇✉❝❤❡r✱ ❊✳✱ ❉❛①✐♥❣❡r✱ ▲✳✱ ❍✉❡tt❡•✱ ❇✳✱ ❇ö❤♠❞♦r❢❡r✱ •✳✱ •r❡❣♦r✱ ❲✳✱ ❑r❡✐•✱ ❉✳ P✳✱ ▼❛t③❦❡✱ ▼✳✱ ❛♥❞ ▼❛t③❦❡✱ ❆✳ ❏✳ ✭✷✵✵✽✮✳ ❆ str✉❝t✉r❛•✲♠❛✐♥t❡♥❛♥❝❡✲♦❢✲❝❤r♦♠♦s♦♠❡s ❤✐♥❣❡ ❞♦♠❛✐♥✲❝♦♥t❛✐♥✐♥❣ ♣r♦t❡✐♥ ✐s r❡q✉✐r❡❞ ❢♦r |◆❆✲❞✐r❡❝t❡❞ ❉◆❆ ♠❡t❤②•❛t✐♦♥✳ ◆❛✲ t✉r❡ •❡♥❡t✐❝s✱ ✹✵✭✺✮✿✻✼✵✕✻✼✺✳ ❑❛♦✱ ❍✳ (✳✱ ❉♦✇♥❡s✱ ▼✳✱ ❖r❞❡♥t•✐❝❤✱ P✳✱ ❛♥❞ ❊✈❛♥s✱ |✳ ▼✳ ✭✷✵✵✵✮✳ ■s♦•❛t✐♦♥ ♦❢ ❛ ♥♦✈❡• ❤✐s✲ t♦♥❡ ❞❡❛❝❡t②•❛s❡ r❡✈❡❛•s t❤❛t ❝•❛ss ■ ❛♥❞ ❝•❛ss ■■ ❞❡❛❝❡t②•❛s❡s ♣r♦♠♦t❡ ❙▼|❚✲♠❡❞✐❛t❡❞ r❡♣r❡ss✐♦♥✳ •❡♥❡s ❛♥❞ ❉❡✈❡•♦♣♠❡♥t✱ ✶✹✭✶✮✿✺✺✕✻✻✳ ❑❡••❡②✱ ▲✳ ❆✳✱ ▼❡③✉•✐s✱ ❙✳✱ (❛t❡s✱ ❈✳ ▼✳✱ ❲❛ss✱ ▼✳ ◆✳✱ ❛♥❞ ❙t❡r♥❜❡r❣✱ ▼✳ ❏✳ ✭✷✵✶✺✮✳ ❚❤❡ P❤②r❡✷ ✇❡❜ ♣♦rt❛• ❢♦r ♣r♦t❡✐♥ ♠♦❞❡•✐♥❣✱ ♣r❡❞✐❝t✐♦♥ ❛♥❞ ❛♥❛•②s✐s✳ ◆❛t✉r❡ Pr♦t♦❝♦•s✱ ✶✵✭✻✮✿✽✹✺✕✽✺✽✳ ❑❡♠♣❢❡r✱ |✳ ❛♥❞ P♦♠❜♦✱ ❆✳ ✭✷✵✶✾✮✳ ▼❡t❤♦❞s ❢♦r ♠❛♣♣✐♥❣ ✸❉ ❝❤r♦♠♦s♦♠❡ ❛r❝❤✐t❡❝t✉r❡✳ ◆❛t✉r❡ |❡✈✐❡✇s •❡♥❡t✐❝s✱ ✷✶✭✹✮✿✷✵✼✕✷✷✻✳ ❑❡♥✐r②✱ ❆✳✱ •❡❛r✐♥❣✱ ▲✳ ❏✳✱ ❏❛♥s③✱ ◆✳✱ ▲✐✉✱ ❏✳✱ ❍♦•✐❦✱ ❆✳ )✳✱ ❍✐❝❦❡②✱ P✳ ❋✳✱ ❑✐♥❦❡•✱ ❙✳ ❆✳✱ ▼♦♦r❡✱ ❉✳ ▲✳✱ ❇r❡s•✐♥✱ ❑✳✱ ❈❤❡♥✱ ❑✳✱ ▲✐✉✱ |✳✱ P❤✐••✐♣s✱ ❈✳✱ P❛❦✉s❝❤✱ ▼✳✱ ❇✐❜❡♥✱ ❈✳✱ ❙❤❡r✐❞❛♥✱ ❏✳ ▼✳✱ ❑✐•❡✱ ❇✳ ❚✳✱ ❈❛r♠✐❝❤❛❡•✱ ❈✳✱ |✐t❝❤✐❡✱ ▼✳ ❊✳✱ ❍✐•t♦♥✱ ❉✳ ❏✳✱ ❛♥❞ ❇•❡✇✐tt✱ ▼✳ ❊✳ ✭✷✵✶✻✮✳ ❙❡t❞❜✶✲♠❡❞✐❛t❡❞ ❍✸❑✾ ♠❡t❤②•❛t✐♦♥ ✐s ❡♥r✐❝❤❡❞ ♦♥ t❤❡ ✐♥❛❝t✐✈❡ ❳ ❛♥❞ ♣•❛②s ❛ r♦•❡ ✐♥ ✐ts ❡♣✐❣❡♥❡t✐❝ s✐•❡♥❝✐♥❣✳ ❊♣✐❣❡♥❡t✐❝s ❛♥❞ ❈❤r♦♠❛t✐♥✱ ✾✭✶✮✿✶✻✳ ❑✐♠✱ ❏✳✱ ❍❛❦❡✱ ❙✳ ❇✳✱ ❛♥❞ |♦❡❞❡r✱ |✳ •✳ ✭✷✵✵✺✮✳ ❚❤❡ ❤✉♠❛♥ ❤♦♠♦•♦❣ ♦❢ ②❡❛st ❇|❊✶ ❢✉♥❝✲ t✐♦♥s ❛s ❛ tr❛♥s❝r✐♣t✐♦♥❛• ❝♦❛❝t✐✈❛t♦r t❤r♦✉❣❤ ❞✐r❡❝t ❛❝t✐✈❛t♦r ✐♥t❡r❛❝t✐♦♥s✳ ▼♦•❡❝✉•❛r ❈❡••✱ ✷✵✭✺✮✿✼✺✾✕✼✼✵✳ ✶✾✾ |❡❢❡r❡♥❝❡s |❊❋❊|❊◆❈❊❙ ❑✐③❡r✱ ❑✳ ❖✳✱ P❤❛t♥❛♥✐✱ ❍✳ P✳✱ ❙❤✐❜❛t❛✱ (✳✱ ❍❛••✱ ❍✳✱ •r❡❡♥•❡❛❢✱ ❆✳ ▲✳✱ ❛♥❞ ❙tr❛❤•✱ ❇✳ ❉✳ ✭✷✵✵✺✮✳ ❆ ◆♦✈❡• ❉♦♠❛✐♥ ✐♥ ❙❡t✷ ▼❡❞✐❛t❡s |◆❆ P♦•②♠❡r❛s❡ ■■ ■♥t❡r❛❝t✐♦♥ ❛♥❞ ❈♦✉✲ ♣•❡s ❍✐st♦♥❡ ❍✸ ❑✸✻ ▼❡t❤②•❛t✐♦♥ ✇✐t❤ ❚r❛♥s❝r✐♣t ❊•♦♥❣❛t✐♦♥✳ ▼♦•❡❝✉•❛r ❛♥❞ ❈❡••✉•❛r ❇✐♦•♦❣②✱ ✷✺✭✽✮✿✸✸✵✺✕✸✸✶✻✳ ❑•♦❡t✱ ❙✳ ▲✳✱ ❇❛②♠❛③✱ ❍✳ ■✳✱ ▼❛❦♦✇s❦✐✱ ▼✳✱ •r♦❡♥❡✇♦•❞✱ ❱✳✱ ❏❛♥s❡♥✱ P✳ ❲✳✱ ❇❡r❡♥❞s❡♥✱ ▼✳✱ ◆✐❛③✐✱ ❍✳✱ ❑♦♣s✱ •✳ ❏✳✱ ❛♥❞ ❱❡r♠❡✉•❡♥✱ ▼✳ ✭✷✵✶✺✮✳ ❚♦✇❛r❞s ❡•✉❝✐❞❛t✐♥❣ t❤❡ st❛❜✐•✐t②✱ ❞②♥❛♠✐❝s ❛♥❞ ❛r❝❤✐t❡❝t✉r❡ ♦❢ t❤❡ ♥✉❝•❡♦s♦♠❡ r❡♠♦❞❡•✐♥❣ ❛♥❞ ❞❡❛❝❡t②•❛s❡ ❝♦♠♣•❡① ❜② ✉s✐♥❣ q✉❛♥t✐t❛t✐✈❡ ✐♥t❡r❛❝t✐♦♥ ♣r♦t❡♦♠✐❝s✳ ❋❊❇❙ ❏♦✉r♥❛•✱ ✷✽✷✭✾✮✿✶✼✼✹✕✶✼✽✺✳ ❑♥✉❝❦•❡s✱ P✳✱ ▲❡♥❝❡✱ ❚✳✱ ❍❛✉ss♠❛♥♥✱ ■✳ ❯✳✱ ❏❛❝♦❜✱ ❉✳✱ ❑r❡✐♠✱ ◆✳✱ ❈❛r•✱ ❙✳ ❍✳✱ ▼❛s✐❡••♦✱ ■✳✱ ❍❛r❡s✱ ❚✳✱ ❱✐••❛s❡ñ♦r✱ |✳✱ ❍❡ss✱ ❉✳✱ ❆♥❞r❛❞❡✲◆❛✈❛rr♦✱ ▼✳ ❆✳✱ ❇✐❣❣✐♦❣❡r❛✱ ▼✳✱ ❍❡•♠✱ ▼✳✱ ❙♦••❡r✱ ▼✳✱ ❇ü❤•❡r✱ ▼✳✱ ❛♥❞ |♦✐❣♥❛♥t✱ ❏✳ (✳ ✭✷✵✶✽✮✳ )❝✸❤✶✸✴❋•❛❝❝ ✐s r❡q✉✐r❡❞ ❢♦r ❛❞❡♥♦s✐♥❡ ♠❡t❤②•❛t✐♦♥ ❜② ❜r✐❞❣✐♥❣ t❤❡ ♠|◆❆✲❜✐♥❞✐♥❣ ❢❛❝t♦r |❜▼✶✺✴s♣❡♥✐t♦ t♦ t❤❡ ♠✻ ❛ ♠❛❝❤✐♥❡r② ❝♦♠♣♦♥❡♥t ❲t❛♣✴❋•✭✷✮❞✳ •❡♥❡s ❛♥❞ ❉❡✈❡•♦♣♠❡♥t✱ ✸✷✭✺✲✻✮✿✹✶✺✕✹✷✾✳ ❑♦❤•♠❛✐❡r✱ ❆✳✱ ❙❛✈❛r❡s❡✱ ❋✳✱ ▲❛❝❤♥❡r✱ ▼✳✱ ▼❛rt❡♥s✱ ❏✳✱ ❏❡♥✉✇❡✐♥✱ ❚✳✱ ❛♥❞ ❲✉t③✱ ❆✳ ✭✷✵✵✹✮✳ ❆ ❈❤r♦♠♦s♦♠❛• ▼❡♠♦r② ❚r✐❣❣❡r❡❞ ❜② ❳✐st |❡❣✉•❛t❡s ❍✐st♦♥❡ ▼❡t❤②•❛t✐♦♥ ✐♥ ❳ ■♥❛❝t✐✈❛t✐♦♥✳ P▲♦❙ ❇✐♦•♦❣②✱ ✷✭✼✮✿❡✶✼✶✳ ❑♦•♦❞③✐❡•✱ P✳ ❆✳✱ (❡❤ ❏❛♥✱ ▲✳✱ ❛♥❞ ◆✉♥❣ ❏❛♥✱ (✳ ✭✶✾✾✺✮✳ ▼✉t❛t✐♦♥s t❤❛t ❛✛❡❝t t❤❡ •❡♥❣t❤✱ ❢❛s❝✐❝✉•❛t✐♦♥✱ ♦r ✈❡♥tr❛• ♦r✐❡♥t❛t✐♦♥ ♦❢ s♣❡❝✐✜❝ s❡♥s♦r② ❛①♦♥s ✐♥ t❤❡ ❉r♦s♦♣❤✐•❛ ❡♠❜r②♦✳ ◆❡✉r♦♥✱ ✶✺✭✷✮✿✷✼✸✕✷✽✻✳ ❑♦♠❛r♥✐ts❦②✱ P✳✱ ❈❤♦✱ ❊✳ ❏✳✱ ❛♥❞ ❇✉r❛t♦✇s❦✐✱ ❙✳ ✭✷✵✵✵✮✳ ❉✐✛❡r❡♥t ♣❤♦s♣❤♦r②•❛t❡❞ ❢♦r♠s ♦❢ |◆❆ ♣♦•②♠❡r❛s❡ ■■ ❛♥❞ ❛ss♦❝✐❛t❡❞ ♠|◆❆ ♣r♦❝❡ss✐♥❣ ❢❛❝t♦rs ❞✉r✐♥❣ tr❛♥s❝r✐♣t✐♦♥✳ •❡♥❡s ❛♥❞ ❉❡✈❡•♦♣♠❡♥t✱ ✶✹✭✶✾✮✿✷✹✺✷✕✷✹✻✵✳ ❑♦✉③❛r✐❞❡s✱ ❚✳ ✭✷✵✵✼✮✳ ❈❤r♦♠❛t✐♥ ▼♦❞✐✜❝❛t✐♦♥s ❛♥❞ ❚❤❡✐r ❋✉♥❝t✐♦♥✳ ❑r♦❣❛♥✱ ◆✳ ❏✳✱ ❉♦✈❡r✱ ❏✳✱ ❲♦♦❞✱ ❆✳✱ ❙❝❤♥❡✐❞❡r✱ ❏✳✱ ❍❡✐❞t✱ ❏✳✱ ❇♦❛t❡♥❣✱ ▼✳ ❆✳✱ ❉❡❛♥✱ ❑✳✱ |②❛♥✱ ❖✳ ❲✳✱ •♦•s❤❛♥✐✱ ❆✳✱ ❏♦❤♥st♦♥✱ ▼✳✱ •r❡❡♥❜•❛tt✱ ❏✳ ❋✳✱ ❛♥❞ ❙❤✐•❛t✐❢❛r❞✱ ❆✳ ✭✷✵✵✸✮✳ ❚❤❡ P❛❢✶ ❝♦♠♣•❡① ✐s r❡q✉✐r❡❞ ❢♦r ❤✐st♦♥❡ ❍✸ ♠❡t❤②•❛t✐♦♥ ❜② ❈❖▼P❆❙❙ ❛♥❞ ❉♦t✶♣✿ ▲✐♥❦✐♥❣ tr❛♥s❝r✐♣t✐♦♥❛• ❡•♦♥❣❛t✐♦♥ t♦ ❤✐st♦♥❡ ♠❡t❤②•❛t✐♦♥✳ ▼♦•❡❝✉•❛r ❈❡••✱ ✶✶✭✸✮✿✼✷✶✕ ✼✷✾✳ ❑✉❛♥❣✱ ❇✳✱ ❲✉✱ ❙✳ ❈✳✱ ❙❤✐♥✱ (✳✱ ▲✉♦✱ ▲✳✱ ❛♥❞ ❑♦•♦❞③✐❡•✱ P✳ ✭✷✵✵✵✮✳ s♣•✐t ❡♥❞s ❡♥❝♦❞❡s •❛r❣❡ ♥✉❝•❡❛r ♣r♦t❡✐♥s t❤❛t r❡❣✉•❛t❡ ♥❡✉r♦♥❛• ❝❡•• ❢❛t❡ ❛♥❞ ❛①♦♥ ❡①t❡♥s✐♦♥ ✐♥ t❤❡ ❉r♦s♦♣❤✐•❛ ❡♠❜r②♦✳ ❉❡✈❡•♦♣♠❡♥t ✭❈❛♠❜r✐❞❣❡✱ ❊♥❣•❛♥❞✮✱ ✶✷✼✭✼✮✿✶✺✶✼✕✷✾✳ ❑✉r♦❞❛✱ ❑✳✱ ❍❛♥✱ ❍✳✱ ❚❛♥✐✱ ❙✳✱ ❚❛♥✐❣❛❦✐✱ ❑✳✱ ❚✉♥✱ ❚✳✱ ❋✉r✉❦❛✇❛✱ ❚✳✱ ❚❛♥✐❣✉❝❤✐✱ (✳✱ ❑✉r♦♦❦❛✱ ❍✳✱ ❍❛♠❛❞❛✱ (✳✱ ❚♦②♦❦✉♥✐✱ ❙✳✱ ❛♥❞ ❍♦♥•♦✱ ❚✳ ✭✷✵✵✸✮✳ |❡❣✉•❛t✐♦♥ ♦❢ ♠❛r❣✐♥❛• ③♦♥❡ ❇ ❝❡•• ❞❡✈❡•♦♣♠❡♥t ❜② ▼■◆❚✱ ❛ s✉♣♣r❡ss♦r ♦❢ ◆♦t❝❤✴|❇P✲❏ s✐❣♥❛•✐♥❣ ♣❛t❤✇❛②✳ ■♠♠✉♥✐t②✱ ✶✽✭✷✮✿✸✵✶✕✸✶✷✳ ✷✵✵ |❡❢❡r❡♥❝❡s |❊❋❊|❊◆❈❊❙ ▲❛✐✱ ❆✳ (✳ ❛♥❞ ❲❛❞❡✱ P✳ ❆✳ ✭✷✵✶✶✮✳ ❈❛♥❝❡r ❜✐♦•♦❣② ❛♥❞ ◆✉|❉✿ ❆ ♠✉•t✐❢❛❝❡t❡❞ ❝❤r♦♠❛t✐♥ r❡♠♦❞❡••✐♥❣ ❝♦♠♣•❡①✳ ▲❛③❛r✱ ▼✳ ❆✳ ✭✷✵✵✸✮✳ ◆✉❝•❡❛r r❡❝❡♣t♦r ❝♦r❡♣r❡ss♦rs✳ ◆✉❝•❡❛r |❡❝❡♣t♦r ❙✐❣♥❛•✐♥❣✱ ✶✭✶✮✿♥rs✳✵✶✵✵✶✳ ▲❡❡✱ ❏✳ ❚✳ ✭✷✵✶✷✮✳ ❊♣✐❣❡♥❡t✐❝ r❡❣✉•❛t✐♦♥ ❜② •♦♥❣ ♥♦♥❝♦❞✐♥❣ |◆❆s✳ ❙❝✐❡♥❝❡✱ ✸✸✽✭✻✶✶✸✮✿✶✹✸✺✕✶✹✸✾✳ ▲❡❡✱ ❏✳ ❚✳ ❛♥❞ ❇❛rt♦•♦♠❡✐✱ ▼✳ ❙✳ ✭✷✵✶✸✮✳ ❳✲✐♥❛❝t✐✈❛t✐♦♥✱ ✐♠♣r✐♥t✐♥❣✱ ❛♥❞ •♦♥❣ ♥♦♥❝♦❞✐♥❣ |◆❆s ✐♥ ❤❡❛•t❤ ❛♥❞ ❞✐s❡❛s❡✳ ❈❡••✱ ✶✺✷✭✻✮✿✶✸✵✽✕✶✸✷✸✳ ▲❡❡✱ ❏✳ ❚✳✱ ❉❛✈✐❞♦✇✱ ▲✳ ❙✳✱ ❛♥❞ ❲❛rs❤❛✇s❦②✱ ❉✳ ✭✶✾✾✾✮✳ ❚s✐①✱ ❛ ❣❡♥❡ ❛♥t✐s❡♥s❡ t♦ ❳✐st ❛t t❤❡ ❳✲✐♥❛❝t✐✈❛t✐♦♥ ❝❡♥tr❡✳ ◆❛t✉r❡ •❡♥❡t✐❝s✱ ✷✶✭✹✮✿✹✵✵✕✹✵✹✳ ▲é❣❛ré✱ ❙✳✱ ❈❛✈❛••♦♥❡✱ ▲✳✱ ▼❛♠♦✱ ❆✳✱ ❈❤❛❜♦t✱ ❈✳✱ ❙✐r♦✐s✱ ■✳✱ ▼❛❣•✐♦❝❝♦✱ ❆✳✱ ❑•✐♠♦✇✐❝③✱ ❆✳✱ ❚♦♥✐♥✱ P✳ ◆✳✱ ❇✉❝❤❛♥❛♥✱ ▼✳✱ ❑❡✐•t②✱ ❉✳✱ ❍❛ss❛♥✱ ❙✳✱ ▲❛♣❡rr✐èr❡✱ ❉✳✱ ▼❛❞❡r✱ ❙✳✱ ❆•❡②♥✐❦♦✈❛✱ ❖✳✱ ❛♥❞ ❇❛s✐❦✱ ▼✳ ✭✷✵✶✺✮✳ ❚❤❡ ❡str♦❣❡♥ r❡❝❡♣t♦r ❝♦❢❛❝t♦r ❙P❊◆ ❢✉♥❝t✐♦♥s ❛s ❛ t✉♠♦r s✉♣♣r❡ss♦r ❛♥❞ ❝❛♥❞✐❞❛t❡ ❜✐♦♠❛r❦❡r ♦❢ ❞r✉❣ r❡s♣♦♥s✐✈❡♥❡ss ✐♥ ❤♦r♠♦♥❡✲ ❞❡♣❡♥❞❡♥t ❜r❡❛st ❝❛♥❝❡rs✳ ❈❛♥❝❡r |❡s❡❛r❝❤✱ ✼✺✭✷✵✮✿✹✸✺✶✕✹✸✻✸✳ ▲é❣❛ré✱ ❙✳✱ ❈❤❛❜♦t✱ ❈✳✱ ❛♥❞ ❇❛s✐❦✱ ▼✳ ✭✷✵✶✼✮✳ ❙P❊◆✱ ❛ ♥❡✇ ♣•❛②❡r ✐♥ ♣r✐♠❛r② ❝✐•✐❛ ❢♦r♠❛t✐♦♥ ❛♥❞ ❝❡•• ♠✐❣r❛t✐♦♥ ✐♥ ❜r❡❛st ❝❛♥❝❡r✳ ❇r❡❛st ❈❛♥❝❡r |❡s❡❛r❝❤✱ ✶✾✭✶✮✿✶✵✹✳ ▲✐✱ ❳✳ ❛♥❞ ❋✉✱ ❳✳ ❉✳ ✭✷✵✶✾✮✳ ❈❤r♦♠❛t✐♥✲❛ss♦❝✐❛t❡❞ |◆❆s ❛s ❢❛❝✐•✐t❛t♦rs ♦❢ ❢✉♥❝t✐♦♥❛• ❣❡♥♦♠✐❝ ✐♥t❡r❛❝t✐♦♥s✳ ▲✐♥✱ ❍✳✱ •✉♣t❛✱ ❱✳✱ ❱❡r♠✐•②❡❛✱ ▼✳ ❉✳✱ ❋❛•❝✐❛♥✐✱ ❋✳✱ ▲❡❡✱ ❏✳ ❚✳✱ ❖✬◆❡✐••✱ ▲✳ P✳✱ ❛♥❞ ❚✉r♥❡r✱ ❇✳ ▼✳ ✭✷✵✵✼✮✳ ❉♦s❛❣❡ ❝♦♠♣❡♥s❛t✐♦♥ ✐♥ t❤❡ ♠♦✉s❡ ❜❛•❛♥❝❡s ✉♣✲r❡❣✉•❛t✐♦♥ ❛♥❞ s✐•❡♥❝✐♥❣ ♦❢ ❳✲•✐♥❦❡❞ ❣❡♥❡s✳ P▲♦❙ ❇✐♦•♦❣②✱ ✺✭✶✷✮✿✷✽✵✾✕✷✽✷✵✳ ▲✐♥✱ ❍✳ ❱✳✱ ❉♦r♦q✉❡③✱ ❉✳ ❇✳✱ ❈❤♦✱ ❙✳✱ ❈❤❡♥✱ ❋✳✱ |❡❜❛②✱ ■✳✱ ❛♥❞ ❈❛❞✐❣❛♥✱ ❑✳ ▼✳ ✭✷✵✵✸✮✳ ❙♣•✐ts ❡♥❞s ✐s ❛ t✐ss✉❡✴♣r♦♠♦t❡r s♣❡❝✐✜❝ r❡❣✉•❛t♦r ♦❢ ❲✐♥❣•❡ss s✐❣♥❛•✐♥❣✳ ❉❡✈❡•♦♣♠❡♥t✱ ✶✸✵✭✶✹✮✿✸✶✷✺✕✸✶✸✺✳ ▲✐✉✱ ❇✳✱ ▼✐t❛♥✐✱ (✳✱ |❛♦✱ ❳✳✱ )❛❢❡r❡♦✱ ▼✳✱ )❤❛♥❣✱ ❏✳✱ )❤❛♥❣✱ ❏✳✱ ❋✉tr❡❛•✱ P✳ ❆✳✱ ▲♦③❛♥♦✱ •✳✱ ❛♥❞ ❊•✲◆❛❣❣❛r✱ ❆✳ ❑✳ ✭✷✵✶✼✮✳ ❙♣❛t✐♦✲❚❡♠♣♦r❛• •❡♥♦♠✐❝ ❍❡t❡r♦❣❡♥❡✐t②✱ P❤②•♦❣❡♥②✱ ❛♥❞ ▼❡t❛st❛t✐❝ ❊✈♦•✉t✐♦♥ ✐♥ ❙❛•✐✈❛r② ❆❞❡♥♦✐❞ ❈②st✐❝ ❈❛r❝✐♥♦♠❛✳ ❏♦✉r♥❛• ♦❢ t❤❡ ◆❛t✐♦♥❛• ❈❛♥❝❡r ■♥st✐t✉t❡✱ ✶✵✾✭✶✵✮✳ ▲♦❞❛✱ ❆✳✱ ❇r❛♥❞s♠❛✱ ❏✳ ❍✳✱ ❱❛ss✐•❡✈✱ ■✳✱ ❙❡r✈❛♥t✱ ◆✳✱ ▲♦♦s✱ ❋✳✱ ❆♠✐r♥❛sr✱ ❆✳✱ ❙♣•✐♥t❡r✱ ❊✳✱ ❇❛r✐••♦t✱ ❊✳✱ P♦♦t✱ |✳ ❆✳✱ ❍❡❛r❞✱ ❊✳✱ ❛♥❞ •r✐❜♥❛✉✱ ❏✳ ✭✷✵✶✼✮✳ •❡♥❡t✐❝ ❛♥❞ ❡♣✐❣❡♥❡t✐❝ ❢❡❛t✉r❡s ❞✐r❡❝t ❞✐✛❡r❡♥t✐❛• ❡✣❝✐❡♥❝② ♦❢ ❳✐st✲♠❡❞✐❛t❡❞ s✐•❡♥❝✐♥❣ ❛t ❳✲❝❤r♦♠♦s♦♠❛• ❛♥❞ ❛✉t♦s♦♠❛• •♦❝❛t✐♦♥s✳ ◆❛t✉r❡ ❈♦♠♠✉♥✐❝❛t✐♦♥s✱ ✽✭✶✮✳ ✷✵✶ |❡❢❡r❡♥❝❡s |❊❋❊|❊◆❈❊❙ ▲♦❞❛✱ ❆✳ ❛♥❞ ❍❡❛r❞✱ ❊✳ ✭✷✵✶✾✮✳ ❳✐st |◆❆ ✐♥ ❛❝t✐♦♥✿ P❛st✱ ♣r❡s❡♥t✱ ❛♥❞ ❢✉t✉r❡✳ ▲♦s✱ •✳ ❱✳✱ ❊♥❝❡••✱ ▲✳ P✳✱ ▼❝❉♦✉❣❛••✱ ▼✳ •✳✱ ❍❛rt③❡••✱ ❉✳ ❉✳✱ ❑❛r❛ss✐♥❛✱ ◆✳✱ )✐♠♣r✐❝❤✱ ❈✳✱ ❲♦♦❞✱ ▼✳ •✳✱ ▲❡❛r✐s❤✱ |✳✱ ❖❤❛♥❛✱ |✳ ❋✳✱ ❯r❤✱ ▼✳✱ ❙✐♠♣s♦♥✱ ❉✳✱ ▼❡♥❞❡③✱ ❏✳✱ )✐♠♠❡r♠❛♥✱ ❑✳✱ ❖tt♦✱ P✳✱ ❱✐❞✉❣✐r✐s✱ •✳✱ )❤✉✱ ❏✳✱ ❉❛r③✐♥s✱ ❆✳✱ ❑•❛✉❜❡rt✱ ❉✳ ❍✳✱ ❇✉••❡✐t✱ |✳ ❋✳✱ ❛♥❞ ❲♦♦❞✱ ❑✳ ❱✳ ✭✷✵✵✽✮✳ ❍❛•♦❚❛❣✿ ❆ ◆♦✈❡• Pr♦t❡✐♥ ▲❛❜❡•✐♥❣ ❚❡❝❤♥♦•♦❣② ❢♦r ❈❡•• ■♠❛❣✐♥❣ ❛♥❞ Pr♦t❡✐♥ ❆♥❛•②s✐s✳ ❆❈❙ ❈❤❡♠✐❝❛• ❇✐♦•♦❣②✱ ✸✭✻✮✿✸✼✸✕✸✽✷✳ ▲✉✱ ❏✳✱ ❏❡♦♥❣✱ ❍✳✲❲✳✱ ❑♦♥❣✱ ◆✳✱ (❛♥❣✱ (✳✱ ❈❛rr♦••✱ ❏✳✱ ▲✉♦✱ ❍✳ |✳✱ ❙✐•❜❡rst❡✐♥✱ ▲✳ ❊✳✱ (✉♣♦▼❛✱ ❛♥❞ ❈❤❛✐✱ ▲✳ ✭✷✵✵✾✮✳ ❈♦rr❡❝t✐♦♥✿ ❙t❡♠ ❈❡•• ❋❛❝t♦r ❙❆▲▲✹ |❡♣r❡ss❡s t❤❡ ❚r❛♥s❝r✐♣t✐♦♥s ♦❢ P❚❊◆ ❛♥❞ ❙❆▲▲✶ t❤r♦✉❣❤ ❛♥ ❊♣✐❣❡♥❡t✐❝ |❡♣r❡ss♦r ❈♦♠♣•❡①✳ P▲♦❙ ❖◆❊✱ ✹✭✼✮✳ ▲✉✱ )✳✱ )❤❛♥❣✱ ◗✳ ❈✳✱ ▲❡❡✱ ❇✳✱ ❋•②♥♥✱ |✳ ❆✳✱ ❙♠✐t❤✱ ▼✳ ❆✳✱ |♦❜✐♥s♦♥✱ ❏✳ ❚✳✱ ❉❛✈✐❞♦✈✐❝❤✱ ❈✳✱ •♦♦❞✐♥❣✱ ❆✳ |✳✱ •♦♦❞r✐❝❤✱ ❑✳ ❏✳✱ ▼❛tt✐❝❦✱ ❏✳ ❙✳✱ ▼❡s✐r♦✈✱ ❏✳ P✳✱ ❈❡❝❤✱ ❚✳ |✳✱ ❛♥❞ ❈❤❛♥❣✱ ❍✳ (✳ ✭✷✵✶✻✮✳ |◆❆ ❉✉♣•❡① ▼❛♣ ✐♥ ▲✐✈✐♥❣ ❈❡••s |❡✈❡❛•s ❍✐❣❤❡r✲❖r❞❡r ❚r❛♥s❝r✐♣t♦♠❡ ❙tr✉❝t✉r❡✳ ❈❡••✱ ✶✻✺✭✺✮✿✶✷✻✼✕✶✷✼✾✳ ▲✉❝❝❤❡s✐✱ ❏✳ ❈✳✱ ❑❡••②✱ ❲✳ •✳✱ ❛♥❞ P❛♥♥✐♥❣✱ ❇✳ ✭✷✵✵✺✮✳ ❈❤r♦♠❛t✐♥ |❡♠♦❞❡•✐♥❣ ✐♥ ❉♦s❛❣❡ ❈♦♠♣❡♥s❛t✐♦♥✳ ❆♥♥✉❛• |❡✈✐❡✇ ♦❢ •❡♥❡t✐❝s✱ ✸✾✭✶✮✿✻✶✺✕✻✺✶✳ ▲✉❞❡✇✐❣✱ ❆✳ ❍✳✱ ❑♦❜❡r✲❊✐s❡r♠❛♥♥✱ ❈✳✱ ❲❡✐t③❡•✱ ❈✳✱ ❇❡t❤❦❡✱ ❆✳✱ ◆❡✉❜❡rt✱ ❑✳✱ •❡r✐s❝❤✱ ❇✳✱ ❍✉tt❡r✱ ❍✳✱ ❛♥❞ ❆♥t❡❜✐✱ ❆✳ ✭✷✵✵✹✮✳ ❆ ♥♦✈❡• ♥✉❝•❡❛r r❡❝❡♣t♦r✴❝♦r❡❣✉•❛t♦r ❝♦♠✲ ♣•❡① ❝♦♥tr♦•s ❈✳ ❡•❡❣❛♥s •✐♣✐❞ ♠❡t❛❜♦•✐s♠✱ •❛r✈❛• ❞❡✈❡•♦♣♠❡♥t✱ ❛♥❞ ❛❣✐♥❣✳ •❡♥❡s ❛♥❞ ❉❡✈❡•♦♣♠❡♥t✱ ✶✽✭✶✼✮✿✷✶✷✵✕✷✶✸✸✳ ▲✉✐❦❡♥❤✉✐s✱ ❙✳✱ ❲✉t③✱ ❆✳✱ ❛♥❞ ❏❛❡♥✐s❝❤✱ |✳ ✭✷✵✵✶✮✳ ❆♥t✐s❡♥s❡ ❚r❛♥s❝r✐♣t✐♦♥ t❤r♦✉❣❤ t❤❡ ❳✐st ▲♦❝✉s ▼❡❞✐❛t❡s ❚s✐① ❋✉♥❝t✐♦♥ ✐♥ ❊♠❜r②♦♥✐❝ ❙t❡♠ ❈❡••s✳ ▼♦•❡❝✉•❛r ❛♥❞ ❈❡••✉•❛r ❇✐♦•♦❣②✱ ✷✶✭✷✹✮✿✽✺✶✷✕✽✺✷✵✳ ▲✉♥❞❡✱ ❇✳ ▼✳✱ |❡✐❝❤♦✇✱ ❙✳ ▲✳✱ ❑✐♠✱ ▼✳✱ ❙✉❤✱ ❍✳✱ ▲❡❡♣❡r✱ ❚✳ ❈✳✱ (❛♥❣✱ ❋✳✱ ▼✉ts❝❤•❡r✱ ❍✳✱ ❇✉r❛t♦✇s❦✐✱ ❙✳✱ ▼❡✐♥❤❛rt✱ ❆✳✱ ❛♥❞ ❱❛r❛♥✐✱ •✳ ✭✷✵✶✵✮✳ ❈♦♦♣❡r❛t✐✈❡ ✐♥t❡r❛❝t✐♦♥ ♦❢ tr❛♥s❝r✐♣t✐♦♥ t❡r♠✐♥❛t✐♦♥ ❢❛❝t♦rs ✇✐t❤ t❤❡ |◆❆ ♣♦•②♠❡r❛s❡ ■■ ❈✲t❡r♠✐♥❛• ❞♦♠❛✐♥✳ ◆❛t✉r❡ ❙tr✉❝t✉r❛• ❛♥❞ ▼♦•❡❝✉•❛r ❇✐♦•♦❣②✱ ✶✼✭✶✵✮✿✶✶✾✺✕✶✷✵✶✳ ▲②♦♥✱ ▼✳ ❋✳ ✭✶✾✻✶✮✳ •❡♥❡ ❛❝t✐♦♥ ✐♥ t❤❡ ❳✲❝❤r♦♠♦s♦♠❡ ♦❢ t❤❡ ♠♦✉s❡ ✭♠✉s ♠✉s❝✉•✉s ▲✳✮✳ ◆❛t✉r❡✱ ✶✾✵✭✹✼✼✸✮✿✸✼✷✕✸✼✸✳ ▲②♦♥✱ ▼✳ ❋✳ ✭✶✾✻✷✮✳ ❙❡① ❝❤r♦♠❛t✐♥ ❛♥❞ ❣❡♥❡ ❛❝t✐♦♥ ✐♥ t❤❡ ♠❛♠♠❛•✐❛♥ ❳✲❝❤r♦♠♦s♦♠❡✳ ❆♠❡r✐❝❛♥ •♦✉r♥❛• ♦❢ ❤✉♠❛♥ ❣❡♥❡t✐❝s✱ ✶✹✿✶✸✺✕✶✹✽✳ ▼❛✱ ❍✳✱ ❙♦♥❣✱ ❇✳✱ •✉♦✱ ❙✳✱ ▲✐✱ •✳✱ ❛♥❞ ❏✐♥✱ •✳ ✭✷✵✷✵✮✳ ■❞❡♥t✐✜❝❛t✐♦♥ ♦❢ ❣❡r♠•✐♥❡ ❛♥❞ s♦✲ ♠❛t✐❝ ♠✉t❛t✐♦♥s ✐♥ ♣❛♥❝r❡❛t✐❝ ❛❞❡♥♦sq✉❛♠♦✉s ❝❛r❝✐♥♦♠❛ ✉s✐♥❣ ✇❤♦•❡ ❡①♦♠❡ s❡q✉❡♥❝✲ ✐♥❣✳ ❈❛♥❝❡r ❇✐♦♠❛r❦❡rs✱ ✷✼✭✸✮✿✸✽✾✕✸✾✼✳ ✷✵✷ |❡❢❡r❡♥❝❡s |❊❋❊|❊◆❈❊❙ ▼❛❝❡✱ ❑✳ ❛♥❞ ❚✉❣♦r❡s✱ ❆✳ ✭✷✵✵✹✮✳ ❚❤❡ ♣r♦❞✉❝t ♦❢ t❤❡ s♣•✐t ❡♥❞s ❣❡♥❡ ✐s r❡q✉✐r❡❞ ❢♦r t❤❡ ♠❛✐♥t❡♥❛♥❝❡ ♦❢ ♣♦s✐t✐♦♥❛• ✐♥❢♦r♠❛t✐♦♥ ❞✉r✐♥❣ ❉r♦s♦♣❤✐•❛ ❞❡✈❡•♦♣♠❡♥t✳ ❇▼❈ ❉❡✈❡•♦♣✲ ♠❡♥t❛• ❇✐♦•♦❣②✱ ✹✿✶✺✳ ▼❛❦✱ ❲✳✱ ❇❛①t❡r✱ ❏✳✱ ❙✐•✈❛✱ ❏✳✱ ◆❡✇❛••✱ ❆✳ ❊✳✱ ❛♥❞ ❖tt❡✱ ❆✳ P✳ ✭✷✵✵✷✮✳ ▼✐t♦t✐❝❛••② st❛❜•❡ ❛ss♦❝✐❛t✐♦♥ ♦❢ ♣♦•②❝♦♠❜ ❣r♦✉♣ ♣r♦t❡✐♥s ❊❡❞ ❛♥❞ ❊♥①✶ ✇✐t❤ t❤❡ ✐♥❛❝t✐✈❡ ❳ ❝❤r♦♠♦s♦♠❡ ✐♥ tr♦♣❤♦❜•❛st st❡♠ ❝❡••s✳ ❈✉rr❡♥t ❇✐♦•♦❣②✱ ✶✷✭✶✷✮✿✶✵✶✻✕✶✵✷✵✳ ▼❛❦✱ ❲✳✱ ◆❡st❡r♦✈❛✱ ❚✳ ❇✳✱ ❉❡ ◆❛♣♦•❡s✱ ▼✳✱ ❆♣♣❛♥❛❤✱ |✳✱ (❛♠❛♥❛❦❛✱ ❙✳✱ ❖tt❡✱ ❆✳ P✳✱ ❛♥❞ ❇r♦❝❦❞♦r✛✱ ◆✳ ✭✷✵✵✹✮✳ |❡❛❝t✐✈❛t✐♦♥ ♦❢ t❤❡ P❛t❡r♥❛• ❳ ❈❤r♦♠♦s♦♠❡ ✐♥ ❊❛r•② ▼♦✉s❡ ❊♠❜r②♦s✳ ❙❝✐❡♥❝❡✱ ✸✵✸✭✺✻✺✽✮✿✻✻✻✕✻✻✾✳ ▼❛r❛❤r❡♥s✱ (✳✱ P❛♥♥✐♥❣✱ ❇✳✱ ❉❛✉s♠❛♥✱ ❏✳✱ ❙tr❛✉ss✱ ❲✳✱ ❛♥❞ ❏❛❡♥✐s❝❤✱ |✳ ✭✶✾✾✼✮✳ ❳✐st✲ ❞❡✜❝✐❡♥t ♠✐❝❡ ❛r❡ ❞❡❢❡❝t✐✈❡ ✐♥ ❞♦s❛❣❡ ❝♦♠♣❡♥s❛t✐♦♥ ❜✉t ♥♦t s♣❡r♠❛t♦❣❡♥❡s✐s✳ •❡♥❡s ❛♥❞ ❉❡✈❡•♦♣♠❡♥t✱ ✶✶✭✷✮✿✶✺✻✕✶✻✻✳ ▼❛r❦❛❦✐✱ (✳✱ •❛♥ ❈❤♦♥❣✱ ❏✳✱ ▲✉♦♥❣✱ ❈✳✱ ❚❛♥✱ ❙✳ (✳✱ ❲❛♥❣✱ (✳✱ ❏❛❝♦❜s♦♥✱ ❊✳ ❈✳✱ ▼❛❡str✐♥✐✱ ❉✳✱ ❉r♦r✱ ■✳✱ ▼✐str②✱ ❇✳ ❆✳✱ ❙❝❤ö♥❡❜❡r❣✱ ❏✳✱ ❇❛♥❡r•❡❡✱ ❆✳✱ •✉tt♠❛♥✱ ▼✳✱ ❈❤♦✉✱ ❚✳✱ ❛♥❞ P•❛t❤✱ ❑✳ ✭✷✵✷✵✮✳ ❳✐st✲s❡❡❞❡❞ ♥✉❝•❡❛t✐♦♥ s✐t❡s ❢♦r♠ •♦❝❛• ❝♦♥❝❡♥tr❛t✐♦♥ ❣r❛❞✐❡♥ts ♦❢ s✐•❡♥❝✐♥❣ ♣r♦t❡✐♥s t♦ ✐♥❛❝t✐✈❛t❡ t❤❡ ❳✲❝❤r♦♠♦s♦♠❡ ❊q✉❛• ❝♦♥tr✐❜✉t✐♦♥✳ ❜✐♦|①✐✈✱ ♣❛❣❡ ✷✵✷✵✳✶✶✳✷✷✳✸✾✸✺✹✻✳ ▼❛r❦s✱ ❍✳✱ ❑❡rst❡♥s✱ ❍✳ ❍✳ ❉✳✱ ❇❛r❛❦❛t✱ ❚✳ ❙✳✱ ❙♣•✐♥t❡r✱ ❊✳✱ ❉✐r❦s✱ |✳ ❆✳ ▼✳✱ ✈❛♥ ▼✐❡r•♦✱ •✳✱ ❏♦s❤✐✱ ❖✳✱ ❲❛♥❣✱ ❙✳✲(✳✱ ❇❛❜❛❦✱ ❚✳✱ ❆•❜❡rs✱ ❈✳ ❆✳✱ ❑❛•❦❛♥✱ ❚✳✱ ❙♠✐t❤✱ ❆✳✱ ❏♦✉♥❡❛✉✱ ❆✳✱ ❞❡ ▲❛❛t✱ ❲✳✱ •r✐❜♥❛✉✱ ❏✳✱ ❛♥❞ ❙t✉♥♥❡♥❜❡r❣✱ ❍✳ •✳ ✭✷✵✶✺✮✳ ❉②♥❛♠✐❝s ♦❢ ❣❡♥❡ s✐•❡♥❝✲ ✐♥❣ ❞✉r✐♥❣ ❳ ✐♥❛❝t✐✈❛t✐♦♥ ✉s✐♥❣ ❛••❡•❡✲s♣❡❝✐✜❝ |◆❆✲s❡q✳ •❡♥♦♠❡ ❇✐♦•♦❣②✱ ✶✻✭✶✮✿✶✹✾✳ ▼❛rs❤❛•• •r❛✈❡s✱ ❏✳ ❆✳ ❛♥❞ ❙❤❡tt②✱ ❙✳ ✭✷✵✵✶✮✳ ❙❡① ❢r♦♠ ❲ t♦ )✿ ❊✈♦•✉t✐♦♥ ♦❢ ✈❡rt❡❜r❛t❡ s❡① ❝❤r♦♠♦s♦♠❡s ❛♥❞ s❡① ❞❡t❡r♠✐♥✐♥❣ ❣❡♥❡s✳ ■♥ ❏♦✉r♥❛• ♦❢ ❊①♣❡r✐♠❡♥t❛• )♦♦•♦❣②✱ ✈♦•✉♠❡ ✷✾✵✱ ♣❛❣❡s ✹✹✾✕✹✻✷✳ ▼❛s✉✐✱ ❖✳✱ ❍❡❛r❞✱ ❊✳✱ ❛♥❞ ❑♦s❡❦✐✱ ❍✳ ✭✷✵✶✽✮✳ ▲✐✈❡ ■♠❛❣✐♥❣ ♦❢ ❳✐st |◆❆✳ ♣❛❣❡s ✻✼✕✼✷✳ ▼❛✉r✉s✱ ❑✳✱ ❆♣♣❡♥③❡••❡r✱ ❙✳✱ |♦t❤✱ ❙✳✱ ❑✉♣❡r✱ ❏✳✱ |♦st✱ ❙✳✱ ▼❡✐❡r•♦❤❛♥♥✱ ❙✳✱ ❆r❛♠♣❛t③✐✱ P✳✱ •♦❡❜❡•❡r✱ ▼✳✱ |♦s❡♥✇❛•❞✱ ❆✳✱ •❡✐ss✐♥❣❡r✱ ❊✳✱ ❛♥❞ ❲♦❜s❡r✱ ▼✳ ✭✷✵✶✽✮✳ P❛♥❡• ❙❡✲ q✉❡♥❝✐♥❣ ❙❤♦✇s |❡❝✉rr❡♥t •❡♥❡t✐❝ ❋❆❙ ❆•t❡r❛t✐♦♥s ✐♥ Pr✐♠❛r② ❈✉t❛♥❡♦✉s ▼❛r❣✐♥❛• )♦♥❡ ▲②♠♣❤♦♠❛✳ ❏♦✉r♥❛• ♦❢ ■♥✈❡st✐❣❛t✐✈❡ ❉❡r♠❛t♦•♦❣②✱ ✶✸✽✭✼✮✿✶✺✼✸✕✶✺✽✶✳ ▼❝❈r❛❝❦❡♥✱ ❙✳✱ ❋♦♥❣✱ ◆✳✱ |♦s♦♥✐♥❛✱ ❊✳✱ (❛♥❦✉•♦✈✱ ❑✳✱ ❇r♦t❤❡rs✱ •✳✱ ❙✐❞❡r♦✈s❦✐✱ ❉✳✱ ❍❡ss❡•✱ ❆✳✱ ❋♦st❡r✱ ❙✳✱ ❙❤✉♠❛♥✱ ❙✳✱ ❛♥❞ ❇❡♥t•❡②✱ ❉✳ ▲✳ ✭✶✾✾✼✮✳ ✺✬✲❈❛♣♣✐♥❣ ❡♥③②♠❡s ❛r❡ t❛r❣❡t❡❞ t♦ ♣r❡✲♠|◆❆ ❜② ❜✐♥❞✐♥❣ t♦ t❤❡ ♣❤♦s♣❤♦r②•❛t❡❞ ❝❛r❜♦①②✲t❡r♠✐♥❛• ❞♦♠❛✐♥ ♦❢ |◆❆ ♣♦•②♠❡r❛s❡ ■■✳ •❡♥❡s ❛♥❞ ❉❡✈❡•♦♣♠❡♥t✱ ✶✶✭✷✹✮✿✸✸✵✻✕✸✸✶✽✳ ✷✵✸ |❡❢❡r❡♥❝❡s |❊❋❊|❊◆❈❊❙ ▼❝❍✉❣❤✱ ❈✳ ❆✳✱ ❈❤❡♥✱ ❈✳✲❑✳✱ ❈❤♦✇✱ ❆✳✱ ❙✉r❦❛✱ ❈✳ ❋✳✱ ❚r❛♥✱ ❈✳✱ ▼❝❉♦♥❡•✱ P✳✱ P❛♥❞②❛✲ ❏♦♥❡s✱ ❆✳✱ ❇•❛♥❝♦✱ ▼✳✱ ❇✉r❣❤❛r❞✱ ❈✳✱ ▼♦r❛❞✐❛♥✱ ❆✳✱ ❙✇❡r❡❞♦s❦✐✱ ▼✳ ❏✳✱ ❙❤✐s❤❦✐♥✱ ❆✳ ❆✳✱ ❙✉✱ ❏✳✱ ▲❛♥❞❡r✱ ❊✳ ❙✳✱ ❍❡ss✱ ❙✳✱ P•❛t❤✱ ❑✳✱ ❛♥❞ •✉tt♠❛♥✱ ▼✳ ✭✷✵✶✺✮✳ ❚❤❡ ❳✐st •♥❝|◆❆ ✐♥t❡r❛❝ts ❞✐r❡❝t•② ✇✐t❤ ❙❍❆|P t♦ s✐•❡♥❝❡ tr❛♥s❝r✐♣t✐♦♥ t❤r♦✉❣❤ ❍❉❆❈✸✳ ◆❛t✉r❡✱ ✺✷✶✭✼✺✺✶✮✿✷✸✷✕✷✸✻✳ ▼❡✐♥❤❛rt✱ ❆✳ ❛♥❞ ❈r❛♠❡r✱ P✳ ✭✷✵✵✹✮✳ |❡❝♦❣♥✐t✐♦♥ ♦❢ |◆❆ ♣♦•②♠❡r❛s❡ ■■ ❝❛r❜♦①②✲t❡r♠✐♥❛• ❞♦♠❛✐♥ ❜② ✸ ′ ✲|◆❆✲♣r♦❝❡ss✐♥❣ ❢❛❝t♦rs✳ ◆❛t✉r❡✱ ✹✸✵✭✻✾✾✻✮✿✷✷✸✕✷✷✻✳ ▼✐❣❡♦♥✱ ❇✳ |✳ ✭✷✵✶✻✮✳ ❆♥ ♦✈❡r✈✐❡✇ ♦❢ ❳ ✐♥❛❝t✐✈❛t✐♦♥ ❜❛s❡❞ ♦♥ s♣❡❝✐❡s ❞✐✛❡r❡♥❝❡s✳ ❙❡♠✐✲ ♥❛rs ✐♥ ❈❡•• ❛♥❞ ❉❡✈❡•♦♣♠❡♥t❛• ❇✐♦•♦❣②✱ ✺✻✿✶✶✶✕✶✶✻✳ ▼✐❦❛♠✐✱ ❙✳✱ ❑❛♥❛❜❛✱ ❚✳✱ ❚❛❦✐③❛✇❛✱ ◆✳✱ ❑♦❜❛②❛s❤✐✱ ❆✳✱ ▼❛❡s❛❦✐✱ |✳✱ ❋✉•✐✇❛r❛✱ ❚✳✱ ■t♦✱ (✳✱ ❛♥❞ ▼✐s❤✐♠❛✱ ▼✳ ✭✷✵✶✹✮✳ ❙tr✉❝t✉r❛• ✐♥s✐❣❤ts ✐♥t♦ t❤❡ r❡❝r✉✐t♠❡♥t ♦❢ ❙▼|❚ ❜② t❤❡ ❝♦r❡♣r❡ss♦r ❙❍❆|P ✉♥❞❡r ♣❤♦s♣❤♦r②•❛t✐✈❡ r❡❣✉•❛t✐♦♥✳ ❙tr✉❝t✉r❡✱ ✷✷✭✶✮✿✸✺✕✹✻✳ ▼✐••❡r✱ ❆✳✱ |❛•s❡r✱ ▼✳✱ ❑•♦❡t✱ ❙✳ ▲✳✱ ▲♦♦s✱ |✳✱ ◆✐s❤✐♥❛❦❛♠✉r❛✱ |✳✱ ❇❡rt♦♥❡✱ P✳✱ ❱❡r♠❡✉•❡♥✱ ▼✳✱ ❛♥❞ ❍❡♥❞r✐❝❤✱ ❇✳ ✭✷✵✶✻✮✳ ❙❛••✹ ❝♦♥tr♦•s ❞✐✛❡r❡♥t✐❛t✐♦♥ ♦❢ ♣•✉r✐♣♦t❡♥t ❝❡••s ✐♥❞❡♣❡♥✲ ❞❡♥t•② ♦❢ t❤❡ ♥✉❝•❡♦s♦♠❡ r❡♠♦❞❡••✐♥❣ ❛♥❞ ❞❡❛❝❡t②•❛t✐♦♥ ✭◆✉|❉✮ ❝♦♠♣•❡①✳ ❉❡✈❡•♦♣♠❡♥t ✭❈❛♠❜r✐❞❣❡✮✱ ✶✹✸✭✶✼✮✿✸✵✼✹✕✸✵✽✹✳ ▼✐♥❛•✐❣✐✱ ❆✳✱ ❋r♦❜❡r❣✱ ❏✳ ❊✳✱ ❲❡✐✱ ❈✳✱ ❙✉♥✇♦♦✱ ❍✳✱ ❑❡s♥❡r✱ ❇✳✱ ❈♦•♦❣♥♦r✐✱ ❉✳✱ ▲❡ss✐♥❣✱ ❉✳✱ P❛②❡r✱ ❇✳✱ ❇♦✉❦❤❛•✐✱ ▼✳✱ ❍❛❛s✱ ❲✳✱ ❛♥❞ ▲❡❡✱ ❏✳ ❚✳ ✭✷✵✶✺✮✳ ❆ ❝♦♠♣r❡❤❡♥s✐✈❡ ❳✐st ✐♥t❡r❛❝t♦♠❡ r❡✈❡❛•s ❝♦❤❡s✐♥ r❡♣✉•s✐♦♥ ❛♥❞ ❛♥ |◆❆✲❞✐r❡❝t❡❞ ❝❤r♦♠♦s♦♠❡ ❝♦♥❢♦r♠❛t✐♦♥✳ ❙❝✐❡♥❝❡✱ ✸✹✾✭✻✷✹✺✮✿❛❛❜✷✷✼✻✕❛❛❜✷✷✼✻✳ ▼♦✐♥❞r♦t✱ ❇✳✱ ❈❡r❛s❡✱ ❆✳✱ ❈♦❦❡r✱ ❍✳✱ ▼❛s✉✐✱ ❖✳✱ •r✐•③❡♥❤♦✉t✱ ❆✳✱ P✐♥t❛❝✉❞❛✱ •✳✱ ❙❝❤❡r✲ ♠❡••❡❤✱ ▲✳✱ ◆❡st❡r♦✈❛✱ ❚✳ ❇✳✱ ❛♥❞ ❇r♦❝❦❞♦r✛✱ ◆✳ ✭✷✵✶✺✮✳ ❆ P♦♦•❡❞ s❤|◆❆ ❙❝r❡❡♥ ■❞❡♥t✐✜❡s |❜♠✶✺✱ ❙♣❡♥✱ ❛♥❞ ❲t❛♣ ❛s ❋❛❝t♦rs |❡q✉✐r❡❞ ❢♦r ❳✐st |◆❆✲▼❡❞✐❛t❡❞ ❙✐•❡♥❝✲ ✐♥❣✳ ❈❡•• r❡♣♦rts✱ ✶✷✭✹✮✿✺✻✷✕✼✷✳ ▼♦♥❢♦rt✱ ❆✳✱ ❉✐ ▼✐♥✐♥✱ •✳✱ P♦st•♠❛②r✱ ❆✳✱ ❋r❡✐♠❛♥♥✱ |✳✱ ❆r✐❡t✐✱ ❋✳✱ ❚❤♦r❡✱ ❙✳✱ ❛♥❞ ❲✉t③✱ ❆✳ ✭✷✵✶✺✮✳ ■❞❡♥t✐✜❝❛t✐♦♥ ♦❢ ❙♣❡♥ ❛s ❛ ❈r✉❝✐❛• ❋❛❝t♦r ❢♦r ❳✐st ❋✉♥❝t✐♦♥ t❤r♦✉❣❤ ❋♦r✇❛r❞ •❡♥❡t✐❝ ❙❝r❡❡♥✐♥❣ ✐♥ ❍❛♣•♦✐❞ ❊♠❜r②♦♥✐❝ ❙t❡♠ ❈❡••s✳ ❈❡•• |❡♣♦rts✱ ✶✷✭✹✮✿✺✺✹✕✺✻✶✳ ▼♦♥❦✱ ▼✳ ❛♥❞ ❍❛r♣❡r✱ ▼✳ ■✳ ✭✶✾✼✾✮✳ ❙❡q✉❡♥t✐❛• ❳ ❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥ ❝♦✉♣•❡❞ ✇✐t❤ ❝❡••✉•❛r ❞✐✛❡r❡♥t✐❛t✐♦♥ ✐♥ ❡❛r•② ♠♦✉s❡ ❡♠❜r②♦s ❬✶✽(✳ ◆❛t✉r❡✱ ✷✽✶✭✺✼✷✾✮✿✸✶✶✕✸✶✸✳ ▼♦r❡②✱ ▲✳ ❛♥❞ ❍❡•✐♥✱ ❑✳ ✭✷✵✶✵✮✳ P♦•②❝♦♠❜ ❣r♦✉♣ ♣r♦t❡✐♥✲♠❡❞✐❛t❡❞ r❡♣r❡ss✐♦♥ ♦❢ tr❛♥✲ s❝r✐♣t✐♦♥✳ ❚r❡♥❞s ✐♥ ❇✐♦❝❤❡♠✐❝❛• ❙❝✐❡♥❝❡s✱ ✸✺✭✻✮✿✸✷✸✕✸✸✷✳ ▼♦rr✐s✱ ❉✳ P✳ ❛♥❞ •r❡❡♥•❡❛❢✱ ❆✳ ▲✳ ✭✷✵✵✵✮✳ ❚❤❡ s♣•✐❝✐♥❣ ❢❛❝t♦r✱ Pr♣✹✵✱ ❜✐♥❞s t❤❡ ♣❤♦s✲ ♣❤♦r②•❛t❡❞ ❝❛r❜♦①②•✲t❡r♠✐♥❛• ❞♦♠❛✐♥ ♦❢ |◆❆ P♦•②♠❡r❛s❡ ■■✳ ❏♦✉r♥❛• ♦❢ ❇✐♦•♦❣✐❝❛• ❈❤❡♠✐str②✱ ✷✼✺✭✺✶✮✿✸✾✾✸✺✕✸✾✾✹✸✳ ✷✵✹ |❡❢❡r❡♥❝❡s |❊❋❊|❊◆❈❊❙ ▼✉••❡r✱ ❍✳ ❏✳ ✭✶✾✶✽✮✳ •❡♥❡t✐❝ ❱❛r✐❛❜✐•✐t②✱ ❚✇✐♥ ❍②❜r✐❞s ❛♥❞ ❈♦♥st❛♥t ❍②❜r✐❞s✱ ✐♥ ❛ ❈❛s❡ ♦❢ ❇❛•❛♥❝❡❞ ▲❡t❤❛• ❋❛❝t♦rs✳ •❡♥❡t✐❝s✱ ✸✭✺✮✿✹✷✷✕✾✾✳ ◆❛❣②✱ ▲✳✱ ❑❛♦✱ ❍✳ (✳✱ ❈❤❛❦r❛✈❛rt✐✱ ❉✳✱ ▲✐♥✱ |✳ ❏✳✱ ❍❛ss✐❣✱ ❈✳ ❆✳✱ ❆②❡r✱ ❉✳ ❊✳✱ ❙❝❤r❡✐❜❡r✱ ❙✳ ▲✳✱ ❛♥❞ ❊✈❛♥s✱ |✳ ▼✳ ✭✶✾✾✼✮✳ ◆✉❝•❡❛r r❡❝❡♣t♦r r❡♣r❡ss✐♦♥ ♠❡❞✐❛t❡❞ ❜② ❛ ❝♦♠♣•❡① ❝♦♥t❛✐♥✐♥❣ ❙▼|❚✱ ♠❙✐♥✸❆✱ ❛♥❞ ❤✐st♦♥❡ ❞❡❛❝❡t②•❛s❡✳ ❈❡••✱ ✽✾✭✸✮✿✸✼✸✕✸✽✵✳ ◆❛✈❛rr♦✱ P✳✱ ❈❤❛♠❜❡rs✱ ■✳✱ ❑❛r✇❛❝❦✐✲◆❡✐s✐✉s✱ ❱✳✱ ❈❤✉r❡❛✉✱ ❈✳✱ ▼♦r❡②✱ ❈✳✱ |♦✉❣❡✉••❡✱ ❈✳✱ ❛♥❞ ❆✈♥❡r✱ P✳ ✭✷✵✵✽✮✳ ▼♦•❡❝✉•❛r ❝♦✉♣•✐♥❣ ♦❢ ❳✐st r❡❣✉•❛t✐♦♥ ❛♥❞ ♣•✉r✐♣♦t❡♥❝②✳ ❙❝✐❡♥❝❡✱ ✸✷✶✭✺✽✾✻✮✿✶✻✾✸✕✶✻✾✺✳ ◆❛✈❛rr♦✱ P✳✱ ❖•❞✜❡•❞✱ ❆✳✱ ▲❡❣♦✉♣✐✱ ❏✳✱ ❋❡st✉❝❝✐❛✱ ◆✳✱ ❉✉❜♦✐s✱ ❆✳ ❙✳✱ ❆tt✐❛✱ ▼✳✱ ❙❝❤♦♦r•❡♠✲ ♠❡r✱ ❏✳✱ |♦✉❣❡✉••❡✱ ❈✳✱ ❈❤❛♠❜❡rs✱ ■✳✱ ❛♥❞ ❆✈♥❡r✱ P✳ ✭✷✵✶✵✮✳ ▼♦•❡❝✉•❛r ❝♦✉♣•✐♥❣ ♦❢ ❚s✐① r❡❣✉•❛t✐♦♥ ❛♥❞ ♣•✉r✐♣♦t❡♥❝②✳ ◆❛t✉r❡✱ ✹✻✽✭✼✸✷✷✮✿✹✺✼✕✹✻✵✳ ◆❡st❡r♦✈❛✱ ❚✳ ❇✳✱ ❲❡✐✱ •✳✱ ❈♦❦❡r✱ ❍✳✱ P✐♥t❛❝✉❞❛✱ •✳✱ ❇♦✇♥❡ss✱ ❏✳ ❙✳✱ )❤❛♥❣✱ ❚✳✱ ❆•♠❡✐❞❛✱ ▼✳✱ ❇•♦❡❝❤•✱ ❇✳✱ ▼♦✐♥❞r♦t✱ ❇✳✱ ❈❛rt❡r✱ ❊✳ ❏✳✱ ❆•✈❛r❡③ |♦❞r✐❣♦✱ ■✳✱ P❛♥✱ ◗✳✱ ❇✐✱ (✳✱ ❙♦♥❣✱ ❈✳ ❳✳✱ ❛♥❞ ❇r♦❝❦❞♦r✛✱ ◆✳ ✭✷✵✶✾✮✳ ❙②st❡♠❛t✐❝ ❛••❡•✐❝ ❛♥❛•②s✐s ❞❡✜♥❡s t❤❡ ✐♥t❡r♣•❛② ♦❢ ❦❡② ♣❛t❤✇❛②s ✐♥ ❳ ❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥✳ ◆❛t✉r❡ ❈♦♠♠✉♥✐❝❛t✐♦♥s✱ ✶✵✭✶✮✿✸✶✷✾✳ ◆❡✇❜❡rr②✱ ❊✳ P✳✱ ▲❛t✐✜✱ ❚✳✱ ❛♥❞ ❚♦✇•❡r✱ ❉✳ ❆✳ ✭✶✾✾✾✮✳ ❚❤❡ ||▼ ❞♦♠❛✐♥ ♦❢ ▼■◆❚✱ ❛ ♥♦✈❡• ▼s①✷ ❜✐♥❞✐♥❣ ♣r♦t❡✐♥✱ r❡❝♦❣♥✐③❡s ❛♥❞ r❡❣✉•❛t❡s t❤❡ r❛t ♦st❡♦❝❛•❝✐♥ ♣r♦♠♦t❡r✳ ❇✐♦❝❤❡♠✐str②✱ ✸✽✭✸✸✮✿✶✵✻✼✽✕✶✵✻✾✵✳ ◆❣✱ ❍✳ ❍✳✱ |♦❜❡rt✱ ❋✳✱ (♦✉♥❣✱ |✳ ❆✳✱ ❛♥❞ ❙tr✉❤•✱ ❑✳ ✭✷✵✵✸✮✳ ❚❛r❣❡t❡❞ r❡❝r✉✐t♠❡♥t ♦❢ ❙❡t✶ ❤✐st♦♥❡ ♠❡t❤②•❛s❡ ❜② ❡•♦♥❣❛t✐♥❣ P♦• ■■ ♣r♦✈✐❞❡s ❛ •♦❝❛•✐③❡❞ ♠❛r❦ ❛♥❞ ♠❡♠♦r② ♦❢ r❡❝❡♥t tr❛♥s❝r✐♣t✐♦♥❛• ❛❝t✐✈✐t②✳ ▼♦•❡❝✉•❛r ❈❡••✱ ✶✶✭✸✮✿✼✵✾✕✼✶✾✳ ◆✐❦♦•❛❦❛❦✐✱ ❊✳✱ ▼②•♦♥✐s✱ ■✳✱ ❛♥❞ •✐❛♥♥❛❦♦✉r♦s✱ ❚✳ ✭✷✵✶✼✮✳ ▲❛♠✐♥ ❇ |❡❝❡♣t♦r✿ ■♥t❡r♣•❛② ❜❡t✇❡❡♥ ❙tr✉❝t✉r❡✱ ❋✉♥❝t✐♦♥ ❛♥❞ ▲♦❝❛•✐③❛t✐♦♥✳ ❈❡••s✱ ✻✭✸✮✿✷✽✳ ◆✐s❤✐♠✉r❛✱ ❑✳✱ ❋✉❦❛❣❛✇❛✱ ❚✳✱ ❚❛❦✐s❛✇❛✱ ❍✳✱ ❑❛❦✐♠♦t♦✱ ❚✳✱ ❛♥❞ ❑❛♥❡♠❛❦✐✱ ▼✳ ✭✷✵✵✾✮✳ ❆♥ ❛✉①✐♥✲❜❛s❡❞ ❞❡❣r♦♥ s②st❡♠ ❢♦r t❤❡ r❛♣✐❞ ❞❡♣•❡t✐♦♥ ♦❢ ♣r♦t❡✐♥s ✐♥ ♥♦♥♣•❛♥t ❝❡••s✳ ◆❛t✉r❡ ▼❡t❤♦❞s✱ ✻✭✶✷✮✿✾✶✼✕✾✷✷✳ ◆♦r❛✱ ❊✳ P✳✱ •♦•♦❜♦r♦❞❦♦✱ ❆✳✱ ❱❛•t♦♥✱ ❆✳ ▲✳✱ •✐❜❝✉s✱ ❏✳ ❍✳✱ ❯❡❜❡rs♦❤♥✱ ❆✳✱ ❆❜❞❡♥♥✉r✱ ◆✳✱ ❉❡❦❦❡r✱ ❏✳✱ ▼✐r♥②✱ ▲✳ ❆✳✱ ❛♥❞ ❇r✉♥❡❛✉✱ ❇✳ •✳ ✭✷✵✶✼✮✳ ❚❛r❣❡t❡❞ ❉❡❣r❛❞❛t✐♦♥ ♦❢ ❈❚❈❋ ❉❡❝♦✉♣•❡s ▲♦❝❛• ■♥s✉•❛t✐♦♥ ♦❢ ❈❤r♦♠♦s♦♠❡ ❉♦♠❛✐♥s ❢r♦♠ •❡♥♦♠✐❝ ❈♦♠♣❛rt♠❡♥t❛•✲ ✐③❛t✐♦♥✳ ❈❡••✱ ✶✻✾✭✺✮✿✾✸✵✕✾✹✹✳❡✷✷✳ ◆♦r❛✱ ❊✳ P✳✱ ▲❛•♦✐❡✱ ❇✳ |✳✱ ❙❝❤✉•③✱ ❊✳ •✳✱ •✐♦r❣❡tt✐✱ ▲✳✱ ❖❦❛♠♦t♦✱ ■✳✱ ❙❡r✈❛♥t✱ ◆✳✱ P✐♦•♦t✱ ❚✳✱ ❱❛♥ ❇❡r❦✉♠✱ ◆✳ ▲✳✱ ▼❡✐s✐❣✱ ❏✳✱ ❙❡❞❛t✱ ❏✳✱ •r✐❜♥❛✉✱ ❏✳✱ ❇❛r✐••♦t✱ ❊✳✱ ❇•üt❤❣❡♥✱ ◆✳✱ ❉❡❦❦❡r✱ ❏✳✱ ❛♥❞ ❍❡❛r❞✱ ❊✳ ✭✷✵✶✷✮✳ ❙♣❛t✐❛• ♣❛rt✐t✐♦♥✐♥❣ ♦❢ t❤❡ r❡❣✉•❛t♦r② •❛♥❞s❝❛♣❡ ♦❢ t❤❡ ❳✲✐♥❛❝t✐✈❛t✐♦♥ ❝❡♥tr❡✳ ◆❛t✉r❡✱ ✹✽✺✭✼✸✾✽✮✿✸✽✶✕✸✽✺✳ ✷✵✺ |❡❢❡r❡♥❝❡s |❊❋❊|❊◆❈❊❙ ◆♦r♠❛♥♥♦✱ ◆✳✱ ❉❡ ▲✉❝❛✱ ❆✳✱ ❇✐❛♥❝♦✱ ❈✳✱ ❙tr✐③③✐✱ ▲✳✱ ▼❛♥❝✐♥♦✱ ▼✳✱ ▼❛✐❡••♦✱ ▼✳ |✳✱ ❈❛r♦t❡♥✉t♦✱ ❆✳✱ ❉❡ ❋❡♦✱ •✳✱ ❈❛♣♦♥✐❣r♦✱ ❋✳✱ ❛♥❞ ❙❛•♦♠♦♥✱ ❉✳ ❙✳ ✭✷✵✵✻✮✳ ❊♣✐❞❡r♠❛• ❣r♦✇t❤ ❢❛❝t♦r r❡❝❡♣t♦r ✭❊•❋|✮ s✐❣♥❛•✐♥❣ ✐♥ ❝❛♥❝❡r✳ •❡♥❡✱ ✸✻✻✭✶✮✿✷✕✶✻✳ ◆♦rr✐s✱ ❉✳ P✳✱ ❇r♦❝❦❞♦r✛✱ ◆✳✱ ❛♥❞ |❛st❛♥✱ ❙✳ ✭✶✾✾✶✮✳ ▼❡t❤②•❛t✐♦♥ st❛t✉s ♦❢ ❈♣•✲r✐❝❤ ✐s•❛♥❞s ♦♥ ❛❝t✐✈❡ ❛♥❞ ✐♥❛❝t✐✈❡ ♠♦✉s❡ ❳ ❝❤r♦♠♦s♦♠❡s✳ ▼❛♠♠❛•✐❛♥ •❡♥♦♠❡✱ ✶✭✷✮✿✼✽✕ ✽✸✳ ❖❤♥♦✱ ❙✳ ✭✶✾✻✼✮✳ ❙❡① ❝❤r♦♠♦s♦♠❡s ❛♥❞ s❡① •✐♥❦❡❞ ❣❡♥❡s✳ ❇❡r•✐♥❙♣r✐♥❣❡r✳ ❖❤♥♦✱ ❙✳✱ ❑❛♣•❛♥✱ ❲✳ ❉✳✱ ❛♥❞ ❑✐♥♦s✐t❛✱ |✳ ✭✶✾✺✽✮✳ ❙♦♠❛t✐❝ ❛ss♦❝✐❛t✐♦♥ ♦❢ t❤❡ ♣♦s✐t✐✈❡•② ❤❡t❡r♦♣②❝♥♦t✐❝ ❳✲❝❤r♦♠♦s♦♠❡s ✐♥ ❢❡♠❛•❡ ♠✐❝❡ ✭▼✉s ♠✉s❝✉•✉s✮✳ ❊①♣❡r✐♠❡♥t❛• ❈❡•• |❡s❡❛r❝❤✱ ✶✺✭✸✮✿✻✶✻✕✻✶✽✳ ❖❤♥♦✱ ❙✳✱ ❑❛♣•❛♥✱ ❲✳ ❉✳✱ ❛♥❞ ❑✐♥♦s✐t❛✱ |✳ ✭✶✾✺✾✮✳ ❋♦r♠❛t✐♦♥ ♦❢ t❤❡ s❡① ❝❤r♦♠❛t✐♥ ❜② ❛ s✐♥❣•❡ ❳✲❝❤r♦♠♦s♦♠❡ ✐♥ •✐✈❡r ❝❡••s ♦❢ |❛tt✉s ♥♦r✈❡❣✐❝✉s✳ ❊①♣❡r✐♠❡♥t❛• ❈❡•• |❡s❡❛r❝❤✱ ✶✽✭✷✮✿✹✶✺✕✹✶✽✳ ❖❦❛♠♦t♦✱ ■✳✱ ❆r♥❛✉❞✱ ❉✳✱ ▲❡ ❇❛❝❝♦♥✱ P✳✱ ❖tt❡✱ ❆✳ P✳✱ ❉✐st❡❝❤❡✱ ❈✳ ▼✳✱ ❆✈♥❡r✱ P✳✱ ❛♥❞ ❍❡❛r❞✱ ❊✳ ✭✷✵✵✺✮✳ ❊✈✐❞❡♥❝❡ ❢♦r ❞❡ ♥♦✈♦ ✐♠♣r✐♥t❡❞ ❳✲❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥ ✐♥❞❡♣❡♥✲ ❞❡♥t ♦❢ ♠❡✐♦t✐❝ ✐♥❛❝t✐✈❛t✐♦♥ ✐♥ ♠✐❝❡✳ ◆❛t✉r❡✱ ✹✸✽✭✼✵✻✻✮✿✸✻✾✕✸✼✸✳ ❖❦❛♠♦t♦✱ ■✳✱ ❖tt❡✱ ❆✳ P✳✱ ❆••✐s✱ ❈✳ ❉✳✱ |❡✐♥❜❡r❣✱ ❉✳✱ ❛♥❞ ❍❡❛r❞✱ ❊✳ ✭✷✵✵✹✮✳ ❊♣✐❣❡♥❡t✐❝ ❉②♥❛♠✐❝s ♦❢ ■♠♣r✐♥t❡❞ ❳ ■♥❛❝t✐✈❛t✐♦♥ ❉✉r✐♥❣ ❊❛r•② ▼♦✉s❡ ❉❡✈❡•♦♣♠❡♥t✳ ❙❝✐❡♥❝❡✱ ✸✵✸✭✺✻✺✽✮✿✻✹✹✕✻✹✾✳ ❖❦❛♠♦t♦✱ ■✳✱ P❛tr❛t✱ ❈✳✱ ❚❤é♣♦t✱ ❉✳✱ P❡②♥♦t✱ ◆✳✱ ❋❛✉q✉❡✱ P✳✱ ❉❛♥✐❡•✱ ◆✳✱ ❉✐❛❜❛♥❣♦✉❛②❛✱ P✳✱ ❲♦•❢✱ ❏✳✲P✳✱ |❡♥❛r❞✱ ❏✳✲P✳✱ ❉✉r❛♥t❤♦♥✱ ❱✳✱ ❛♥❞ ❍❡❛r❞✱ ❊✳ ✭✷✵✶✶✮✳ ❊✉t❤❡r✐❛♥ ♠❛♠✲ ♠❛•s ✉s❡ ❞✐✈❡rs❡ str❛t❡❣✐❡s t♦ ✐♥✐t✐❛t❡ ❳✲❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥ ❞✉r✐♥❣ ❞❡✈❡•♦♣♠❡♥t✳ ◆❛t✉r❡✱ ✹✼✷✭✼✸✹✸✮✿✸✼✵✕✸✼✹✳ ❖s✇❛•❞✱ ❋✳✱ ❑♦st❡③❦❛✱ ❯✳✱ ❆str❛❤❛♥ts❡✛✱ ❑✳✱ ❇♦✉rt❡❡•❡✱ ❙✳✱ ❉✐••✐♥❣❡r✱ ❑✳✱ )❡❝❤♥❡r✱ ❯✳✱ ▲✉❞✇✐❣✱ ▲✳✱ ❲✐•❞❛✱ ▼✳✱ ❍❛♠❡✐st❡r✱ ❍✳✱ ❑♥ö❝❤❡•✱ ❲✳✱ ▲✐♣t❛②✱ ❙✳✱ ❛♥❞ ❙❝❤♠✐❞✱ |✳ ▼✳ ✭✷✵✵✷✮✳ ❙❍❆|P ✐s ❛ ♥♦✈❡• ❝♦♠♣♦♥❡♥t ♦❢ t❤❡ ◆♦t❝❤✴|❇P✲❏κ s✐❣♥❛••✐♥❣ ♣❛t❤✇❛②✳ ❊▼❇❖ ❏♦✉r♥❛•✱ ✷✶✭✷✵✮✿✺✹✶✼✕✺✹✷✻✳ ❖s✇❛•❞✱ ❋✳✱ |♦❞r✐❣✉❡③✱ P✳✱ •✐❛✐♠♦✱ ❇✳ ❉✳✱ ❆♥t♦♥❡••♦✱ )✳ ❆✳✱ ▼✐r❛✱ ▲✳✱ ▼✐tt•❡r✱ •✳✱ ❚❤✐❡•✱ ❱✳ ◆✳✱ ❈♦••✐♥s✱ ❑✳ ❏✳✱ ❚❛❜❛•❛✱ ◆✳✱ ❈✐③❡•s❦②✱ ❲✳✱ |♦t❤❡✱ ▼✳✱ ❑ü❤•✱ ❙✳ ❏✳✱ ❑ü❤•✱ ▼✳✱ ❋❡rr❛♥t❡✱ ❋✳✱ ❍❡✐♥✱ ❑✳✱ ❑♦✈❛••✱ |✳ ❆✳✱ ❉♦♠✐♥❣✉❡③✱ ▼✳✱ ❛♥❞ ❇♦r❣❣r❡❢❡✱ ❚✳ ✭✷✵✶✻✮✳ ❆ ♣❤♦s♣❤♦✲❞❡♣❡♥❞❡♥t ♠❡❝❤❛♥✐s♠ ✐♥✈♦•✈✐♥❣ ◆❈♦| ❛♥❞ ❑▼❚✷❉ ❝♦♥tr♦•s ❛ ♣❡r♠✐ss✐✈❡ ❝❤r♦♠❛t✐♥ st❛t❡ ❛t ◆♦t❝❤ t❛r❣❡t ❣❡♥❡s✳ ◆✉❝•❡✐❝ ❆❝✐❞s |❡s❡❛r❝❤✱ ✹✹✭✶✵✮✿✹✼✵✸✕✹✼✷✵✳ ✷✵✻ |❡❢❡r❡♥❝❡s |❊❋❊|❊◆❈❊❙ P❛t✐•✱ ❉✳ P✳✱ ❈❤❡♥✱ ❈✳✲❑✳✱ P✐❝❦❡r✐♥❣✱ ❇✳ ❋✳✱ ❈❤♦✇✱ ❆✳✱ ❏❛❝❦s♦♥✱ ❈✳✱ •✉tt♠❛♥✱ ▼✳✱ ❛♥❞ ❏❛✛r❡②✱ ❙✳ |✳ ✭✷✵✶✻✮✳ ♠✻❆ |◆❆ ♠❡t❤②•❛t✐♦♥ ♣r♦♠♦t❡s ❳■❙❚✲♠❡❞✐❛t❡❞ tr❛♥s❝r✐♣t✐♦♥❛• r❡♣r❡ss✐♦♥✳ ◆❛t✉r❡✱ ✺✸✼✭✼✻✷✵✮✿✸✻✾✕✸✼✸✳ P❛tr❛t✱ ❈✳✱ ❖❦❛♠♦t♦✱ ■✳✱ ❉✐❛❜❛♥❣♦✉❛②❛✱ P✳✱ ❱✐❛•♦♥✱ ❱✳✱ ▲❡ ❇❛❝❝♦♥✱ P✳✱ ❈❤♦✇✱ ❏✳✱ ❛♥❞ ❍❡❛r❞✱ ❊✳ ✭✷✵✵✾✮✳ ❉②♥❛♠✐❝ ❝❤❛♥❣❡s ✐♥ ♣❛t❡r♥❛• ❳✲❝❤r♦♠♦s♦♠❡ ❛❝t✐✈✐t② ❞✉r✐♥❣ ✐♠✲ ♣r✐♥t❡❞ ❳✲❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥ ✐♥ ♠✐❝❡✳ Pr♦❝❡❡❞✐♥❣s ♦❢ t❤❡ ◆❛t✐♦♥❛• ❆❝❛❞❡♠② ♦❢ ❙❝✐❡♥❝❡s✱ ✶✵✻✭✶✸✮✿✺✶✾✽✕✺✷✵✸✳ P❡♥♥②✱ •✳ ❉✳✱ ❑❛②✱ •✳ ❋✳✱ ❙❤❡❛r❞♦✇♥✱ ❙✳ ❆✳✱ |❛st❛♥✱ ❙✳✱ ❛♥❞ ❇r♦❝❦❞♦r✛✱ ◆✳ ✭✶✾✾✻✮✳ |❡q✉✐r❡♠❡♥t ❢♦r ❳✐st ✐♥ ❳ ❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥✳ ◆❛t✉r❡✱ ✸✼✾✭✻✺✻✶✮✿✶✸✶✕✶✸✼✳ P❡tr♦♣♦✉•♦s✱ ❙✳✱ ❊❞s❣är❞✱ ❉✳✱ |❡✐♥✐✉s✱ ❇✳✱ ❉❡♥❣✱ ◗✳✱ P❛♥✉•❛✱ ❙✳ P✳✱ ❈♦❞❡•✉♣♣✐✱ ❙✳✱ P•❛③❛ |❡②❡s✱ ❆✳✱ ▲✐♥♥❛rss♦♥✱ ❙✳✱ ❙❛♥❞❜❡r❣✱ |✳✱ ❛♥❞ ▲❛♥♥❡r✱ ❋✳ ✭✷✵✶✻✮✳ ❙✐♥❣•❡✲❈❡•• |◆❆✲❙❡q |❡✈❡❛•s ▲✐♥❡❛❣❡ ❛♥❞ ❳ ❈❤r♦♠♦s♦♠❡ ❉②♥❛♠✐❝s ✐♥ ❍✉♠❛♥ Pr❡✐♠♣•❛♥t❛t✐♦♥ ❊♠❜r②♦s✳ ❈❡••✱ ✶✻✺✭✹✮✿✶✵✶✷✕✶✵✷✻✳ P✐♥t❛❝✉❞❛✱ •✳✱ ❲❡✐✱ •✳✱ |♦✉st❛♥✱ ❈✳✱ ❑✐r♠✐③✐t❛s✱ ❇✳ ❆✳✱ ❙♦•❝❛♥✱ ◆✳✱ ❈❡r❛s❡✱ ❆✳✱ ❈❛st❡••♦✱ ❆✳✱ ▼♦❤❛♠♠❡❞✱ ❙✳✱ ▼♦✐♥❞r♦t✱ ❇✳✱ ◆❡st❡r♦✈❛✱ ❚✳ ❇✳✱ ❛♥❞ ❇r♦❝❦❞♦r✛✱ ◆✳ ✭✷✵✶✼✮✳ ❤♥✲ |◆P❑ |❡❝r✉✐ts P❈•❋✸✴✺✲P|❈✶ t♦ t❤❡ ❳✐st |◆❆ ❇✲|❡♣❡❛t t♦ ❊st❛❜•✐s❤ P♦•②❝♦♠❜✲ ▼❡❞✐❛t❡❞ ❈❤r♦♠♦s♦♠❛• ❙✐•❡♥❝✐♥❣✳ ▼♦•❡❝✉•❛r ❈❡••✱ ✻✽✭✺✮✿✾✺✺✕✾✻✾✳❡✶✵✳ P✐♥t❡r✱ ❙✳ ❋✳✱ ❙❛❞r❡②❡✈✱ |✳ ■✳✱ (✐•❞✐r✐♠✱ ❊✳✱ ❏❡♦♥✱ (✳✱ ❖❤s✉♠✐✱ ❚✳ ❑✳✱ ❇♦r♦✇s❦②✱ ▼✳✱ ❛♥❞ ▲❡❡✱ ❏✳ ❚✳ ✭✷✵✶✷✮✳ ❙♣r❡❛❞✐♥❣ ♦❢ ❳ ❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥ ✈✐❛ ❛ ❤✐❡r❛r❝❤② ♦❢ ❞❡✜♥❡❞ P♦•②❝♦♠❜ st❛t✐♦♥s✳ •❡♥♦♠❡ |❡s❡❛r❝❤✱ ✷✷✭✶✵✮✿✶✽✻✹✕✶✽✼✻✳ P•❛t❤✱ ❑✳✱ ❋❛♥❣✱ ❏✳✱ ▼•②♥❛r❝③②❦✲❊✈❛♥s✱ ❙✳ ❑✳✱ ❈❛♦✱ |✳✱ ❲♦rr✐♥❣❡r✱ ❑✳ ❆✳✱ ❲❛♥❣✱ ❍✳✱ ❉❡ •❛ ❈r✉③✱ ❈✳ ❈✳✱ ❖tt❡✱ ❆✳ P✳✱ P❛♥♥✐♥❣✱ ❇✳✱ ❛♥❞ )❤❛♥❣✱ (✳ ✭✷✵✵✸✮✳ |♦•❡ ♦❢ ❤✐st♦♥❡ ❍✸ •②s✐♥❡ ✷✼ ♠❡t❤②•❛t✐♦♥ ✐♥ ❳ ✐♥❛❝t✐✈❛t✐♦♥✳ ❙❝✐❡♥❝❡✱ ✸✵✵✭✺✻✶✻✮✿✶✸✶✕✶✸✺✳ P♦♥t✐❡r✱ ❉✳ ❇✳ ❛♥❞ •r✐❜♥❛✉✱ ❏✳ ✭✷✵✶✶✮✳ ❳✐st r❡❣✉•❛t✐♦♥ ❛♥❞ ❢✉♥❝t✐♦♥ ❡❳♣•♦r❡❞✳ ❍✉♠❛♥ •❡♥❡t✐❝s✱ ✶✸✵✭✷✮✿✷✷✸✕✷✸✻✳ P♦✉••❡t✱ P✳✱ ❈❛r♣❡♥t✐❡r✱ ❙✳✱ ❛♥❞ ❇❛r✐••♦t✱ ❊✳ ✭✷✵✵✼✮✳ ♠②Pr♦▼❙✱ ❛ ✇❡❜ s❡r✈❡r ❢♦r ♠❛♥✲ ❛❣❡♠❡♥t ❛♥❞ ✈❛•✐❞❛t✐♦♥ ♦❢ ♠❛ss s♣❡❝tr♦♠❡tr②✲❜❛s❡❞ ♣r♦t❡♦♠✐❝ ❞❛t❛✳ Pr♦t❡♦♠✐❝s✱ ✼✭✶✺✮✿✷✺✺✸✕✷✺✺✻✳ ◗✉❡r❡♥❡t✱ ▼✳✱ •♦✉❜❛r❞✱ ❱✳✱ ❈❤❛t❡•❛✐♥✱ •✳✱ ❉❛✈♦✉st✱ ◆✳✱ ❛♥❞ ▼♦••❡r❡❛✉✱ ❇✳ ✭✷✵✶✺✮✳ ❙♣❡♥ ✐s r❡q✉✐r❡❞ ❢♦r ♣✐❣♠❡♥t ❝❡•• s✉r✈✐✈❛• ❞✉r✐♥❣ ♣✉♣❛• ❞❡✈❡•♦♣♠❡♥t ✐♥ ❉r♦s♦♣❤✐•❛✳ ❉❡✈❡•♦♣✲ ♠❡♥t❛• ❇✐♦•♦❣②✱ ✹✵✷✭✷✮✿✷✵✽✕✷✶✺✳ |❛♦✱ ❙✳ ❙✳✱ ❍✉♥t•❡②✱ ▼✳ ❍✳✱ ❉✉r❛♥❞✱ ◆✳ ❈✳✱ ❙t❛♠❡♥♦✈❛✱ ❊✳ ❑✳✱ ❇♦❝❤❦♦✈✱ ■✳ ❉✳✱ |♦❜✐♥s♦♥✱ ❏✳ ❚✳✱ ❙❛♥❜♦r♥✱ ❆✳ ▲✳✱ ▼❛❝❤♦•✱ ■✳✱ ❖♠❡r✱ ❆✳ ❉✳✱ ▲❛♥❞❡r✱ ❊✳ ❙✳✱ ❛♥❞ ❆✐❞❡♥✱ ❊✳ ▲✳ ✭✷✵✶✹✮✳ ✷✵✼ |❡❢❡r❡♥❝❡s |❊❋❊|❊◆❈❊❙ ❆ ✸❉ ♠❛♣ ♦❢ t❤❡ ❤✉♠❛♥ ❣❡♥♦♠❡ ❛t ❦✐•♦❜❛s❡ r❡s♦•✉t✐♦♥ r❡✈❡❛•s ♣r✐♥❝✐♣•❡s ♦❢ ❝❤r♦♠❛t✐♥ •♦♦♣✐♥❣✳ ❈❡••✱ ✶✺✾✭✼✮✿✶✻✻✺✕✶✻✽✵✳ |❛st❛♥✱ ❙✳ ✭✶✾✽✷✮✳ ❚✐♠✐♥❣ ♦❢ ❳✲❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥ ✐♥ ♣♦st✐♠♣•❛♥t❛t✐♦♥ ♠♦✉s❡ ❡♠❜r②♦s✳ ❏♦✉r♥❛• ♦❢ ❡♠❜r②♦•♦❣② ❛♥❞ ❡①♣❡r✐♠❡♥t❛• ♠♦r♣❤♦•♦❣②✱ ✼✶✿✶✶✕✷✹✳ |❛st❛♥✱ ❙✳ ✭✶✾✽✸✮✳ ◆♦♥✲r❛♥❞♦♠ ❳✲❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥ ✐♥ ♠♦✉s❡ ❳✲❛✉t♦s♦♠❡ tr❛♥s•♦✲ ❝❛t✐♦♥ ❡♠❜r②♦s ✲ •♦❝❛t✐♦♥ ♦❢ t❤❡ ✐♥❛❝t✐✈❛t✐♦♥ ❝❡♥tr❡✳ ❏♦✉r♥❛• ♦❢ ❊♠❜r②♦•♦❣② ❛♥❞ ❊①✲ ♣❡r✐♠❡♥t❛• ▼♦r♣❤♦•♦❣②✱ ❱❖▲✳ ✼✽✿✶✕✷✷✳ |❛st❛♥✱ ❙✳ ❛♥❞ |♦❜❡rts♦♥✱ ❊✳ ❏✳ ✭✶✾✽✺✮✳ ❳✲❝❤r♦♠♦s♦♠❡ ❞❡•❡t✐♦♥s ✐♥ ❡♠❜r②♦✲❞❡r✐✈❡❞ ✭❊❑✮ ❝❡•• •✐♥❡s ❛ss♦❝✐❛t❡❞ ✇✐t❤ •❛❝❦ ♦❢ ❳✲❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥✳ ❏♦✉r♥❛• ♦❢ ❡♠❜r②♦•♦❣② ❛♥❞ ❡①♣❡r✐♠❡♥t❛• ♠♦r♣❤♦•♦❣②✱ ✾✵✿✸✼✾✕✽✽✳ |❛tt❦❛✱ ▼✳✱ ❲❡st♣❤❛•✱ ❙✳✱ •❛❤r✱ ❇✳ ▼✳✱ ❏✉st✱ ❙✳✱ ❛♥❞ |♦tt❜❛✉❡r✱ ❲✳ ✭✷✵✷✶✮✳ ❙♣❡♥ ❞❡✜❝✐❡♥❝② ✐♥t❡r❢❡r❡s ✇✐t❤ ❈♦♥♥❡①✐♥ ✹✸ ❡①♣r❡ss✐♦♥ ❛♥❞ •❡❛❞s t♦ ❤❡❛rt ❢❛✐•✉r❡ ✐♥ ③❡❜r❛✜s❤✳ ❏♦✉r♥❛• ♦❢ ♠♦•❡❝✉•❛r ❛♥❞ ❝❡••✉•❛r ❝❛r❞✐♦•♦❣②✳ |❡❜❛②✱ ■✳✱ ❈❤❡♥✱ ❋✳✱ ❍s✐❛♦✱ ❋✳✱ ❑♦•♦❞③✐❡•✱ P✳ ❆✳✱ ❑✉❛♥❣✱ ❇✳ ❍✳✱ ▲❛✈❡rt②✱ ❚✳✱ ❙✉❤✱ ❈✳✱ ❱♦❛s✱ ▼✳✱ ❲✐••✐❛♠s✱ ❆✳✱ ❛♥❞ |✉❜✐♥✱ •✳ ▼✳ ✭✷✵✵✵✮✳ ❆ ❣❡♥❡t✐❝ s❝r❡❡♥ ❢♦r ♥♦✈❡• ❝♦♠♣♦♥❡♥ts ♦❢ t❤❡ |❛s✴▼✐t♦❣❡♥✲❛❝t✐✈❛t❡❞ ♣r♦t❡✐♥ ❦✐♥❛s❡ s✐❣♥❛•✐♥❣ ♣❛t❤✇❛② t❤❛t ✐♥t❡r❛❝t ✇✐t❤ t❤❡ ②❛♥ ❣❡♥❡ ♦❢ ❉r♦s♦♣❤✐•❛ ✐❞❡♥t✐✜❡s s♣•✐t ❡♥❞s✱ ❛ ♥❡✇ |◆❆ r❡❝♦❣♥✐t✐♦♥ ♠♦t✐❢✲❝♦♥t❛✐♥✐♥❣ ♣r♦t❡✐♥✳ •❡♥❡t✐❝s✱ ✶✺✹✭✷✮✿✻✾✺✕✼✶✷✳ |❡❣♦✱ ❆✳✱ ❙✐♥❝•❛✐r✱ P✳ ❇✳✱ ❚❛♦✱ ❲✳✱ ❑✐r❡❡✈✱ ■✳✱ ❛♥❞ ❇❡•♠♦♥t✱ ❆✳ ❙✳ ✭✷✵✵✽✮✳ ❚❤❡ ❢❛❝✉•t❛t✐✈❡ ❤❡t❡r♦❝❤r♦♠❛t✐♥ ♦❢ t❤❡ ✐♥❛❝t✐✈❡ ❳ ❝❤r♦♠♦s♦♠❡ ❤❛s ❛ ❞✐st✐♥❝t✐✈❡ ❝♦♥❞❡♥s❡❞ ✉•tr❛str✉❝✲ t✉r❡✳ ❏♦✉r♥❛• ♦❢ ❈❡•• ❙❝✐❡♥❝❡✱ ✶✷✶✭✼✮✿✶✶✶✾✕✶✶✷✼✳ |✐❝❡✱ ❲✳ |✳ ✭✶✾✽✼✮✳ •❡♥❡t✐❝ ❤✐t❝❤❤✐❦✐♥❣ ❛♥❞ t❤❡ ❡✈♦•✉t✐♦♥ ♦❢ r❡❞✉❝❡❞ ❣❡♥❡t✐❝ ❛❝t✐✈✐t② ♦❢ t❤❡ ( s❡① ❝❤r♦♠♦s♦♠❡✳ •❡♥❡t✐❝s✱ ✶✶✻✭✶✮✿✶✻✶✕✶✻✼✳ |✐❝❡✱ ❲✳ |✳ ✭✶✾✾✻✮✳ ❊✈♦•✉t✐♦♥ ♦❢ t❤❡ ( ❙❡① ❈❤r♦♠♦s♦♠❡ ✐♥ ❆♥✐♠❛•s✳ ❇✐♦❙❝✐❡♥❝❡✱ ✹✻✭✺✮✿✸✸✶✕✸✹✸✳ |✐❞✐♥❣s✲❋✐❣✉❡r♦❛✱ |✳✱ ❙t❡✇❛rt✱ ❊✳ |✳✱ ◆❡st❡r♦✈❛✱ ❚✳ ❇✳✱ ❈♦❦❡r✱ ❍✳✱ P✐♥t❛❝✉❞❛✱ •✳✱ •♦❞✇✐♥✱ ❏✳✱ ❲✐•s♦♥✱ |✳✱ ❍❛s•❛♠✱ ❆✳✱ ▲✐••❡②✱ ❋✳✱ |✉✐❣r♦❦✱ |✳✱ ❇❛❣❡❣❤♥✐✱ ❙✳ ❆✳✱ ❆•❜❛❞r❛♥✐✱ •✳✱ ▼❛♥s✜❡•❞✱ ❲✳✱ |♦✉•s♦♥✱ ❏✳ ❆✳✱ ❇r♦❝❦❞♦r✛✱ ◆✳✱ ❆✐♥s❝♦✉❣❤✱ ❏✳ ❋✳✱ ❛♥❞ ❈♦✈❡r•❡②✱ ❉✳ ✭✷✵✶✼✮✳ ❚❤❡ ♥✉❝•❡❛r ♠❛tr✐① ♣r♦t❡✐♥ ❈■)✶ ❢❛❝✐•✐t❛t❡s •♦❝❛•✐③❛t✐♦♥ ♦❢ ❳✐st |◆❆ t♦ t❤❡ ✐♥❛❝t✐✈❡ ❳✲❝❤r♦♠♦s♦♠❡ t❡rr✐t♦r②✳ •❡♥❡s ❛♥❞ ❉❡✈❡•♦♣♠❡♥t✱ ✸✶✭✾✮✿✽✼✻✕✽✽✽✳ |✐✐s✐♥❣✱ ❊✳ ▼✳✱ ❈♦♠❡t✱ ■✳✱ ▲❡❜•❛♥❝✱ ❇✳✱ ❲✉✱ ❳✳✱ ❏♦❤❛♥s❡♥✱ ❏✳ ❱✳✱ ❛♥❞ ❍❡•✐♥✱ ❑✳ ✭✷✵✶✹✮✳ •❡♥❡ s✐•❡♥❝✐♥❣ tr✐❣❣❡rs ♣♦•②❝♦♠❜ r❡♣r❡ss✐✈❡ ❝♦♠♣•❡① ✷ r❡❝r✉✐t♠❡♥t t♦ ❈♣• ■s•❛♥❞s ❣❡♥♦♠❡ ✇✐❞❡✳ ▼♦•❡❝✉•❛r ❈❡••✱ ✺✺✭✸✮✿✸✹✼✕✸✻✵✳ ✷✵✽ |❡❢❡r❡♥❝❡s |❊❋❊|❊◆❈❊❙ |♦❜❡rt✲❋✐♥❡str❛✱ ❚✳✱ ❚❛♥✱ ❇✳ ❋✳✱ ▼✐r❛✲❇♦♥t❡♥❜❛•✱ ❍✳✱ ❚✐♠♠❡rs✱ ❊✳✱ •♦♥t❛♥✲P❛r❞♦✱ ❈✳✱ ▼❡r③♦✉❦✱ ❙✳✱ •✐❛✐♠♦✱ ❇✳ ❉✳✱ ❉♦ss✐♥✱ ❋✳✱ ✈❛♥ ■❏❝❦❡♥✱ ❲✳ ❋✳ ❏✳✱ ▼❛rt❡♥s✱ ❏✳ ❲✳ ▼✳✱ ❇♦r❣❣r❡❢❡✱ ❚✳✱ ❍❡❛r❞✱ ❊✳✱ ❛♥❞ •r✐❜♥❛✉✱ ❏✳ ✭✷✵✷✵✮✳ ❙P❊◆ ✐s |❡q✉✐r❡❞ ❢♦r ❳✐st ❯♣r❡❣✉✲ •❛t✐♦♥ ❞✉r✐♥❣ ■♥✐t✐❛t✐♦♥ ♦❢ ❳ ❈❤r♦♠♦s♦♠❡ ■♥❛❝t✐✈❛t✐♦♥✳ ❜✐♦|①✐✈✳ |♦ss✐✱ ❉✳✱ ❚r✐❢♦♥♦✈✱ ❱✳✱ ❋❛♥❣❛③✐♦✱ ▼✳✱ ❇r✉s❝❛❣❣✐♥✱ ❆✳✱ |❛s✐✱ ❙✳✱ ❙♣✐♥❛✱ ❱✳✱ ▼♦♥t✐✱ ❙✳✱ ❱❛✐s✐tt✐✱ ❚✳✱ ❆rr✉❣❛✱ ❋✳✱ ❋❛♠à✱ |✳✱ ❈✐❛r❞✉••♦✱ ❈✳✱ •r❡❝♦✱ ▼✳✱ ❈r❡st❛✱ ❙✳✱ P✐r❛♥❞❛✱ ❉✳✱ ❍♦•♠❡s✱ ❆✳✱ ❋❛❜❜r✐✱ •✳✱ ▼❡ss✐♥❛✱ ▼✳✱ |✐♥❛•❞✐✱ ❆✳✱ ❲❛♥❣✱ ❏✳✱ ❆❣♦st✐♥❡••✐✱ ❈✳✱ P✐❝❝❛•✉❣❛✱ P✳ P✳✱ ▲✉❝✐♦♥✐✱ ▼✳✱ ❚❛❜❜ò✱ ❋✳✱ ❙❡rr❛✱ |✳✱ ❋r❛♥❝❡s❝❤❡tt✐✱ ❙✳✱ ❉❡❛♠❜r♦❣✐✱ ❈✳✱ ❉❛♥✐❡•❡✱ •✳✱ •❛tt❡✐✱ ❱✳✱ ▼❛r❛s❝❛✱ |✳✱ ❋❛❝❝❤❡tt✐✱ ❋✳✱ ❆r❝❛✐♥✐✱ ▲✳✱ ■♥❣❤✐r❛♠✐✱ •✳✱ ❇❡rt♦♥✐✱ ❋✳✱ P✐•❡r✐✱ ❙✳ ❆✳✱ ❉❡❛❣•✐♦✱ ❙✳✱ ❋♦à✱ |✳✱ ❉❛••❛✲❋❛✈❡r❛✱ |✳✱ P❛sq✉❛•✉❝❝✐✱ ▲✳✱ |❛❜❛❞❛♥✱ |✳✱ ❛♥❞ •❛✐❞❛♥♦✱ •✳ ✭✷✵✶✷✮✳ ❚❤❡ ❝♦❞✐♥❣ ❣❡♥♦♠❡ ♦❢ s♣•❡♥✐❝ ♠❛r❣✐♥❛• ③♦♥❡ •②♠♣❤♦♠❛✿ ❆❝t✐✈❛t✐♦♥ ♦❢ ◆❖❚❈❍✷ ❛♥❞ ♦t❤❡r ♣❛t❤✇❛②s r❡❣✉•❛t✐♥❣ ♠❛r❣✐♥❛• ③♦♥❡ ❞❡✈❡•♦♣♠❡♥t✳ ❏♦✉r♥❛• ♦❢ ❊①♣❡r✐♠❡♥t❛• ▼❡❞✐❝✐♥❡✱ ✷✵✾✭✾✮✿✶✺✸✼✕✶✺✺✶✳ |♦✉♥❞tr❡❡✱ ■✳ ❆✳✱ ❊✈❛♥s✱ ▼✳ ❊✳✱ P❛♥✱ ❚✳✱ ❛♥❞ ❍❡✱ ❈✳ ✭✷✵✶✼✮✳ ❉②♥❛♠✐❝ |◆❆ ▼♦❞✐✜❝❛t✐♦♥s ✐♥ •❡♥❡ ❊①♣r❡ss✐♦♥ |❡❣✉•❛t✐♦♥✳ ❙❛❞♦✱ ❚✳✱ ❋❡♥♥❡r✱ ▼✳ ❍✳✱ ❚❛♥✱ ❙✳ ❙✳✱ ❚❛♠✱ P✳✱ ❙❤✐♦❞❛✱ ❚✳✱ ❛♥❞ ▲✐✱ ❊✳ ✭✷✵✵✵✮✳ ❳ ✐♥❛❝t✐✈❛✲ t✐♦♥ ✐♥ t❤❡ ♠♦✉s❡ ❡♠❜r②♦ ❞❡✜❝✐❡♥t ❢♦r ❉♥♠t✶✿ ❉✐st✐♥❝t ❡✛❡❝t ♦❢ ❤②♣♦♠❡t❤②•❛t✐♦♥ ♦♥ ✐♠♣r✐♥t❡❞ ❛♥❞ r❛♥❞♦♠ ❳ ✐♥❛❝t✐✈❛t✐♦♥✳ ❉❡✈❡•♦♣♠❡♥t❛• ❇✐♦•♦❣②✱ ✷✷✺✭✷✮✿✷✾✹✕✸✵✸✳ ❙❛❦❛t❛✱ (✳✱ ◆❛❣❛♦✱ ❑✳✱ ❍♦❦✐✱ (✳✱ ❙❛s❛❦✐✱ ❍✳✱ ❖❜✉s❡✱ ❈✳✱ ❛♥❞ ❙❛❞♦✱ ❚✳ ✭✷✵✶✼✮✳ ❉❡❢❡❝ts ✐♥ ❞♦s❛❣❡ ❝♦♠♣❡♥s❛t✐♦♥ ✐♠♣❛❝t ❣•♦❜❛• ❣❡♥❡ r❡❣✉•❛t✐♦♥ ✐♥ t❤❡ ♠♦✉s❡ tr♦♣❤♦❜•❛st✳ ❉❡✈❡•✲ ♦♣♠❡♥t ✭❈❛♠❜r✐❞❣❡✮✱ ✶✹✹✭✶✺✮✿✷✼✽✹✕✷✼✾✼✳ ❙❛❦❤❞❛r✐✱ ❆✳✱ ❖❦✱ ❈✳ (✳✱ P❛t❡•✱ ❑✳ P✳✱ ❑❛♥❛❣❛•✲❙❤❛♠❛♥♥❛✱ |✳✱ (✐♥✱ ❈✳ ❈✳✱ )✉♦✱ )✳✱ ❍✉✱ ❙✳✱ |♦✉t❜♦rt✱ ▼✳ ❏✳✱ ▲✉t❤r❛✱ |✳✱ ▼❡❞❡✐r♦s✱ ▲✳ ❏✳✱ ❑❤♦✉r②✱ ❏✳ ❉✳✱ ❛♥❞ ▲♦❣❤❛✈✐✱ ❙✳ ✭✷✵✶✾✮✳ ❚P✺✸ ♠✉t❛t✐♦♥s ❛r❡ ❝♦♠♠♦♥ ✐♥ ♠❛♥t•❡ ❝❡•• •②♠♣❤♦♠❛✱ ✐♥❝•✉❞✐♥❣ t❤❡ ✐♥❞♦•❡♥t •❡✉❦❡♠✐❝ ♥♦♥✲♥♦❞❛• ✈❛r✐❛♥t✳ ❆♥♥❛•s ♦❢ ❉✐❛❣♥♦st✐❝ P❛t❤♦•♦❣②✱ ✹✶✿✸✽✕✹✷✳ ❙á♥❝❤❡③✲P✉•✐❞♦✱ ▲✳✱ |♦•❛s✱ ❆✳ ▼✳✱ ✈❛♥ ❲❡•②✱ ❑✳ ❍✳✱ ▼❛rt✐♥❡③✲❆✱ ❈✳✱ ❛♥❞ ❱❛•❡♥❝✐❛✱ ❆✳ ✭✷✵✵✹✮✳ ❙P❖❈✿ ❆ ✇✐❞❡•② ❞✐str✐❜✉t❡❞ ❞♦♠❛✐♥ ❛ss♦❝✐❛t❡❞ ✇✐t❤ ❝❛♥❝❡r✱ ❛♣♦♣t♦s✐s ❛♥❞ tr❛♥s❝r✐♣t✐♦♥✳ ❇▼❈ ❇✐♦✐♥❢♦r♠❛t✐❝s✱ ✺✿✾✶✳ ❙❛✈❛r❡s❡✱ ❋✳✱ ❋•❛❤♥❞♦r❢❡r✱ ❑✳✱ ❏❛❡♥✐s❝❤✱ |✳✱ ❇✉ss•✐♥❣❡r✱ ▼✳✱ ❛♥❞ ❲✉t③✱ ❆✳ ✭✷✵✵✻✮✳ ❍❡♠❛t♦♣♦✐❡t✐❝ Pr❡❝✉rs♦r ❈❡••s ❚r❛♥s✐❡♥t•② |❡❡st❛❜•✐s❤ P❡r♠✐ss✐✈❡♥❡ss ❢♦r ❳■♥❛❝t✐✈❛✲ t✐♦♥✳ ▼♦•❡❝✉•❛r ❛♥❞ ❈❡••✉•❛r ❇✐♦•♦❣②✱ ✷✻✭✶✾✮✿✼✶✻✼✕✼✶✼✼✳ ❙❝❤♠✐t③✱ |✳✱ ❲r✐❣❤t✱ •✳ ❲✳✱ ❍✉❛♥❣✱ ❉✳ ❲✳✱ ❏♦❤♥s♦♥✱ ❈✳ ❆✳✱ P❤❡•❛♥✱ ❏✳ ❉✳✱ ❲❛♥❣✱ ❏✳ ◗✳✱ |♦✉••❛♥❞✱ ❙✳✱ ❑❛s❜❡❦❛r✱ ▼✳✱ (♦✉♥❣✱ |✳ ▼✳✱ ❙❤❛✛❡r✱ ❆✳ ▲✳✱ ❍♦❞s♦♥✱ ❉✳ ❏✳✱ ❳✐❛♦✱ ❲✳✱ (✉✱ ❳✳✱ (❛♥❣✱ (✳✱ )❤❛♦✱ ❍✳✱ ❳✉✱ ❲✳✱ ▲✐✉✱ ❳✳✱ )❤♦✉✱ ❇✳✱ ❉✉✱ ❲✳✱ ❈❤❛♥✱ ❲✳ ❈✳✱ ❏❛✛❡✱ ❊✳ ❙✳✱ •❛s❝♦②♥❡✱ |✳ ❉✳✱ ❈♦♥♥♦rs✱ ❏✳ ▼✳✱ ❈❛♠♣♦✱ ❊✳✱ ▲♦♣❡③✲•✉✐••❡r♠♦✱ ❆✳✱ |♦s❡♥✇❛•❞✱ ✷✵✾ |❡❢❡r❡♥❝❡s |❊❋❊|❊◆❈❊❙ ❆✳✱ ❖tt✱ •✳✱ ❉❡•❛❜✐❡✱ ❏✳✱ |✐♠s③❛✱ ▲✳ ▼✳✱ ❚❛② ❑✉❛♥❣ ❲❡✐✱ ❑✳✱ )❡•❡♥❡t③✱ ❆✳ ❉✳✱ ▲❡♦♥❛r❞✱ ❏✳ P✳✱ ❇❛rt•❡tt✱ ◆✳ ▲✳✱ ❚r❛♥✱ ❇✳✱ ❙❤❡tt②✱ ❏✳✱ )❤❛♦✱ (✳✱ ❙♦♣♣❡t✱ ❉✳ |✳✱ P✐tt❛•✉❣❛✱ ❙✳✱ ❲✐•s♦♥✱ ❲✳ ❍✳✱ ❛♥❞ ❙t❛✉❞t✱ ▲✳ ▼✳ ✭✷✵✶✽✮✳ •❡♥❡t✐❝s ❛♥❞ ♣❛t❤♦❣❡♥❡s✐s ♦❢ ❞✐✛✉s❡ •❛r❣❡ ❇✲❈❡•• •②♠♣❤♦♠❛✳ ◆❡✇ ❊♥❣•❛♥❞ ❏♦✉r♥❛• ♦❢ ▼❡❞✐❝✐♥❡✱ ✸✼✽✭✶✺✮✿✶✸✾✻✕✶✹✵✼✳ ❙❝❤♦❡❢t♥❡r✱ ❙✳✱ ❙❡♥❣✉♣t❛✱ ❆✳ ❑✳✱ ❑✉❜✐❝❡❦✱ ❙✳✱ ▼❡❝❤t•❡r✱ ❑✳✱ ❙♣❛❤♥✱ ▲✳✱ ❑♦s❡❦✐✱ ❍✳✱ ❏❡♥✉✇❡✐♥✱ ❚✳✱ ❛♥❞ ❲✉t③✱ ❆✳ ✭✷✵✵✻✮✳ |❡❝r✉✐t♠❡♥t ♦❢ P|❈✶ ❢✉♥❝t✐♦♥ ❛t t❤❡ ✐♥✐t✐❛t✐♦♥ ♦❢ ❳ ✐♥❛❝t✐✈❛t✐♦♥ ✐♥❞❡♣❡♥❞❡♥t ♦❢ P|❈✷ ❛♥❞ s✐•❡♥❝✐♥❣✳ ❚❤❡ ❊▼❇❖ ❏♦✉r♥❛•✱ ✷✺✭✶✸✮✿✸✶✶✵✕ ✸✶✷✷✳ ❙❝❤ü••❡r✱ |✳✱ ❋♦r♥é✱ ■✳✱ ❙tr❛✉❜✱ ❚✳✱ ❙❝❤r❡✐❡❝❦✱ ❆✳✱ ❚❡①✐❡r✱ (✳✱ ❙❤❛❤✱ ◆✳✱ ❉❡❝❦❡r✱ ❚✳ ▼✳✱ ❈r❛♠❡r✱ P✳✱ ■♠❤♦❢✱ ❆✳✱ ❛♥❞ ❊✐❝❦✱ ❉✳ ✭✷✵✶✻✮✳ ❍❡♣t❛❞✲❙♣❡❝✐✜❝ P❤♦s♣❤♦r②•❛t✐♦♥ ♦❢ |◆❆ P♦•②♠❡r❛s❡ ■■ ❈❚❉✳ ▼♦•❡❝✉•❛r ❈❡••✱ ✻✶✭✷✮✿✸✵✺✕✸✶✹✳ ❙❝❤✉•③✱ ❊✳ •✳✱ ▼❡✐s✐❣✱ ❏✳✱ ◆❛❦❛♠✉r❛✱ ❚✳✱ ❖❦❛♠♦t♦✱ ■✳✱ ❙✐❡❜❡r✱ ❆✳✱ P✐❝❛r❞✱ ❈✳✱ ❇♦r❡♥s③t❡✐♥✱ ▼✳✱ ❙❛✐t♦✉✱ ▼✳✱ ❇•üt❤❣❡♥✱ ◆✳✱ ❛♥❞ ❍❡❛r❞✱ ❊✳ ✭✷✵✶✹✮✳ ❚❤❡ t✇♦ ❛❝t✐✈❡ ❳ ❝❤r♦♠♦s♦♠❡s ✐♥ ❢❡♠❛•❡ ❊❙❈s ❜•♦❝❦ ❡①✐t ❢r♦♠ t❤❡ ♣•✉r✐♣♦t❡♥t st❛t❡ ❜② ♠♦❞✉•❛t✐♥❣ t❤❡ ❊❙❈ s✐❣♥❛•✐♥❣ ♥❡t✇♦r❦✳ ❈❡•• ❙t❡♠ ❈❡••✱ ✶✹✭✷✮✿✷✵✸✕✷✶✻✳ ❙❝❤✇❛•❜✱ ❇✳✱ ▼✐❝❤❡•✱ ▼✳✱ )❛❝❤❡r✱ ❇✳✱ ❍❛✉❢✱ ❑✳ ❋✳✱ ❉❡♠❡•✱ ❈✳✱ ❚r❡s❝❤✱ ❆✳✱ •❛❣♥❡✉r✱ ❏✳✱ ❛♥❞ ❈r❛♠❡r✱ P✳ ✭✷✵✶✻✮✳ ❚❚✲s❡q ♠❛♣s t❤❡ ❤✉♠❛♥ tr❛♥s✐❡♥t tr❛♥s❝r✐♣t♦♠❡✳ ❙❝✐❡♥❝❡✱ ✸✺✷✭✻✷✾✵✮✿✶✷✷✺✕✶✷✷✽✳ ❙❤✐✱ (✳✱ ❉♦✇♥❡s✱ ▼✳✱ ❳✐❡✱ ❲✳✱ ❑❛♦✱ ❍✳ (✳✱ ❖r❞❡♥t•✐❝❤✱ P✳✱ ❚s❛✐✱ ❈✳ ❈✳✱ ❍♦♥✱ ▼✳✱ ❛♥❞ ❊✈❛♥s✱ |✳ ▼✳ ✭✷✵✵✶✮✳ ❙❤❛r♣✱ ❛♥ ✐♥❞✉❝✐❜•❡ ❝♦❢❛❝t♦r t❤❛t ✐♥t❡❣r❛t❡s ♥✉❝•❡❛r r❡❝❡♣t♦r r❡♣r❡ss✐♦♥ ❛♥❞ ❛❝t✐✈❛t✐♦♥✳ •❡♥❡s ✫ ❞❡✈❡•♦♣♠❡♥t✱ ✶✺✭✾✮✿✶✶✹✵✕✺✶✳ ❙✐•✈❛✱ ❏✳✱ ▼❛❦✱ ❲✳✱ )✈❡t❦♦✈❛✱ ■✳✱ ❆♣♣❛♥❛❤✱ |✳✱ ◆❡st❡r♦✈❛✱ ❚✳ ❇✳✱ ❲❡❜st❡r✱ )✳✱ P❡t❡rs✱ ❆✳ ❍✳✱ ❏❡♥✉✇❡✐♥✱ ❚✳✱ ❖tt❡✱ ❆✳ P✳✱ ❛♥❞ ❇r♦❝❦❞♦r✛✱ ◆✳ ✭✷✵✵✸✮✳ ❊st❛❜•✐s❤♠❡♥t ♦❢ ❤✐st♦♥❡ ❍✸ ♠❡t❤②•❛t✐♦♥ ♦♥ t❤❡ ✐♥❛❝t✐✈❡ ❳ ❝❤r♦♠♦s♦♠❡ r❡q✉✐r❡s tr❛♥s✐❡♥t r❡❝r✉✐t♠❡♥t ♦❢ ❊❡❞✲ ❊♥①✶ ♣♦•②❝♦♠❜ ❣r♦✉♣ ❝♦♠♣•❡①❡s✳ ❉❡✈❡•♦♣♠❡♥t❛• ❈❡••✱ ✹✭✹✮✿✹✽✶✕✹✾✺✳ ❙❦❡♥❡✱ P✳ ❏✳ ❛♥❞ ❍❡♥✐❦♦✛✱ ❙✳ ✭✷✵✶✼✮✳ ❆♥ ❡✣❝✐❡♥t t❛r❣❡t❡❞ ♥✉❝•❡❛s❡ str❛t❡❣② ❢♦r ❤✐❣❤✲ r❡s♦•✉t✐♦♥ ♠❛♣♣✐♥❣ ♦❢ ❉◆❆ ❜✐♥❞✐♥❣ s✐t❡s✳ ❡▲✐❢❡✱ ✻✳ ❙♠❡❡ts✱ ❉✳✱ ▼❛r❦❛❦✐✱ (✳✱ ❙❝❤♠✐❞✱ ❱✳ ❏✳✱ ❑r❛✉s✱ ❋✳✱ ❚❛tt❡r♠✉s❝❤✱ ❆✳✱ ❈❡r❛s❡✱ ❆✳✱ ❙t❡rr✱ ▼✳✱ ❋✐❡❞•❡r✱ ❙✳✱ ❉❡♠♠❡r•❡✱ ❏✳✱ P♦♣❦❡♥✱ ❏✳✱ ▲❡♦♥❤❛r❞t✱ ❍✳✱ ❇r♦❝❦❞♦r✛✱ ◆✳✱ ❈r❡♠❡r✱ ❚✳✱ ❙❝❤❡r♠❡••❡❤✱ ▲✳✱ ❛♥❞ ❈r❡♠❡r✱ ▼✳ ✭✷✵✶✹✮✳ ❚❤r❡❡✲❞✐♠❡♥s✐♦♥❛• s✉♣❡r✲r❡s♦•✉t✐♦♥ ♠✐✲ ❝r♦s❝♦♣② ♦❢ t❤❡ ✐♥❛❝t✐✈❡ ❳ ❝❤r♦♠♦s♦♠❡ t❡rr✐t♦r② r❡✈❡❛•s ❛ ❝♦••❛♣s❡ ♦❢ ✐ts ❛❝t✐✈❡ ♥✉❝•❡❛r ❝♦♠♣❛rt♠❡♥t ❤❛r❜♦r✐♥❣ ❞✐st✐♥❝t ❳✐st |◆❆ ❢♦❝✐✳ ❊♣✐❣❡♥❡t✐❝s ❛♥❞ ❈❤r♦♠❛t✐♥✱ ✼✭✶✮✿✽✳ ❙ø❣❛❛r❞✱ ❚✳ ▼✳ ▼✳ ❛♥❞ ❙✈❡•str✉♣✱ ❏✳ ◗✳ ✭✷✵✵✼✮✳ ❍②♣❡r♣❤♦s♣❤♦r②•❛t✐♦♥ ♦❢ t❤❡ ❈✲t❡r♠✐♥❛• r❡♣❡❛t ❞♦♠❛✐♥ ♦❢ |◆❆ ♣♦•②♠❡r❛s❡ ■■ ❢❛❝✐•✐t❛t❡s ❞✐ss♦❝✐❛t✐♦♥ ♦❢ ✐ts ❝♦♠♣•❡① ✇✐t❤ ♠❡❞✐✲ ❛t♦r✳ ❏♦✉r♥❛• ♦❢ ❇✐♦•♦❣✐❝❛• ❈❤❡♠✐str②✱ ✷✽✷✭✶✾✮✿✶✹✶✶✸✕✶✹✶✷✵✳ ✷✶✵ |❡❢❡r❡♥❝❡s |❊❋❊|❊◆❈❊❙ ❙♦♥♠❡③✱ ❈✳✱ ❇ä✉r•❡✱ ■✳✱ ▼❛❣✉s✐♥✱ ❆✳✱ ❉r❡♦s✱ |✳✱ ▲❛✉❜✐♥❣❡r✱ ❙✳✱ ❲❡✐❣❡•✱ ❉✳✱ ❛♥❞ ❉❡❛♥✱ ❈✳ ✭✷✵✶✶✮✳ |◆❆ ✸ ′ ♣r♦❝❡ss✐♥❣ ❢✉♥❝t✐♦♥s ♦❢ ❆r❛❜✐❞♦♣s✐s ❋❈❆ ❛♥❞ ❋P❆ •✐♠✐t ✐♥t❡r❣❡♥✐❝ tr❛♥s❝r✐♣t✐♦♥✳ Pr♦❝❡❡❞✐♥❣s ♦❢ t❤❡ ◆❛t✐♦♥❛• ❆❝❛❞❡♠② ♦❢ ❙❝✐❡♥❝❡s ♦❢ t❤❡ ❯♥✐t❡❞ ❙t❛t❡s ♦❢ ❆♠❡r✐❝❛✱ ✶✵✽✭✷✵✮✿✽✺✵✽✕✽✺✶✸✳ ❙♦✉✐•❤♦•✱ ❈✳✱ ❈♦r♠✐❡r✱ ❙✳✱ ❚❛♥✐❣❛❦✐✱ ❑✳✱ ❇❛❜✐♥❡t✱ ❈✳✱ ❛♥❞ ❈♦❤❡♥✲❚❛♥♥♦✉❞•✐✱ ▼✳ ✭✷✵✵✻✮✳ |❇P✲❏κ✲❉❡♣❡♥❞❡♥t ◆♦t❝❤ ❙✐❣♥❛•✐♥❣ ■s ❉✐s♣❡♥s❛❜•❡ ❢♦r ▼♦✉s❡ ❊❛r•② ❊♠❜r②♦♥✐❝ ❉❡✈❡•✲ ♦♣♠❡♥t✳ ▼♦•❡❝✉•❛r ❛♥❞ ❈❡••✉•❛r ❇✐♦•♦❣②✱ ✷✻✭✶✸✮✿✹✼✻✾✕✹✼✼✹✳ ❙♣•✐♥t❡r✱ ❊✳✱ ❞❡ ❲✐t✱ ❊✳✱ ◆♦r❛✱ ❊✳ P✳✱ ❑•♦✉s✱ P✳✱ ✈❛♥ ❞❡ ❲❡r❦❡♥✱ ❍✳ ❏✳✱ )❤✉✱ (✳✱ ❑❛❛✐•✱ ▲✳ ❏✳✱ ✈❛♥ ■•❝❦❡♥✱ ❲✳✱ •r✐❜♥❛✉✱ ❏✳✱ ❍❡❛r❞✱ ❊✳✱ ❛♥❞ ❞❡ ▲❛❛t✱ ❲✳ ✭✷✵✶✶✮✳ ❚❤❡ ✐♥❛❝t✐✈❡ ❳ ❝❤r♦♠♦s♦♠❡ ❛❞♦♣ts ❛ ✉♥✐q✉❡ t❤r❡❡✲❞✐♠❡♥s✐♦♥❛• ❝♦♥❢♦r♠❛t✐♦♥ t❤❛t ✐s ❞❡♣❡♥❞❡♥t ♦♥ ❳✐st |◆❆✳ •❡♥❡s ❛♥❞ ❉❡✈❡•♦♣♠❡♥t✱ ✷✺✭✶✸✮✿✶✸✼✶✕✶✸✽✸✳ ❙t❛✈r♦♣♦✉•♦s✱ ◆✳✱ ▲✉✱ ◆✳✱ ❛♥❞ ▲❡❡✱ ❏✳ ❚✳ ✭✷✵✵✶✮✳ ❆ ❢✉♥❝t✐♦♥❛• r♦•❡ ❢♦r ❚s✐① tr❛♥s❝r✐♣t✐♦♥ ✐♥ ❜•♦❝❦✐♥❣ ❳✐st |◆❆ ❛❝❝✉♠✉•❛t✐♦♥ ❜✉t ♥♦t ✐♥ ❳✲❝❤r♦♠♦s♦♠❡ ❝❤♦✐❝❡✳ Pr♦❝❡❡❞✐♥❣s ♦❢ t❤❡ ◆❛t✐♦♥❛• ❆❝❛❞❡♠② ♦❢ ❙❝✐❡♥❝❡s ♦❢ t❤❡ ❯♥✐t❡❞ ❙t❛t❡s ♦❢ ❆♠❡r✐❝❛✱ ✾✽✭✶✽✮✿✶✵✷✸✷✕✶✵✷✸✼✳ ❙t❡♣❤❡♥s✱ P✳ ❏✳✱ ❉❛✈✐❡s✱ ❍✳ |✳✱ ▼✐t❛♥✐✱ (✳✱ ❱❛♥ ▲♦♦✱ P✳✱ ❙❤•✐❡♥✱ ❆✳✱ ❚❛r♣❡②✱ P✳ ❙✳✱ P❛✲ ♣❛❡♠♠❛♥✉✐•✱ ❊✳✱ ❈❤❡✈❡rt♦♥✱ ❆✳✱ ❇✐❣♥❡••✱ •✳ |✳✱ ❇✉t•❡r✱ ❆✳ P✳✱ •❛♠❜•❡✱ ❏✳✱ •❛♠❜•❡✱ ❙✳✱ ❍❛r❞②✱ ❈✳✱ ❍✐♥t♦♥✱ ❏✳✱ ❏✐❛✱ ▼✳✱ ❏❛②❛❦✉♠❛r✱ ❆✳✱ ❏♦♥❡s✱ ❉✳✱ ▲❛t✐♠❡r✱ ❈✳✱ ▼❝▲❛r❡♥✱ ❙✳✱ ▼❝❇r✐❞❡✱ ❉✳ ❏✳✱ ▼❡♥③✐❡s✱ ❆✳✱ ▼✉❞✐❡✱ ▲✳✱ ▼❛❞❞✐s♦♥✱ ▼✳✱ |❛✐♥❡✱ ❑✳✱ ◆✐❦✲)❛✐♥❛•✱ ❙✳✱ ❖✬▼❡❛r❛✱ ❙✳✱ ❚❡❛❣✉❡✱ ❏✳ ❲✳✱ ❱❛r❡•❛✱ ■✳✱ ❲❡❞❣❡✱ ❉✳ ❈✳✱ ❲❤✐t♠♦r❡✱ ■✳✱ ▲✐♣♣♠❛♥✱ ❙✳ ▼✳✱ ▼❝❉❡r♠♦tt✱ ❯✳✱ ❙tr❛tt♦♥✱ ▼✳ |✳✱ ❈❛♠♣❜❡••✱ P✳ ❏✳✱ ❊•✲◆❛❣❣❛r✱ ❆✳ ❑✳✱ ❛♥❞ ❋✉tr❡❛•✱ P✳ ❆✳ ✭✷✵✶✸✮✳ ❲❤♦•❡ ❡①♦♠❡ s❡q✉❡♥❝✐♥❣ ♦❢ ❛❞❡♥♦✐❞ ❝②st✐❝ ❝❛r❝✐♥♦♠❛✳ ❏♦✉r♥❛• ♦❢ ❈•✐♥✐❝❛• ■♥✈❡st✐❣❛t✐♦♥✱ ✶✷✸✭✼✮✿✷✾✻✺✕✷✾✻✽✳ ❙✉♥✇♦♦✱ ❍✳✱ ❈♦•♦❣♥♦r✐✱ ❉✳✱ ❋r♦❜❡r❣✱ ❏✳ ❊✳✱ ❏❡♦♥✱ (✳✱ ❛♥❞ ▲❡❡✱ ❏✳ ❚✳ ✭✷✵✶✼✮✳ |❡♣❡❛t ❊ ❛♥❝❤♦rs ❳✐st |◆❆ t♦ t❤❡ ✐♥❛❝t✐✈❡ ❳ ❝❤r♦♠♦s♦♠❛• ❝♦♠♣❛rt♠❡♥t t❤r♦✉❣❤ ❈❉❑◆✶❆✲ ✐♥t❡r❛❝t✐♥❣ ♣r♦t❡✐♥ ✭❈■)✶✮✳ Pr♦❝❡❡❞✐♥❣s ♦❢ t❤❡ ◆❛t✐♦♥❛• ❆❝❛❞❡♠② ♦❢ ❙❝✐❡♥❝❡s ♦❢ t❤❡ ❯♥✐t❡❞ ❙t❛t❡s ♦❢ ❆♠❡r✐❝❛✱ ✶✶✹✭✹✵✮✿✶✵✻✺✹✕✶✵✻✺✾✳ ❚❛❦❛❣✐✱ ◆✳ ❛♥❞ ❙❛s❛❦✐✱ ▼✳ ✭✶✾✼✺✮✳ Pr❡❢❡r❡♥t✐❛• ✐♥❛❝t✐✈❛t✐♦♥ ♦❢ t❤❡ ♣❛t❡r♥❛••② ❞❡r✐✈❡❞ ❳ ❝❤r♦♠♦s♦♠❡ ✐♥ t❤❡ ❡①tr❛❡♠❜r②♦♥✐❝ ♠❡♠❜r❛♥❡s ♦❢ t❤❡ ♠♦✉s❡✳ ◆❛t✉r❡✱ ✷✺✻✭✺✺✶✾✮✿✻✹✵✕ ✻✹✷✳ ❚❛♥✱ ❳✳✱ ❈❛•❞❡r♦♥✲❱✐••❛•♦❜♦s✱ ▲✳ ■✳ ❆✳✱ ❙❤❛r♦♥✱ ▼✳✱ )❤❡♥❣✱ ❈✳✱ |♦❜✐♥s♦♥✱ ❈✳ ❱✳✱ ❊st❡••❡✱ ▼✳✱ ❛♥❞ )❤❡♥❣✱ ◆✳ ✭✷✵✵✼✮✳ ▼❡❝❤❛♥✐s♠ ♦❢ ❛✉①✐♥ ♣❡r❝❡♣t✐♦♥ ❜② t❤❡ ❚■|✶ ✉❜✐q✉✐t✐♥ •✐❣❛s❡✳ ◆❛t✉r❡✱ ✹✹✻✭✼✶✸✻✮✿✻✹✵✕✻✹✺✳ ❚❛♥❣✱ ❋✳✱ ❇❛r❜❛❝✐♦r✉✱ ❈✳✱ ◆♦r❞♠❛♥✱ ❊✳✱ ▲✐✱ ❇✳✱ ❳✉✱ ◆✳✱ ❇❛s❤❦✐r♦✈✱ ❱✳ ■✳✱ ▲❛♦✱ ❑✳✱ ❛♥❞ ❙✉r❛♥✐✱ ▼✳ ❆✳ ✭✷✵✶✵✮✳ |◆❆✲❙❡q ❛♥❛•②s✐s t♦ ❝❛♣t✉r❡ t❤❡ tr❛♥s❝r✐♣t♦♠❡ •❛♥❞s❝❛♣❡ ♦❢ ❛ s✐♥❣•❡ ❝❡••✳ ◆❛t✉r❡ Pr♦t♦❝♦•s✱ ✺✭✸✮✿✺✶✻✕✺✸✺✳ ✷✶✶ |❡❢❡r❡♥❝❡s |❊❋❊|❊◆❈❊❙ ❚✐❛♥✱ ❉✳✱ ❙✉♥✱ ❙✳✱ ❛♥❞ ▲❡❡✱ ❏✳ ❚✳ ✭✷✵✶✵✮✳ ❚❤❡ •♦♥❣ ♥♦♥❝♦❞✐♥❣ |◆❆✱ ❏♣①✱ ■s ❛ ♠♦•❡❝✉•❛r s✇✐t❝❤ ❢♦r ❳ ❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥✳ ❈❡••✱ ✶✹✸✭✸✮✿✸✾✵✕✹✵✸✳ ❚♦♥❣✱ ❏✳ ❑✳✱ ❍❛ss✐❣✱ ❈✳ ❆✳✱ ❙❝❤♥✐t③•❡r✱ •✳ |✳✱ ❑✐♥❣st♦♥✱ |✳ ❊✳✱ ❛♥❞ ❙❝❤r❡✐❜❡r✱ ❙✳ ▲✳ ✭✶✾✾✽✮✳ ❈❤r♦♠❛t✐♥ ❞❡❛❝❡t②•❛t✐♦♥ ❜② ❛♥ ❆❚P✲❞❡♣❡♥❞❡♥t ♥✉❝•❡♦s♦♠❡ r❡♠♦❞❡••✐♥❣ ❝♦♠♣•❡①✳ ◆❛✲ t✉r❡✱ ✸✾✺✭✻✼✵✺✮✿✾✶✼✕✾✷✶✳ ❚s✉•✐✱ ▼✳✱ ❙❤✐♥❦✉r❛✱ |✳✱ ❑✉r♦❞❛✱ ❑✳✱ (❛❜❡✱ ❉✳✱ ❛♥❞ ❍♦♥•♦✱ ❚✳ ✭✷✵✵✼✮✳ ▼s①✷✲✐♥t❡r❛❝t✐♥❣ ♥✉❝•❡❛r t❛r❣❡t ♣r♦t❡✐♥ ✭▼✐♥t✮ ❞❡✜❝✐❡♥❝② r❡✈❡❛•s ♥❡❣❛t✐✈❡ r❡❣✉•❛t✐♦♥ ♦❢ ❡❛r•② t❤②♠♦❝②t❡ ❞✐✛❡r❡♥t✐❛t✐♦♥ ❜② ◆♦t❝❤✴|❇P✲❏ s✐❣♥❛•✐♥❣✳ Pr♦❝❡❡❞✐♥❣s ♦❢ t❤❡ ◆❛t✐♦♥❛• ❆❝❛❞❡♠② ♦❢ ❙❝✐❡♥❝❡s ♦❢ t❤❡ ❯♥✐t❡❞ ❙t❛t❡s ♦❢ ❆♠❡r✐❝❛✱ ✶✵✹✭✺✮✿✶✻✶✵✕✶✻✶✺✳ ❱❛••♦t✱ ❈✳✱ P❛tr❛t✱ ❈✳✱ ❈♦••✐❡r✱ ❆✳ ❏✳✱ ❍✉r❡t✱ ❈✳✱ ❈❛s❛♥♦✈❛✱ ▼✳✱ ▲✐②❛❦❛t ❆•✐✱ ❚✳ ▼✳✱ ❚♦s♦•✐♥✐✱ ▼✳✱ ❋r②❞♠❛♥✱ ◆✳✱ ❍❡❛r❞✱ ❊✳✱ |✉❣❣✲•✉♥♥✱ P✳ ❏✳✱ ❛♥❞ |♦✉❣❡✉••❡✱ ❈✳ ✭✷✵✶✼✮✳ ❳❆❈❚ ◆♦♥❝♦❞✐♥❣ |◆❆ ❈♦♠♣❡t❡s ✇✐t❤ ❳■❙❚ ✐♥ t❤❡ ❈♦♥tr♦• ♦❢ ❳ ❈❤r♦♠♦s♦♠❡ ❆❝t✐✈✐t② ❞✉r✐♥❣ ❍✉♠❛♥ ❊❛r•② ❉❡✈❡•♦♣♠❡♥t✳ ❈❡•• ❙t❡♠ ❈❡••✱ ✷✵✭✶✮✿✶✵✷✕✶✶✶✳ ❱❛•♦t✱ ❇✳✱ ▲❛♥❣❡••❛✱ ❖✳✱ ◆❛♥♦✱ ❊✳✱ ❛♥❞ )✐✈②✱ ▼✳ ✭✷✵✶✶✮✳ ▼❛ss❈❤r♦◗✿ ❆ ✈❡rs❛t✐•❡ t♦♦• ❢♦r ♠❛ss s♣❡❝tr♦♠❡tr② q✉❛♥t✐✜❝❛t✐♦♥✳ Pr♦t❡♦♠✐❝s✱ ✶✶✭✶✼✮✿✸✺✼✷✕✸✺✼✼✳ ✈❛♥ ❇❡♠♠❡•✱ ❏✳ •✳✱ ▼✐r❛✲❇♦♥t❡♥❜❛•✱ ❍✳✱ ❛♥❞ •r✐❜♥❛✉✱ ❏✳ ✭✷✵✶✻✮✳ ❈✐s✲ ❛♥❞ tr❛♥s✲r❡❣✉•❛t✐♦♥ ✐♥ ❳ ✐♥❛❝t✐✈❛t✐♦♥✳ ❈❤r♦♠♦s♦♠❛✱ ✶✷✺✭✶✮✿✹✶✕✺✵✳ ❱❛♥❞❡r✇✐❡•❡♥✱ ❇✳ ❉✳✱ (✉❛♥✱ )✳✱ ❋r✐❡❞♠❛♥♥✱ ❉✳ |✳✱ ❛♥❞ ❑♦✈❛••✱ |✳ ❆✳ ✭✷✵✶✶✮✳ ❚r❛♥s❝r✐♣✲ t✐♦♥❛• r❡♣r❡ss✐♦♥ ✐♥ t❤❡ ◆♦t❝❤ ♣❛t❤✇❛②✿ ❚❤❡r♠♦❞②♥❛♠✐❝ ❝❤❛r❛❝t❡r✐③❛t✐♦♥ ♦❢ ❈❙▲✲ ▼■◆❚ ✭▼s①✷✲✐♥t❡r❛❝t✐♥❣ ♥✉❝•❡❛r t❛r❣❡t ♣r♦t❡✐♥✮ ❝♦♠♣•❡①❡s✳ ❏♦✉r♥❛• ♦❢ ❇✐♦•♦❣✐❝❛• ❈❤❡♠✐str②✱ ✷✽✻✭✶✼✮✿✶✹✽✾✷✕✶✹✾✵✷✳ ❱❛s✐••❡✈❛✱ ▲✳✱ ❑✐♠✱ ▼✳✱ ▼✉ts❝❤•❡r✱ ❍✳✱ ❇✉r❛t♦✇s❦✐✱ ❙✳✱ ❛♥❞ ▼❡✐♥❤❛rt✱ ❆✳ ✭✷✵✵✽✮✳ ❚❤❡ ◆r❞✶✲◆❛❜✸✲❙❡♥✶ t❡r♠✐♥❛t✐♦♥ ❝♦♠♣•❡① ✐♥t❡r❛❝ts ✇✐t❤ t❤❡ ❙❡r✺✲♣❤♦s♣❤♦r②•❛t❡❞ |◆❆ ♣♦•②♠❡r❛s❡ ■■ ❈✲t❡r♠✐♥❛• ❞♦♠❛✐♥✳ ◆❛t✉r❡ ❙tr✉❝t✉r❛• ❛♥❞ ▼♦•❡❝✉•❛r ❇✐♦•♦❣②✱ ✶✺✭✽✮✿✼✾✺✕ ✽✵✹✳ ❱✐❝♦s♦✱ ❇✳ ❛♥❞ ❈❤❛r•❡s✇♦rt❤✱ ❇✳ ✭✷✵✵✻✮✳ ❊✈♦•✉t✐♦♥ ♦♥ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡✿ ✉♥✉s✉❛• ♣❛tt❡r♥s ❛♥❞ ♣r♦❝❡ss❡s✳ ◆❛t✉r❡ |❡✈✐❡✇s •❡♥❡t✐❝s✱ ✼✭✽✮✿✻✹✺✕✻✺✸✳ ❱♦•♥✐❝✱ ❊✳✱ ❙✐♠♦♥✱ ❇✳✱ ❙tr❛❤•✱ ❇✳ ❉✳✱ ❙❛tt•❡r✱ ▼✳✱ ❛♥❞ ❈r❛♠❡r✱ P✳ ✭✷✵✵✻✮✳ ❙tr✉❝t✉r❡ ❛♥❞ ❝❛r❜♦①②•✲t❡r♠✐♥❛• ❞♦♠❛✐♥ ✭❈❚❉✮ ❜✐♥❞✐♥❣ ♦❢ t❤❡ ❙❡t✷ ❙|■ ❞♦♠❛✐♥ t❤❛t ❝♦✉♣•❡s ❤✐st♦♥❡ ❍✸ ▲②s✸✻ ♠❡t❤②•❛t✐♦♥ t♦ tr❛♥s❝r✐♣t✐♦♥✳ ❏♦✉r♥❛• ♦❢ ❇✐♦•♦❣✐❝❛• ❈❤❡♠✐str②✱ ✷✽✶✭✶✮✿✶✸✕✶✺✳ ❲❛❞❡✱ P✳ ❆✳✱ ❏♦♥❡s✱ P✳ ▲✳✱ ❱❡r♠❛❛❦✱ ❉✳✱ ❛♥❞ ❲♦•✛❡✱ ❆✳ P✳ ✭✶✾✾✽✮✳ ❆ ♠✉•t✐♣•❡ s✉❜✉♥✐t ▼✐✲✷ ❤✐st♦♥❡ ❞❡❛❝❡t②•❛s❡ ❢r♦♠ ❳❡♥♦♣✉s •❛❡✈✐s ❝♦❢r❛❝t✐♦♥❛t❡s ✇✐t❤ ❛♥ ❛ss♦❝✐❛t❡❞ ❙♥❢✷ s✉♣❡r❢❛♠✐•② ❆❚P❛s❡✳ ❈✉rr❡♥t ❇✐♦•♦❣②✱ ✽✭✶✹✮✿✽✹✸✕✽✹✽✳ ✷✶✷ |❡❢❡r❡♥❝❡s |❊❋❊|❊◆❈❊❙ ❲❛♥❣✱ ❏✳✱ ▼❛❣❡r✱ ❏✳✱ ❈❤❡♥✱ (✳✱ ❙❝❤♥❡✐❞❡r✱ ❊✳✱ ❈r♦ss✱ ❏✳ ❈✳✱ ◆❛❣②✱ ❆✳✱ ❛♥❞ ▼❛❣♥✉s♦♥✱ ❚✳ ✭✷✵✵✶✮✳ ■♠♣r✐♥t❡❞ ❳ ✐♥❛❝t✐✈❛t✐♦♥ ♠❛✐♥t❛✐♥❡❞ ❜② ❛ ♠♦✉s❡ P♦•②❝♦♠❜ ❣r♦✉♣ ❣❡♥❡✳ ◆❛t✉r❡ •❡♥❡t✐❝s✱ ✷✽✭✹✮✿✸✼✶✕✸✼✺✳ ❲❡st✱ ❏✳ ❉✳✱ ❋r❡•s✱ ❲✳ ■✳✱ ❈❤❛♣♠❛♥✱ ❱✳ ▼✳✱ ❛♥❞ P❛♣❛✐♦❛♥♥♦✉✱ ❱✳ ❊✳ ✭✶✾✼✼✮✳ Pr❡❢❡r❡♥✲ t✐❛• ❡①♣r❡ss✐♦♥ ♦❢ t❤❡ ♠❛t❡r♥❛••② ❞❡r✐✈❡❞ ❳ ❝❤r♦♠♦s♦♠❡ ✐♥ t❤❡ ♠♦✉s❡ ②♦•❦ s❛❝✳ ❈❡••✱ ✶✷✭✹✮✿✽✼✸✕✽✽✷✳ ❲✐❡••❡tt❡✱ ❊✳ ▲✳✱ ❍❛r❞✐♥❣✱ ❑✳ ❲✳✱ ▼❛❝❡✱ ❑✳ ❆✳✱ |♦♥s❤❛✉❣❡♥✱ ▼✳ |✳✱ ❲❛♥❣✱ ❋✳ (✳✱ ❛♥❞ ▼❝•✐♥♥✐s✱ ❲✳ ✭✶✾✾✾✮✳ s♣❡♥ ❡♥❝♦❞❡s ❛♥ |◆P ♠♦t✐❢ ♣r♦t❡✐♥ t❤❛t ✐♥t❡r❛❝ts ✇✐t❤ ❍♦① ♣❛t❤✇❛②s t♦ r❡♣r❡ss t❤❡ ❞❡✈❡•♦♣♠❡♥t ♦❢ ❤❡❛❞✲•✐❦❡ s❝•❡r✐t❡s ✐♥ t❤❡ ❉r♦s♦♣❤✐•❛ tr✉♥❦✳ ❉❡✈❡•♦♣♠❡♥t✱ ✶✷✻✭✷✸✮✿✺✸✼✸✕✺✸✽✺✳ ❲✉t③✱ ❆✳ ❛♥❞ ❏❛❡♥✐s❝❤✱ |✳ ✭✷✵✵✵✮✳ ❆ s❤✐❢t ❢r♦♠ r❡✈❡rs✐❜•❡ t♦ ✐rr❡✈❡rs✐❜•❡ ❳ ✐♥❛❝t✐✈❛t✐♦♥ ✐s tr✐❣❣❡r❡❞ ❞✉r✐♥❣ ❊❙ ❝❡•• ❞✐✛❡r❡♥t✐❛t✐♦♥✳ ▼♦•❡❝✉•❛r ❝❡••✱ ✺✭✹✮✿✻✾✺✕✼✵✺✳ ❲✉t③✱ ❆✳✱ |❛s♠✉ss❡♥✱ ❚✳ P✳✱ ❛♥❞ ❏❛❡♥✐s❝❤✱ |✳ ✭✷✵✵✷✮✳ ❈❤r♦♠♦s♦♠❛• s✐•❡♥❝✐♥❣ ❛♥❞ •♦❝❛•✐③❛✲ t✐♦♥ ❛r❡ ♠❡❞✐❛t❡❞ ❜② ❞✐✛❡r❡♥t ❞♦♠❛✐♥s ♦❢ ❳✐st |◆❆✳ ◆❛t✉r❡ •❡♥❡t✐❝s✱ ✸✵✭✷✮✿✶✻✼✕✶✼✹✳ ❳✉❡✱ (✳✱ ❲♦♥❣✱ ❏✳✱ ▼♦r❡♥♦✱ •✳ ❚✳✱ (♦✉♥❣✱ ▼✳ ❑✳✱ ❈ôté✱ ❏✳✱ ❛♥❞ ❲❛♥❣✱ ❲✳ ✭✶✾✾✽✮✳ ◆❯|❉✱ ❛ ♥♦✈❡• ❝♦♠♣•❡① ✇✐t❤ ❜♦t❤ ❆❚P✲❞❡♣❡♥❞❡♥t ❝❤r♦♠❛t✐♥✲r❡♠♦❞❡•✐♥❣ ❛♥❞ ❤✐st♦♥❡ ❞❡❛❝❡t②•❛s❡ ❛❝t✐✈✐t✐❡s✳ ▼♦•❡❝✉•❛r ❈❡••✱ ✷✭✻✮✿✽✺✶✕✽✻✶✳ (❛❜❡✱ ❉✳✱ ❋✉❦✉❞❛✱ ❍✳✱ ❆♦❦✐✱ ▼✳✱ (❛♠❛❞❛✱ ❙✳✱ ❚❛❦❡❜❛②❛s❤✐✱ ❙✳✱ ❙❤✐♥❦✉r❛✱ |✳✱ (❛♠❛♠♦t♦✱ ◆✳✱ ❛♥❞ ❍♦♥•♦✱ ❚✳ ✭✷✵✵✼✮✳ •❡♥❡r❛t✐♦♥ ♦❢ ❛ ❝♦♥❞✐t✐♦♥❛• ❦♥♦❝❦♦✉t ❛••❡•❡ ❢♦r ♠❛♠♠❛•✐❛♥ s♣❡♥ ♣r♦t❡✐♥ ▼✐♥t✴❙❍❆|P✳ •❡♥❡s✐s✱ ✹✺✭✺✮✿✸✵✵✕✸✵✻✳ (❛♠❛❞❛✱ ◆✳✱ ❍❛s❡❣❛✇❛✱ (✳✱ (✉❡✱ ▼✳✱ ❍❛♠❛❞❛✱ ❚✳✱ ◆❛❦❛❣❛✇❛✱ ❙✳✱ ❛♥❞ ❖❣❛✇❛✱ (✳ ✭✷✵✶✺✮✳ ❳✐st ❊①♦♥ ✼ ❈♦♥tr✐❜✉t❡s t♦ t❤❡ ❙t❛❜•❡ ▲♦❝❛•✐③❛t✐♦♥ ♦❢ ❳✐st |◆❆ ♦♥ t❤❡ ■♥❛❝t✐✈❡ ❳✲ ❈❤r♦♠♦s♦♠❡✳ P▲♦❙ •❡♥❡t✐❝s✱ ✶✶✭✽✮✿❡✶✵✵✺✹✸✵✳ (❛♥❣✱ ❋✳✱ ❇❛❜❛❦✱ ❚✳✱ ❙❤❡♥❞✉r❡✱ ❏✳✱ ❛♥❞ ❉✐st❡❝❤❡✱ ❈✳ ▼✳ ✭✷✵✶✵✮✳ ••♦❜❛• s✉r✈❡② ♦❢ ❡s❝❛♣❡ ❢r♦♠ ❳ ✐♥❛❝t✐✈❛t✐♦♥ ❜② |◆❆✲s❡q✉❡♥❝✐♥❣ ✐♥ ♠♦✉s❡✳ •❡♥♦♠❡ |❡s❡❛r❝❤✱ ✷✵✭✺✮✿✻✶✹✕✻✷✷✳ (✐•❞✐r✐♠✱ ❊✳✱ ❑✐r❜②✱ ❏✳ ❊✳✱ ❇r♦✇♥✱ ❉✳ ❊✳✱ ▼❡r❝✐❡r✱ ❋✳ ❊✳✱ ❙❛❞r❡②❡✈✱ |✳ ■✳✱ ❙❝❛❞❞❡♥✱ ❉✳ ❚✳✱ ❛♥❞ ▲❡❡✱ ❏✳ ❚✳ ✭✷✵✶✸✮✳ ❳✐st |◆❆ ✐s ❛ ♣♦t❡♥t s✉♣♣r❡ss♦r ♦❢ ❤❡♠❛t♦•♦❣✐❝ ❝❛♥❝❡r ✐♥ ♠✐❝❡✳ ❈❡••✱ ✶✺✷✭✹✮✿✼✷✼✕✼✹✷✳ (♦❤✱ ❙✳ ▼✳✱ ❈❤♦✱ ❍✳✱ P✐❝❦•❡✱ ▲✳✱ ❊✈❛♥s✱ |✳ ▼✳✱ ❛♥❞ ❏♦♥❡s✱ ❑✳ ❆✳ ✭✷✵✵✼✮✳ ❚❤❡ ❙♣t✻ ❙❍✷ ❞♦♠❛✐♥ ❜✐♥❞s ❙❡r✷✲P |◆❆P■■ t♦ ❞✐r❡❝t ■✇s✶✲❞❡♣❡♥❞❡♥t ♠|◆❆ s♣•✐❝✐♥❣ ❛♥❞ ❡①♣♦rt✳ •❡♥❡s ❛♥❞ ❉❡✈❡•♦♣♠❡♥t✱ ✷✶✭✷✮✿✶✻✵✕✶✼✹✳ (✉✱ ❇✳✱ ◗✐✱ (✳✱ ▲✐✱ |✳✱ ❙❤✐✱ ◗✳✱ ❙❛t♣❛t❤②✱ ❆✳ ❚✳✱ ❛♥❞ ❈❤❛♥❣✱ ❍✳ (✳ ✭✷✵✷✶✮✳ ❇ ❝❡••✲s♣❡❝✐✜❝ ❳■❙❚ ❝♦♠♣•❡① ❡♥❢♦r❝❡s ❳✲✐♥❛❝t✐✈❛t✐♦♥ ❛♥❞ r❡str❛✐♥s ❛t②♣✐❝❛• ❇ ❝❡••s✳ ❈❡••✱ ✵✭✵✮✳ ✷✶✸ |❡❢❡r❡♥❝❡s |❊❋❊|❊◆❈❊❙ (✉✱ ❳✳✱ ▼❛rt✐♥✱ P✳ •✳✱ ❛♥❞ ▼✐❝❤❛❡•s✱ ❙✳ ❉✳ ✭✷✵✶✾✮✳ ❇❖|❉❊| ♣r♦t❡✐♥s ♣r♦t❡❝t ❡①♣r❡ss✐♦♥ ♦❢ ♥❡✐❣❤❜♦r✐♥❣ ❣❡♥❡s ❜② ♣r♦♠♦t✐♥❣ ✸ ′ P♦• ■■ ♣❛✉s✐♥❣ ✐♥ ♣•❛♥ts✳ ◆❛t✉r❡ ❈♦♠♠✉♥✐❝❛t✐♦♥s✱ ✶✵✭✶✮✿✶✕✶✺✳ (✉❛♥✱ )✳✱ ❱❛♥❞❡r❲✐❡•❡♥✱ ❇✳ ❉✳✱ •✐❛✐♠♦✱ ❇✳ ❉✳✱ P❛♥✱ ▲✳✱ ❈♦••✐♥s✱ ❈✳ ❊✳✱ ❚✉r❦✐❡✇✐❝③✱ ❆✳✱ ❍❡✐♥✱ ❑✳✱ ❖s✇❛•❞✱ ❋✳✱ ❇♦r❣❣r❡❢❡✱ ❚✳✱ ❛♥❞ ❑♦✈❛••✱ |✳ ❆✳ ✭✷✵✶✾✮✳ ❙tr✉❝t✉r❛• ❛♥❞ ❋✉♥❝t✐♦♥❛• ❙t✉❞✐❡s ♦❢ t❤❡ |❇P❏✲❙❍❆|P ❈♦♠♣•❡① |❡✈❡❛• ❛ ❈♦♥s❡r✈❡❞ ❈♦r❡♣r❡ss♦r ❇✐♥❞✐♥❣ ❙✐t❡✳ ❈❡•• |❡♣♦rts✱ ✷✻✭✹✮✿✽✹✺✕✽✺✹✳❡✻✳ )❛❝❝❛r❛✱ ❙✳✱ |✐❡s✱ |✳ ❏✳✱ ❛♥❞ ❏❛✛r❡②✱ ❙✳ |✳ ✭✷✵✶✾✮✳ |❡❛❞✐♥❣✱ ✇r✐t✐♥❣ ❛♥❞ ❡r❛s✐♥❣ ♠|◆❆ ♠❡t❤②•❛t✐♦♥✳ ◆❛t✉r❡ |❡✈✐❡✇s ▼♦•❡❝✉•❛r ❈❡•• ❇✐♦•♦❣②✱ ✷✵✭✶✵✮✿✻✵✽✕✻✷✹✳ )❤❛♥✱ ❚✳✱ |✐♥❞t♦r✛✱ ◆✳✱ ❛♥❞ ❇♦✉tr♦s✱ ▼✳ ✭✷✵✶✼✮✳ ❲♥t s✐❣♥❛•✐♥❣ ✐♥ ❝❛♥❝❡r✳ ❖♥❝♦❣❡♥❡✱ ✸✻✭✶✶✮✿✶✹✻✶✕✶✹✼✸✳ )❤❛♥❣✱ ▲✳ ❋✳✱ ❍✉②♥❤✱ ❑✳ ❉✳✱ ❛♥❞ ▲❡❡✱ ❏✳ ❚✳ ✭✷✵✵✼✮✳ P❡r✐♥✉❝•❡♦•❛r ❚❛r❣❡t✐♥❣ ♦❢ t❤❡ ■♥❛❝t✐✈❡ ❳ ❞✉r✐♥❣ ❙ P❤❛s❡✿ ❊✈✐❞❡♥❝❡ ❢♦r ❛ |♦•❡ ✐♥ t❤❡ ▼❛✐♥t❡♥❛♥❝❡ ♦❢ ❙✐•❡♥❝✐♥❣✳ ❈❡••✱ ✶✷✾✭✹✮✿✻✾✸✕✼✵✻✳ )❤❛♥❣✱ (✳✱ ■r❛t♥✐✱ |✳✱ ❊r❞•✉♠❡♥t✲❇r♦♠❛❣❡✱ ❍✳✱ ❚❡♠♣st✱ P✳✱ ❛♥❞ |❡✐♥❜❡r❣✱ ❉✳ ✭✶✾✾✼✮✳ ❍✐st♦♥❡ ❞❡❛❝❡t②•❛s❡s ❛♥❞ ❙❆P✶✽✱ ❛ ♥♦✈❡• ♣♦•②♣❡♣t✐❞❡✱ ❛r❡ ❝♦♠♣♦♥❡♥ts ♦❢ ❛ ❤✉♠❛♥ ❙✐♥✸ ❝♦♠♣•❡①✳ ❈❡••✱ ✽✾✭✸✮✿✸✺✼✕✸✻✹✳ )❤❛♥❣✱ (✳✱ ▲❡|♦②✱ •✳✱ ❙❡❡•✐❣✱ ❍✳ P✳✱ ▲❛♥❡✱ ❲✳ ❙✳✱ ❛♥❞ |❡✐♥❜❡r❣✱ ❉✳ ✭✶✾✾✽✮✳ ❚❤❡ ❞❡r♠❛t♦♠②♦s✐t✐s✲s♣❡❝✐✜❝ ❛✉t♦❛♥t✐❣❡♥ ▼✐✷ ✐s ❛ ❝♦♠♣♦♥❡♥t ♦❢ ❛ ❝♦♠♣•❡① ❝♦♥t❛✐♥✐♥❣ ❤✐s✲ t♦♥❡ ❞❡❛❝❡t②•❛s❡ ❛♥❞ ♥✉❝•❡♦s♦♠❡ r❡♠♦❞❡•✐♥❣ ❛❝t✐✈✐t✐❡s✳ ❈❡••✱ ✾✺✭✷✮✿✷✼✾✕✷✽✾✳ )❤❛♥❣✱ (✳✱ |❛t❛•✱ ❑✳✱ ❙✐♠♣s♦♥✱ •✳ •✳✱ ❛♥❞ ❚♦♥❣✱ ▲✳ ✭✷✵✶✻✮✳ ❈r②st❛• str✉❝t✉r❡ ♦❢ t❤❡ ❙P❖❈ ❞♦♠❛✐♥ ♦❢ t❤❡ ❛r❛❜✐❞♦♣s✐s ✢♦✇❡r✐♥❣ r❡❣✉•❛t♦r ❋P❆✳ P▲♦❙ ❖◆❊✱ ✶✶✭✽✮✳ )❤❛♦✱ ❏✳✱ ❙✉♥✱ ❇✳ ❑✳✱ ❊r✇✐♥✱ ❏✳ ❆✳✱ ❙♦♥❣✱ ❏✳ ❏✳✱ ❛♥❞ ▲❡❡✱ ❏✳ ❚✳ ✭✷✵✵✽✮✳ P♦•②❝♦♠❜ ♣r♦t❡✐♥s t❛r❣❡t❡❞ ❜② ❛ s❤♦rt r❡♣❡❛t |◆❆ t♦ t❤❡ ♠♦✉s❡ ❳ ❝❤r♦♠♦s♦♠❡✳ ❙❝✐❡♥❝❡✱ ✸✷✷✭✺✾✵✷✮✿✼✺✵✕ ✼✺✻✳ )❤✉✱ ❇✳✱ )❤❡♥❣✱ (✳✱ P❤❛♠✱ ❆✳ ❉✳✱ ▼❛♥❞❛•✱ ❙✳ ❙✳✱ ❊r❞•✉♠❡♥t✲❇r♦♠❛❣❡✱ ❍✳✱ ❚❡♠♣st✱ P✳✱ ❛♥❞ |❡✐♥❜❡r❣✱ ❉✳ ✭✷✵✵✺✮✳ ▼♦♥♦✉❜✐q✉✐t✐♥❛t✐♦♥ ♦❢ ❤✉♠❛♥ ❤✐st♦♥❡ ❍✷❇✿ ❚❤❡ ❢❛❝t♦rs ✐♥✈♦•✈❡❞ ❛♥❞ t❤❡✐r r♦•❡s ✐♥ ❍❖❳ ❣❡♥❡ r❡❣✉•❛t✐♦♥✳ ▼♦•❡❝✉•❛r ❈❡••✱ ✷✵✭✹✮✿✻✵✶✕✻✶✶✳ )②•✐❝③✱ ❏✳ ❏✳✱ ❇♦r❡♥s③t❡✐♥✱ ▼✳✱ ❲♦♥❣✱ ❋✳ ❈✳✱ ❍✉❛♥❣✱ (✳✱ ▲❡❡✱ ❈✳✱ ❉✐❡t♠❛♥♥✱ ❙✳✱ ❛♥❞ ❙✉r❛♥✐✱ ▼✳ ❆✳ ✭✷✵✶✽✮✳ •✾❛ r❡❣✉•❛t❡s t❡♠♣♦r❛• ♣r❡✐♠♣•❛♥t❛t✐♦♥ ❞❡✈❡•♦♣♠❡♥t❛• ♣r♦❣r❛♠ ❛♥❞ •✐♥❡❛❣❡ s❡❣r❡❣❛t✐♦♥ ✐♥ ❜•❛st♦❝②st✳ ❡▲✐❢❡✱ ✼✳ ➏②•✐❝③✱ ❏✳ ❏✳✱ ❇♦✉s❛r❞✱ ❆✳✱ ➎✉♠❡r✱ ❑✳✱ ❉♦ss✐♥✱ ❋✳✱ ▼♦❤❛♠♠❛❞✱ ❊✳✱ ❞❛ |♦❝❤❛✱ ❙✳ ❚✳✱ ❙❝❤✇❛•❜✱ ❇✳✱ ❙②①✱ ▲✳✱ ❉✐♥❣•✐✱ ❋✳✱ ▲♦❡✇✱ ❉✳✱ ❈r❛♠❡r✱ P✳✱ ❛♥❞ ❍❡❛r❞✱ ❊✳ ✭✷✵✶✾✮✳ ❚❤❡ ✷✶✹ |❡❢❡r❡♥❝❡s |❊❋❊|❊◆❈❊❙ ■♠♣•✐❝❛t✐♦♥ ♦❢ ❊❛r•② ❈❤r♦♠❛t✐♥ ❈❤❛♥❣❡s ✐♥ ❳ ❈❤r♦♠♦s♦♠❡ ■♥❛❝t✐✈❛t✐♦♥✳ ❈❡••✱ ✶✼✻✭✶✲ ✷✮✿✶✽✷✕✶✾✼✳❡✷✸✳ ✷✶✺ P❛rt ❱■ ❆♣♣❡♥❞✐① ❆♣♣❡♥❞✐① ❆❞❞✐t✐♦♥❛• ♣✉❜•✐❝❛t✐♦♥s ❆♣♣❡♥❞✐① ❆ ❆❞❞✐t✐♦♥❛• ♣✉❜•✐❝❛t✐♦♥s ❆✳✶ ❆rt✐❝•❡ ✸✿ ♠ ✻ ❆ |◆❆ ♠❡t❤②•❛t✐♦♥ r❡❣✉•❛t❡s t❤❡ ❢❛t❡ ♦❢ ❡♥❞♦❣❡♥♦✉s r❡tr♦✈✐r✉s❡s ✭◆❛t✉r❡✳ ✷✵✷✶✮ ❈❤❡•♠✐❝❦✐ ❚✱ |♦❣❡r ❊✱ ❚❡✐ss❛♥❞✐❡r ❆✱ ❉✉r❛ ▼✱ ❇♦♥♥❡✈✐••❡ ▲❆✱ |✉❝•✐ ❙✱ ❉♦ss✐♥ ❋✱ ❋♦✉❛ss✐❡r ❈✱ ▲❛♠❡✐r❛s ❙✱ ❇♦✉r❝✬❤✐s ❉✳ ♠ ✻ ❆ |◆❆ ♠❡t❤②•❛t✐♦♥ r❡❣✉•❛t❡s t❤❡ ❢❛t❡ ♦❢ ❡♥✲ ❞♦❣❡♥♦
Mammalian genomes host millions of retrotransposons, including ERVs that derive from past retroviral infections and have integrated as permanent residents. Over the course of evolution, successive waves of ERVs have multiplied and diversified, providing a fertile ground for genomic innovations. However, ERVs potentially compromise genomic integrity by disrupting genome structure and expression 6 . In laboratory mice, roughly 12% of pathological mutations result from ERV integrations, half of which emanate from a single family of the ERVK class, the IAPs, that comprise approximately 2,800 full-length copies 7 . By contrast, human ERVs are mostly transposition-defective 8 . However, by providing cis-regulatory modules, ERVs can also divert regulatory networks and alter cellular states. Moreover, ERVs generate RNA, cDNA, RNA-DNA hybrid species and proteins, the accumulation of which is associated with and may contribute to senescence, cancer and neurodegenerative diseases 3 . Homeostatic regulation of ERVs is achieved by surveillance at different steps of the ERV life cycle. Notably, chromatin-based silencing by DNA methylation and histone modifications and post-transcriptional control through RNA editing and RNA interference have extensively been characterized 9 . However, these control mechanisms are not active in all cell types or developmental periods. To identify unknown ERV-limiting factors, we performed a CRISPR-Cas9 loss-of-function screen for IAPEz control, a highly active mouse ERV. We engineered mouse embryonic stem (ES) cells to carry constitutively expressed Cas9 and a reporter cassette with IAPEz regulatory elements: IAPEz(5′LTR-UTR+3-60n gag)-GFP-Blast R (Supplementary Table 1), in which 'LTR' denotes long terminal repeat and 'UTR' denotes the untranslated region (Fig. 1a, Extended Data Fig. 1a). Placing a doxycycline (dox)-responsive promoter upstream of the LTR sequence allowed us to test reactivation of the reporter after dox induction, to choose blasticidin-resistance over GFP as a more sensitive marker, and to adjust the blasticidin concentration for selection (Extended Data Fig. 1b,c). We also showed that the IAPEz reporter responded to known IAP repressors, by transducing cells with single guide RNAs (sgRNAs) against KAP1 10 (Extended Data Fig. 1d,e, Supplementary Fig. 1, Supplementary Tables 2,3).

For the screen, we transduced IAPEz-reporter cells with a lentiviral genome-wide sgRNA library at multiplicity of infection (MOI) of 0.2-0.3 (Fig. 1b). The frequencies of sgRNAs after the application of blasticidin (5, 7 and 9 days) versus non-selected conditions were assessed via sequencing, and candidate genes were identified using MAGeCK (model-based analysis of genome-wide CRISPR-Cas9 knockout) 11 . Selection efficiency was verified by dropout of control intergenic sgRNAs, and genes were ranked on the basis of sgRNA P values (Fig. 1c, Supplementary Tables 4,5). Several known IAP-repressing genes were among the top 100 hits: Resf1, Trp53, Daxx, Atrx, Uhrf1, Cbx1 and Dnmt1 [12][13][14][15] . Moreover, we identified several previously unknown candidates for IAP control (Supplementary Table 4). Notably, among the top hits were regulators of the N 6 -methyladenosine (m 6 A) mRNA methylation pathway, such as

Δ 1 L T R g a g KO Δ 1 L T R g a g Δ 1 L T R g a g Δ 1 L T R g a g Δ 1 L T R g a g

Article

Mettl3, Mettl14, Wtap and Zc3h13, the enrichment of which gradually increased with extended blasticidin-mediated pressure (Fig. 1c,d). By repeating the screen under the most stringent selection, we confirmed significant enrichment for Mettl3, Mettl14 and Wtap sgRNAs (Extended Data Fig. 2a,b). m 6 A is the most abundant internal mark on mRNAs, and is crucial for organizing their fate-including export, decay and translation-in an array of biological processes such as development, cell differentiation, stress response and cancer 4 . The deposition of m 6 A is exerted by a nuclear complex with an enzymatically active core formed by methyltransferase-like METTL3 and METTL14 proteins and other calibrating subunits, including WTAP and ZC3H13. METLL3 and METTL14 form a heterodimer, in which METTL3 is the catalytic component and METTL14 facilitates binding to the RNA substrate. WTAP and ZC3H13 are essential for assembling the complex into the nucleus 16,17 .

To confirm that methylation of m 6 A RNA regulates endogenous IAP copies, we generated individual ES cell lines that contained gene knockouts of Mettl3, Mettl14, Wtap and Zc3h13 (Fig. 2a, Extended Data Fig. 3a-d). To avoid differentiation and lethality effects previously reported when m 6 A-depleted ES cells are cultured in metastable conditions (serum plus leukaemia inhibitory factor (LIF)), we derived and cultured ES cells in a medium that stabilizes a ground state of pluripotency ('2i + LIF' medium) 18 . In these conditions, the identity of ES cells was globally preserved in the mutant lines (Extended Data Fig. 3e,f), with a mild reduction in proliferation (Extended Data Fig. 3g). We first confirmed reduced m 6 A RNA methylation levels in the four knockout lines by enzyme-linked immunosorbent assay (ELISA) (Extended Data Fig. 4a). We used quantitative PCR with reverse transcription (qRT-PCR) to confirm that knockout of each of the four m 6 A factors increased the abundance of endogenous IAPEz mRNA by two-to threefold compared to wild-type cells (Fig. 2b). RNA sequencing (RNA-seq) analysis confirmed significant upregulation of IAPEz in m 6 A mutants, as well as close relatives within the ERVK family-such as MMERVK10C, MMERVK10D3, ETnERV2 and Y chromosome-specific IAPEy elementsthat share more than 65% sequence identity with IAPEz (https://www. dfam.org/) (Fig. 2c, Extended Data Fig. 4b-f, Supplementary Table 6). Specific Y-linked elements of the ERV1 family were also more abundant in the knockout lines (MuRVY). Upregulation of ERVK was observed at the subfamily level after random assignment of multi-mapped reads, and confirmed at the level of individual ERVK copies, when considering uniquely assigned reads only (Fig. 2d, Extended Data Fig. 5a). By contrast, MERVL remained globally unaffected and evolutionarily young long interspersed nuclear elements (LINEs or L1s) transcripts showed opposite downregulation in m 6 A-knockout ES cells (Fig. 2c,d, Extended Data Figs. 4e,f,5a). These different responses to the loss of m 6 A mRNA methylation highlight the divergent effects that this pathway may exert depending on the retrotransposon type, with a negative effect on IAP-related ERVK elements, specifically. ERVK-specific upregulation was confirmed in published nuclear RNA-seq datasets from independent Mettl3-knockout ES cells 19 (Extended Data Fig. 5b).

We next evaluated the potential effect of increased IAP transcripts on gene regulation. As previously shown, hundreds of gene transcripts were upregulated in m 6 A mutant ES cells 20 , among which 941 were common between the 4 knockout lines (Extended Data Fig. 6a). However, these upregulated genes did not show correlation with proximity of ERVK annotations (-5 kb to +1 kb from the transcription start site (TSS)) (Extended Data Fig. 6b). Moreover, we did not score increased splicing between exonic sequences and IAP fragments in m 6 A-knockout compared to wild-type ES cells (Extended Data Fig. 6c). As a whole, we conclude that the increased abundance of IAP transcripts after loss of m 6 A loss minimal cis-effects on gene expression. Notably, depletion of m 6 A did not result in downregulation of known retrotransposon repressors (Extended Data Fig. 6d) and did not alter ES cell identity, as demonstrated by expression levels of pluripotency and early differentiation genes (Extended Data Fig. 3e). This provides strong indication that m 6 A RNA methylation directly represses IAP elements.

To verify this, we mapped the abundance and topology of m 6 A methylation on IAPEz transcripts, by m 6 A immunoprecipitation (methylated RNA immunoprecipitation sequencing (MeRIP-seq)) of total RNA from wild-type and Mettl3-knockout ES cells. We scored 15,216 and 4,864 m 6 A peaks in wild-type and Mettl3-knockout ES cells, respectively, with substantially higher m 6 A signal intensities in wild-type cells (Extended Data Fig. 7a). Notably, in addition to the well-characterized enrichment of m 6 A methylation at the 3′ UTR and exons of genic mRNAs [20][21][22][23] (Fig. 3a, Extended Data Fig. 7b,c), we found that a considerable number of METTL3-dependent m 6 A events mapped to retrotransposon annotations, comprising 13% of all peaks, including L1s-as recently reported 19,24 -and ERVK elements (Fig. 3a). When we plotted m 6 A distribution along the IAPEz consensus sequence, we found two distinct regions of METTL3-dependent m 6 A enrichment, predominantly at the 5′UTR-present in the IAPEz reporter-and to a lesser extent on the Pol sequence (Fig. 3b,c, Extended Data Fig. 7d). Enrichment in m 6 A also coincided with the 5′ UTR region of MMERVK10C (Extended Data Fig. 7e,f) and was rather spread across L1MdA_I (Extended Data Fig. 7g,h). The m 6 A RNA methylation mostly occurs on conserved RRACH sequence motifs (in which R denotes A or G, and H denotes A, C or U) 21,23 . Accordingly, we found several RRACH motifs on the 5′ UTR of IAPEz and MMERVK10C consensus sequences (Fig. 3b, Extended Data Fig. 7e,i). By focusing on uniquely mapped copies, we found that m 6 A-dependent repression was proportional to the RRACH content: the more RRACH motifs an IAPEz copy contains in the 5′ UTR, the more upregulated it is in m 6 A-knockout cells (Fig. 3d, Extended Data Fig. 7j). These data demonstrate for the first time, to our knowledge, that IAPs and their ERVK relatives undergo m 6 A RNA methylation, and reveal a new pathway of retrotransposon suppression.

Functions of the m 6 A RNA methylation complex have so far been investigated by conventional gene perturbation techniques-such as knockout or short hairpin RNA (shRNA)-mediated knockdown-which precludes examining the early consequences of m 6 A loss and can also lead to secondary effects after prolonged selection. To address the early and direct IAP responses to m 6 A depletion, we used auxin-inducible degron (AID) 25 to control the depletion of METTL3 and METTL14, individually and in combination (Fig. 4a, Extended Data Fig. 8a,b). The addition of auxin (indole-3-acetic acid (IAA)) resulted in efficient and near-total degradation within 1 h, which persisted over prolonged treatment and was reversible after wash-off (Fig. 4b, Extended Data Fig. 8c). Depletion of METTLs was rapidly followed by substantial and sustainable decrease of m 6 A RNA methylation levels (Extended Data Fig. 8d). Notably, we observed a progressive, time-dependent accumulation of IAP transcripts after the removal of m 6 A (Fig. 4c, Extended Data Fig. 8e), whereas levels of cellular proliferation were unaffected (Extended Data Fig. 8f). A similar trend was observed after degron-mediated depletion of ZC3H13 (Extended Data Fig. 8g-j). After 96 h of m 6 A depletion, levels of IAP mRNA were increased by 7-fold, 5-fold and 15-fold in single METTL3, single METTL14 or METTL3;METTL14 double degron, respectively (Fig. 4c), and this translated into accumulation of IAP-encoded Gag proteins in cytoplasmic speckles (Fig. 4d). Notably, the abundance of IAP mRNA was higher after degron depletion than in established knockouts for individual Mettl genes (only twofold increase in Mettl3 or Mettl14 knockouts) (Fig. 2b), which suggests the implementation of adaptive mechanisms after prolonged m 6 A loss. Moreover, the relative upregulation after simultaneous METTL3 and METTL14 degron-mediated depletion compared to single degrons

Article

of METTL3 and METTL14 highlight their functional synergy in reducing IAP mRNA levels. Finally, re-stabilizing the m 6 A methyltransferase complex after auxin removal resulted in a rapid decline in IAP mRNAs (Fig. 4c, Extended Data Fig. 8e,j). Together, these results suggest that m 6 A RNA methylation dynamically restrains the cellular availability of IAP mRNAs. Increased abundance of IAP mRNA may result from transcriptional and/or post-transcriptional effects of the m 6 A loss. Notably, m 6 A RNA methylation has been shown to affect chromatin-based regulation of transcription 19,26 . We performed CUT&RUN (cleavage under targets and release using nuclease) experiments to profile permissive (H3K4me3 and H3K27ac) and repressive (H3K9me3) histone marks after 96 h of auxin treatment or dimethylsulfoxide (DMSO) control in METTL3;METTL14 double degron ES cells (AID-METTL3;METTL14). On a global scale, the rapid m 6 A removal did not alter the coverage of these histone marks on m 6 A target genes or on different retrotransposon families (Extended Data Fig. 9a-d). When focusing on full-length IAPEz annotations, we observed moderate H3K4me3 gain (Fig. 4e,f), which may contribute to the mRNA upregulation observed after AID-METTL3;METTL14 double degron depletion, although this was not accompanied by a decrease in H3K9me3 or an increase in H3K27ac (Fig. 4e,f). We therefore concluded that acute withdrawal of m 6 A does not strongly modify chromatin-based regulation of ERVKs in the short term.

Owing to its major effect on genic mRNA lifetime 20 , we sought to determine whether m 6 A could destabilize IAP-derived mRNAs. We used METTL3;METTL14 double degron ES cells to monitor IAP RNA levels after the inhibition of transcription with actinomycin D, and then calculated the half-life rate. IAP mRNA levels and half-life rates were substantially higher after degradation of the m 6 A methyltransferase complex than in cells treated with DMSO control (Fig. 4g). Importantly, the rapid reintroduction of m 6 A methylation (by auxin wash-off) restored IAP mRNA degradation (Fig. 4g). The fate of m 6 A-modified mRNAs is determined by a set of readers, among which the YTH-domain containing proteins YTHDF1, YTHDF2 and YTHDF3 have key roles by directing them to cytosolic compartments where they undergo destabilization and decay 5 . Using RNA immunoprecipitation (RIP), we found that YTHDF2 binds to IAP and MMERVK10C mRNAs (Extended Data Fig. 10a-c), and notably, this occurred in an m 6 A-dependent manner (Fig. 4h). Finally, we reasoned that, similarly to depleting the m 6 A writing complex, depleting YTHDF m 6 A readers should increase IAP mRNA abundance. To account for potential redundancy 27,28 , we derived two mutant ES cell lines that exhibited loss of all three YTHDF proteins (Fig. 4i, left, Extended Data Fig. 10d). Depletion of YTHDFs resulted in increased IAP mRNA levels (Fig. 4i, right), which supports the idea that the m 6 A methylation pathway regulates IAP mRNAs in a post-transcriptional and YTHDF-dependent manner, although other mechanisms may also apply.

Our results provide evidence that m 6 A RNA methylation directly affects ERV mRNA abundance by accelerating their clearance. This mechanism appears specific to IAP-related ERVK elements, whereas other retrotransposon types may undergo alternative directions and modes of m 6 A-dependent regulation. Considering the abundance and deleterious effect of ERVKs, this pathway may be particularly relevant in situations of relaxation of chromatin-based control, such as in early mammalian embryos-from which ES cells are derived-or during ageing. The link with YTHDF readers indicates that ERV mRNA decay may occur through phase-partitioning into cytoplasmic processing bodies (P-bodies) 29 , consistent with previous observations that IAP mRNAs localize to these structures 30 . In addition, this pathway may cooperatively prevent m 6 A-modified ERV mRNAs from being reverse transcribed, translated or assembled into protective retrotransposition complexes, providing several layers of control. Notably, m 6 A mostly occurs on the 5′ UTR of ERVKs, a region that contains the tRNA primer-binding site that is essential for reverse transcription. Finally, m 6 A-dependent regulation could also dampen the immunogenic potential of ERV-derived RNA species and their ability to trigger inflammatory responses, as seen in human neurodegenerative diseases 3 .

Online content

Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41586-020-03135-1.

Methods

Data reporting and statistical analysis

No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. All statistical tests, resulting P values and observation numbers are indicated in the figure panels or legends.

Data visualization

Unless stated otherwise, heat maps, violin plots, box plots, Venn diagrams, density plots, dot plots and bar plots that visualize deepsequencing analyses were generated using ggplot2. Box plots always show the median as the centre line, box limits correspond to upper and lower quartiles, and whiskers cover 1.5× the interquartile range. Volcano plots visualizing retrotransposon expression changes from RNA-seq and bar graphs visualizing ELISA and RT-qPCR experiments were generated using Prism 8.3.0. Dot plots and histogram visualizing FACS analyses were generated using NovoExpress software (v.1.2.1).

Cell culture

Mouse embryonic day (E) 14 ES cells were grown in two different media: serum + LIF consisted in Glasgow medium (Sigma), 15% FBS (Gibco), 2 mM l-glutamine, 0.1 mM MEM non-essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), 0.1 mM β-mercaptoethanol, 1,000 U ml -1 leukaemia inhibitory factor (LIF, Miltenyi Biotec); 2i + LIF was made of 50% Neurobasal medium (Gibco), 50% DMEM/F12 (Gibco), 2 mM l-glutamine (Gibco), 0.1 mM β-mercaptoethanol, Ndiff Neuro-2 medium supplement (Millipore), B-27 medium supplement (Gibco), 1,000 U ml -1 LIF, 3 μM GSK3 inhibitor (CT-99021), 1 μM MEK inhibitor (PD0325901). Cells were cultured in 0.2% gelatin-coated flasks at 37 °C with 5% CO 2 . Except for the CRISPR-Cas9 loss-of-function screens that were performed in serum + LIF medium, all experiments were performed in 2i + LIF medium. Mycoplasma-free status of the cell cultures was verified.

Plasmid construction

IAPEz reporter. Plasmids used to target ROSA26 (pEN111) and TIGRE (also known as Igs7) (Addgene 92141) loci, and the ROSA26-and TIGRE-specific sgRNA-encoding plasmids (Addgene 86234 and 92144, respectively) were provided by E. Nora (UCSF). The IAPEz-5′LTR-5′UTR-gag(3-60nt) consensus sequence was obtained from Repeat-Masker and/or from repbase (http://www.repeatmasker.org/) synthetized and cloned into pUC57 by GenScript (Supplementary Table 1). To make the IAPEz reporter (pTCH1), IAPEz-5′LTR-5′UTR-gag(3-60nt) sequence and the GFP-T2A-blasticidin resistance cassette (hereafter denoted as BlastR) were combined using extension PCR and inserted it into the pEN111 backbone using the ClaI site. To insert the Cas9 gene at the TIGRE locus, 3× Flag-NLS-Cas9 was PCR amplified from pX459 expression vector (Addgene 62988) and inserted into plasmid 92141 backbone using BamHI and XhoI sites (pTCH2).

Plasmids for N terminus tagging with AID domain for auxin-inducible degron. The plasmids to target OsTIR1 at the TIGRE locus (Addgene 92141) and the TIGRE-specific sgRNA-encoding plasmid (see above) were provided by E. Nora (UCSF). The plasmids to target AID inserts into the gene-of-interest N terminus were generated as follows: either puromycin resistance (PuroR)-P2A-3×Flag-AID, or BlastR-P2A-3×Flag-AID inserts were cloned into pUC19 backbone (pFD71 with puromycin resistance gene and pFD75 with blasticidin S-resistance gene). Next, homology arms ranging from 320 to 530 bp depending on the gene (flanking both sides, but excluding ATG start codon) for Mettl3, Mettl14 and Zc3h13 were PCR amplified from mouse genomic DNA and inserted into pFD71 or pFD75 surrounding and in frame with AID insert using EcoRI/NcoI sites for upstream homology arms and AgeI/HindII for downstream homology arms. Final expression vectors were used as follows: pTCH3 (BlastR-P2A-3×Flag-AID-METTL3_Nter), pTCH4 (BlastR-P2A-3×Flag-AID-METTL4_Nter) and pFD119 (BlastR-P2A-3×Flag-AID-ZC3H13_Nter) to generate individual endogenous degron ES cell lines for aforementioned genes. The pTCH4 and pTCH5 (PuroR-P2A-3×Flag-AID-Mettl3) were used sequentially to generate the AID-METTL3;-METTL14 double degron ES cell line. For the sgRNA cloning, the pX459 plasmid (Addgene 62988) was digested with BbsI immediately downstream of the U6 promoter and annealed DNA duplex corresponding to the target sgRNA sequences were ligated. sgRNA sequences were chosen to overlap with the gene TSS, so that after introduction of the AID the sgRNA-specific sequences were disrupted. sgRNA sequences used for degron targeting are listed in Supplementary Table 2.

Cell transfection and clone isolation

All transgenic insertions and mutations were performed using Amaxa 4D nucleofector (Lonza). For each nucleofection, 3 × 10 6 -5 × 10 6 cells were electroporated with 1-3 μg of nonlinearized targeting vector and/ or sgRNA/Cas9-encoding plasmids and plated at a low density. Two days later, cells were selected with puromycin (1 μg ml -1 , Life Technologies) or blasticidin S (5 μg m -1 ) for 2 and 5 days, respectively, and individual clones were picked and screened by PCR. Flippase-mediated removal of puromycin-resistance cassettes were performed for the IAPEz reporter cell line (from both ROSA26 and TIGRE loci) and for puromycin resistance cassette for AID-METTL3;METTL14 double degron from the TIGRE locus. For the IAPEz reporter cell line, functionality of the reporter cassette was confirmed by doxycycline (dox) induction followed by FACS and fluorescence microscopy analyses while Cas9 expression and activity was confirmed by Kap1-specific sgRNA introduction (see below) and western blot analysis. To generate Mettl3, Mettl14, Wtap, Zc3h13, Ythdf2 and combined Ythdf mutant ES cells, two sgRNAs for each gene were designed using the online CRISPOR Design Tool 31 to introduce indels and/or deletions. For sgRNA cloning, the pX459 plasmid (Addgene 62988) was digested with BbsI immediately downstream of the U6 promoter and annealed DNA duplex corresponding to the target sgRNA sequences were ligated. The Mettl3-knockout cells were created by deleting part of exon 4; Mettl14-knockout by deleting part of exon 1; Zc3h13-knockout by deleting part of exon 9; and sgRNA targeting of Wtap gene resulted in single nucleotide insertion and premature stop codon in exon 4. Ythdf2-knockout cells were generated by deleting part of exon 4. The mutYthdf1-2-3 and mutYthd2-3 ES cells were obtained by simultaneous introduction of six sgRNAs targeting the Ythdf1, Ythdf2 and Ythdf3 genes. Protein loss was confirmed by western blot for all mutant cell lines; in addition, m 6 A ELISA assays was carried out for Mettl3-, Mettl14-, Wtap-and Zc3h13-knockout lines. For degron lines, proper insertion and AID-fusion protein activities were confirmed by genotyping, western blot analysis and m 6 A ELISA. For sgRNA sequences used for generation of knockout, mutant and degron lines see Supplementary Table 2.

Cell treatments

IAPEz reporter expression was induced after the administration of doxycycline (1 μg ml -1 ). Auxin-mediated depletion of target proteins was achieved by supplementing culture medium with auxin (Sigma) at the recommended concentration of 500 μM. Auxin-containing medium was renewed every 24 h. For auxin wash-off, auxin-containing medium was removed, cells were rinsed twice with PBS, and exposed to auxin-free medium. Blockade of transcription with actinomycin D was achieved by supplementing culture medium with 5 μg ml -1 actinomycin D (Sigma) .

Lentivirus production and lentiviral-based Kap1-specific sgRNA knockout

Two previously described sgRNAs specific to the Kap1 gene 32 (Supplementary Table 2) were incorporated into plentiGuide-puro vector Article (Addgene 52963). For production of lentiviral particles, HEK293FT cells were co-transfected with 3.33 μg of either of the Kap1 lentiGuide-puro constructs, 2.5 μg psPAX2 packaging plasmid and 1 μg pMD2.G envelope plasmid using Lipofectamine 2000 (Invitrogen). Lentiviral supernatant was collected, filtered with 0.45-μm filter, concentrated using Amicon Ultra centrifugal filter (Millipore, 100-kDa cut-off) and added to pre-plated IAPEz reporter cells supplemented with 8 μg ml -1 polybrene (Millipore). Twelve hours after infection, the medium was replaced and supplemented with puromycin (1 μg ml -1 ). After 48 h of puromycin selection the medium was replaced and supplemented with blasticidin S (5 μg ml -1 ) for additional 72 h.

Protein extraction and western blotting

Cells were trypsinized, washed once in medium and once in PBS and pelleted for 5 min at 1,000 rpm. Cell pellets were resuspended in RIPA buffer (1× PBS, 0.5% sodium deoxycholate, 0.1% SDS, 1% Igepal CA-630) containing protease inhibitors (Roche) and incubated on ice for 20 min. Lysates were then centrifuged for 20 min at 16,400 rpm, at 4 °C and supernatants were kept. Protein concentration was determined using Bradford assay and protein extracts were boiled for 10 min in LDS buffer (Life Technologies) containing 200 mM DTT. Equal amounts of protein were loaded on 4-12% Bis-Tris gel (NuPAGE), or 3-8% Tris-Acetate gel (NuPAGE) for ZC3H13 detection. Transfer was performed on a 0.45-μm nitrocellulose membrane (GE Healthcare) using wet-transfer system, blocked with 5% milk in PBS (+Igepal CA-630 to a final concentration of 0.3%) for 1 h at room temperature. Membranes were incubated with primary antibodies (Supplementary Table 3) at 4 °C overnight in 1% milk in PBS (supplemented with 0.3% Igepal CA-630), washed five times with PBS supplemented with 0.3% Igepal CA-630 and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature and washed again five times. Signal was detected using LumiLight Plus Kit (Roche) on the Chemidoc MP imaging system (BioRad). For uncropped images of western blot membranes, see Supplementary Fig. 1.

FACS analysis

Cells were collected, washed with PBS to remove residual medium and proceeded to analyse GFP expression using NovoCyte 2000R (ACEA Biosciences) flow cytometer and NovoExpress software (v1.2.1). The percentage of GFP-positive cells was determined upon definition of three gates: (i) FSC-H vs SSC-H to isolate cells from debris, (ii) SSC-H versus SSC-A to isolate single cells and (iii) SSC-H versus FITC-H for detection of GFP-positive population. For pseudocolour plots and gating strategy see Supplementary Fig. 2.

Genome-wide screen in IAPEz reporter mouse ES cells (screens I and II)

Approximately 300 × 10 6 IAPEz reporter ES cells expressing Cas9 were lentivirally infected with a genome-wide Mouse Two Plasmid Activity-Optimized CRISPR Knockout Library (Addgene 1000000096) as described above, containing 188,509 sgRNAs targeting 18,986 genes and 199 intergenic sgRNAs at a multiplicity of infection of 0.2-0.3 (measured by puromycin-resistance gene co-delivered with the lentiviral vector) and selected for lentiviral integration using puromycin (1 μg ml -1 ) for 3 days. In screen I, the culture was expanded for another 4-8 days. On days 4, 6 and 8 of expansion, 200 × 10 6 cells were split into blasticidin S-selecting conditions (for 9, 7 and 5 days, respectively) and non-selection conditions (9 days). Cells in non-selection conditions were maintained at minimum level of 100 × 10 6 cells and logarithmic growth. After 9 days, 3 × 10 6 -5 × 10 6 cells from selection conditions and 100 × 10 6 non-selection conditions were washed three times with PBS and pelleted by centrifugation for genomic DNA extraction using GeneElute Mammalian Genomic DNA Miniprep kit (Sigma) and Quick-DNA Midiprep Plus kit (Zymo Research), respectively, following the manufacturers guidelines. The sgRNA-encoding insertions were PCR-amplified using Agilent Herculase II Fusion DNA Polymerase (600675). These libraries were then sequenced using Illumina HiSeq 2500 (approximately 5 million-10 million reads with sgRNA sequence per condition; around 40× coverage per library element in non-selection conditions, screen I). As screen I demonstrated that longer blasticidin S treatment resulted in better intergenic sgRNA depletion, we performed screen II in two biological replicates with 9-day-long blasticidin S selection after either 8-day-long or 17-day-long cell culture (early and late selection, respectively). After genomic DNA extraction and library amplification, libraries were sequenced using Illumina HiSeq 2500 (SE65) (approximately 30 million-35 million reads per condition; around 170× coverage per library element in early and late non-selection conditions, screen II. See Supplementary Table 1 for the primer sequences used to amplify the libraries.

Immunofluorescence

Cells were plated on fibronectin-coated (Sigma) glass coverslips. For IAPEz-GFP reporter reactivation control, doxycycline (1 μg ml -1 ) was added for 24 h. The next day, cells were fixed with 3% paraformaldehyde for 10 min at room temperature, rinsed three times with PBS, incubated 3 min in 0.3 μg ml -1 DAPI and rinsed again with PBS. For detection of IAP-GAG, after fixation, cells were permeabilized for 4 min with PBS/0.5× Triton X-100 on ice, blocked with 1% BSA/PBS for 15 min, incubated for 40 min with rabbit anti-mouse IAP-GAG antibody (gift from B. Cullen), 40 min with secondary antibodies and 3 min in 0.3 μg ml -1 DAPI at room temperature. Slides were mounted with VECTASHIELD medium (Vector Laboratories). Images were obtained with an Upright Spinning disk Confocal Microscope (Roper/Zeiss) and processed with Image J.

RT-qPCR analysis

Total RNA was extracted using Trizol (Life Technologies). Genomic DNA was removed by DNase I treatment (Qiagen), precipitated and resuspended in DNase/RNase-free water. Next, 10 μg of RNA was used for a second round of purification using RNeazy Mini columns (Qiagen) and 500 ng RNA was reverse-transcribed using random priming with Superscript III (Life Technologies). Quantitative PCR was performed using the SYBR Green Master Mix on the Viia7 thermal cycling system (Applied Biosystem). Relative expression levels were normalized to Gapdh or Actb (indicated in respective figures) using the ΔΔC t method. For primer sequences, see Supplementary Table 1.

RNA stability assay

For RNA stability assay, 0.5 × 10 6 AID-METTL3;METTL14 dd ES cells treated with either 500 μM auxin or DMSO for 96 h or after 48-h auxin wash-off were re-plated on fibronectin-coated 6-cm plates 24 h before addition of actinomycin D. Next, medium were renewed and supplemented with 5 μg ml -1 actinomycin D (Sigma) to inhibit transcription. Total RNA was extracted at indicated time points and used for RT-qPCR. The half-life of IAPΔ1 was calculated according to the following equation: ln(C i /C 0 ) = -kt i , in which k is the degradation rate, C i is the mRNA value at time i, and t i is the time interval in hours 33 . First, we calculated degradation rate k i from each time point. The half-life t 1/2 is ln(2)/k a , in which k a is the average degradation rate measured across the different time points. m 6 A ELISA m 6 A ELISA was performed using an m 6 A RNA methylation colorimetric assay kit (Abcam, ab185912) according to manufacturer's protocol, and using 200 ng of RNA. After incubation in the last developer solution, the reaction was stopped by adding 100 μl of STOP solution at the moment positive control wells turned medium blue. It is crucial to continuously control the progressive colour change for the positive control; prolonged incubation will result in signal saturation in the experimental wells and potential masking of differences between tested conditions. Absorbance was measured at 450 nm within 5 min using absorbance microplate reader.

RNA immunoprecipitation

For each replicate, 1.5 × 10 7 cells were collected, washed with ice-cold PBS, resuspended in 1 ml of RIP lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.5% Igepal CA-630, containing 40 U ml -1 of RNasin and protease inhibitors (Roche)). Lysates were placed on ice for 20 min and centrifuged at 4 °C to remove cell debris. Supernatants containing RNA-protein complexes were collected and 50 μl was kept for input control. Remaining lysates were precleared with M-280 sheep anti-rabbit IgG magnetic beads (Thermo Fisher Scientific) and incubated with either 5 μg of YTHDF2 antibody or rabbit IgG rotating overnight at 4 °C. Next, 25 μl of beads was added and samples were rotated for additional 2 h at 4 °C. Beads were washed with RIP lysis buffer five times for 10 min and RNA was isolated from the beads as well as input samples using Trizol. Genomic DNA was removed by DNase I treatment (Qiagen), precipitated and resuspended in DNase/ RNase-free water. Next, purified RNA underwent a second round of purification using RNeazy Mini columns (Qiagen). Immunoprecipitated and input RNAs were reverse transcribed using random priming with Superscript III (Life Technologies). Reverse transcription and real-time quantitative PCR were used to measure abundance.

Poly-A RNA sequencing

Total RNA was extracted using Trizol (Life Technologies). Genomic DNA was removed by in solution DNaseI treatment (Qiagen), RNA was precipitated and resuspended in DNase/RNase-free water. Next, 10 μg of RNA was used for a second round of purification using RNeazy Mini columns (Qiagen). RNA integrity was evaluated on TapeStation 4200 (Agilent) using RNA ScreenTape (5067-5576), requiring a minimal integrity number (RIN) of 9. Libraries were prepared according to Illumina's instructions accompanying the TruSeq Stranded mRNA Library Prep Kit. Approximately 800 ng of RNA per replicate was used for library preparation. After library preparation, the length profiles were assessed with the LabChip GX Touch HT system (Perkin Elmer) and equimolar pool from all samples was prepared. Molarity of the pool was quantified by qPCR using KAPA Library Quantification Kit and the CFX96 qPCR system (Biorad) before sequencing. Samples were sequenced using Novaseq 6000 (PE100, approximately 90 million clusters per replicate).

MeRIP-seq

MeRIP-seq was carried out using Magna MeRIP m 6 A Kit (Millipore) according to the manufacturer's instructions. In short, total RNA was extracted using Trizol (Life Technologies). DNaseI-treated RNA samples were chemically fragmented into 100-nucleotide-long fragments and 350 μg of total RNA were subjected to each immunoprecipitation with affinity purified anti-m 6 A antibody in presence of RNase inhibitor. Bound m 6 A-methylated RNA fragments were eluted with free N6-methyladenosine, purified using RNeazy Kit (Qiagen) and processed for library generation using SMARTer Stranded Total RNA-Seq Kit v2 -Pico Input Mammalian (TaKaRa) following the manufacturer's recommendations, but without fragmentation step (9 ng of RNA per replicate). Sequencing was performed using Illumina Novaseq 6000 (PE100, approximately 50 million to 90 million clusters per replicate). The m 6 A IP for wild-type and Mettl3-knockout cells was performed independently three times. Input for each cell line was sequenced as a control.

CUT&RUN

For chromatin profiling, we performed CUT&RUN 34 on AID-METTL3;METTL14 double degron cells treated with either DMSO (control) or auxin for 96 h in two biological replicates. In brief, 2 × 10 5 cells were washed with PBS, three times with wash buffer (20 mM HEPES-KOH pH 7.9, 150 mM NaCl, 0.5 mM spermidine and Protease Inhibitors (Roche)) at room temperature, then resuspended in 1 ml of washing buffer. Next, 10 μl of concanavalin A-coated magnetic beads (Bangs Laboratories, BP531), pre-washed and resuspended in binding buffer (20 mM HEPES-KOH, pH 7.9, 10 mM KCl, 1mM CaCl 2 , 1 mM MnCl 2 ), were added to the cells. After 10 min incubation at room temperature under rotation, bead-bound cells were isolated on a magnetic stand and resuspended in 400 μl of antibody buffer (wash buffer supplemented with 0.02% digitonin (Millipore, 300410) and 2 mM EDTA) containing 2 μg of anti-H3K4me3, -H3K27ac or -H3K9me3 antibody or 2 μg of rabbit IgG (Supplementary Table 3). After 15 min incubation at room temperature under rotation, cells were washed three times in 1 ml of digitonin(dig)-wash buffer (wash buffer supplemented 0.02% digitonin), incubated with pA-MNase (400 μl of dig-wash buffer containing 700 ng ml -1 pA-MNase, produced by the Protein Core Facility of Institut Curie) and washed again three times in 1 ml of dig-wash buffer. Cells were then resuspended in 150 μl of dig-wash buffer, transferred to 1.5 ml tubes, and equilibrated to 0 °C in a metal block for 10 min on ice. To initiate pA-MNase-dependent digestion, CaCl 2 was added to a final 2 mM concentration, incubation was carried out at 0 °C for 30 min, and stopped by adding 150 μl of 2 × STOP solution (200 mM NaCl, 20 mM EDTA, 5 mM EGTA, 0.1% Igepal CA-630, 40 μg ml -1 glycogen). RNase A was added to a final concentration of 50 μg ml -1 and samples were incubated at 37 °C for 20 min. Samples were placed on a magnetic stand, supernatant was transferred to low-binding tubes and SDS and proteinase K were then added to final concentrations of 0.1% and 300 μg ml -1 , respectively, and samples were incubated at 70 °C for 30 min. Total DNA was extracted by phenol/chloroform followed by two rounds of ethanol precipitation, eluted in 40 μl of 1 mM Tris-HCl pH 8.0 and 0.1 mM EDTA, and quantified and analysed using Qubit and TapeStation assays. CUT&RUN libraries were prepared using the Accel-NGS 2S Plus DNA Library Kit (Swift Biosciences) according to the manufacturer's protocol. Samples were sequenced using NovaSeq 6000 (PE50, approximately 60 million clusters per replicate).

Genome-wide CRISPR-Cas9 screen analysis

The sequenced reads were mapped to the sgRNA library. Only reads that contained one sgRNA sequence without mismatch were counted. The MAGeCK 11 test command line (version 0.5.8) was used to rank sgRNAs and genes with following parameters: -norm-method totaladjust-method fdr-remove-zero-threshold 10-gene-lfc-method alphamean-remove-zero both. For screen II, the sequencing primer was oriented in the opposite direction to the sgRNA, therefore the CRISPR mouse pooled library was reverse-complemented before counting.

RNA-seq analysis

Adapters were trimmed using Atropos v.1.1.16 35 . Paired-end read alignment was performed onto the Mouse reference genome (mm10) with STAR v.2.7.0a 36 reporting randomly one position, allowing 6% of mismatches (-outFilterMultimapNmax 5000-outSAMmultNmax 1-outFilterMismatchNmax 999-outFilterMismatchNoverLmax 0.06). Repeat annotation was downloaded from RepeatMasker (http://www. repeatmasker.org/). To reconstruct full-length LTR copies, we used the same strategy as done previously 10 using the perl tool 'one code to find them all' 37 . Reconstructed transposons annotation and basic genes annotation from GENCODE v.18 were merged and used as input for quantification with FeatureCounts v1.5.1 38 . Differential expression analysis was performed using edgeR's normalization combined with voom transformation from limma R package 39,40 . P values were computed using limma and adjusted with the Benjamini-Hochberg correction. Genes and transposon families were declared as differentially expressed if FDR < 5% and log 2 FC > 0.75. Upregulated genes in all four knockout lines were annotated with proximal retrotransposon elements (overlap with promoter regions defined as -5 kb to +1 kb from the TSS). Randomized gene sets were created 100 times and were annotated to proximal retrotransposon elements to compute permutation test using regioneR 41 Zc3h13-knockout cells disappeared (highlighted by an asterisk). This could explain the lower sgRNA ranking for these two genes upon extended selection time in the screen, which was performed in serum + LIF medium (Extended Data Fig. 2b).

Article

RodERV21-int

RLTR41 MMERGLN-int MER34-int MURVY-int RLTR41A2 RMER21A RLTR24 RLTR14-int LTRIS_Mm LTR78 LTR78 LTRIS_Mm MER34-int MERV1_LTR MMERGLN-int MURVY-int RLTR14-int RLTR24 MER89 RLTR4_MM-int LTR78 RLTR14-int RLTR24 MuRRS4-int LTRIS_Mm MURVY-int MLT1G1 MTB_Mm-int ORR1A1-int MLT1G3 MLT1F MLT1E2 ORR1A0-int MTD-int MLT1K MTE2b-int ORR1E-int MTE2b MERVL_2A-int ORR1C1-int LTR50 MERVL-int MLT1F MLT1G1 MLT1G3 MTB_Mm-int ORR1A1-int ORR1C2 MTB_Mm-int RMER15 L1MdF_I L1MdA_I L1MdA_I
Mettl14-KO Zc3h13-KO Wtap-KO Mettl14-KO Zc3h13-KO Wtap-KO Mettl14-KO LTR78 LTRIS_Mm MER34-int MERV1_LTR MMERGLN-int MURVY-int RLTR14 RLTR14-int RLTR24 RMER21A MERVL-int MLT1E2 ORR1A1-int MLT1F MLT1G3 MTB_Mm-int MLT1G1 L1MdA_I L1MdGf_II L1MdTf_I L1MdA_II L1MdA_III L1MdA_IV L1MdTf_II L1MdTf_III L1MdV_I L1ME3Cz -8 -4 -3 -2 -1 01234 -8 -4 -3 -2 -1 01234 -8 -4 -3 -2 -1 01234 Mettl14-KO Zc3h13-KO Wtap-KO Mettl3-KO log2FC (KO/WT) Mettl3-KO Mettl3-KO Mettl3-KO Zc3h13-KO Wtap-KO c d e f -log10(P-value) ETnERV2-int ETnERV3-int IAP-d-int IAP1-MM_I-int IAPEy-int IAPEY4_I-int IAPEz-int IAPLTR1 IAPLTR3-int MMERVK10C RLTR13B2 MMERVK10D3_I-int MLTR11A RLTR11A ERVB7_4-LTR_MM RLTR45-int RLTR10D2 RLTR13D2 -8 -4 -3 -2 -1 01234
-int MMERVK10C-int IAP-d-int ETnERV2-int IAPLTR3-int IAPEY2_LTR RLTR11A RLTR13B2 IAPEY4_I-int MMERVK10D3_I-int MurERV4_19-int RNERVK23-int MLTR11A SRV_MM-int RLTR16C_MM MMERVK9E_I-int MMERVK9C_I-int 15 10 5 0 ETnERV2-int ETnERV3-int IAP-d-int IAP1-MM_I-int IAPEy-int IAPEY4_I-int IAPLTR3-int MMERVK10C-int MMERVK10D3_I-int RMER13A1 RLTR13B2 ERVB4_2-I_MM RLTR45-int RLTRETn_Mm RLTR16B_MM -8 -4 -3 -2 -1 01234 MMERVK9E_I-int MMERVK9C_I-int 15 10 5 0 ETnERV2-int ETnERV3-int IAP-d-int IAP1-MM_I-int IAPEy-int IAPEY4_I-int IAPEz-int IAPLTR1_Mm IAPLTR3-int MMERVK10C-int ERVB7_4-LTR_MM RLTR13B2 RLTR11A MLTR11A SRV_MM-int ERVB7_2B-LTR_MM MMERVK10D3_I-int -8 -4 -3 -2 -1 01234 MMERVK9C_I-int MMERVK9E_I-int 15
-K O M e t t l3 -K O W t a p -K O Z c 3 h 1 3 -K O M e t t l1 4 -K O M e t t l3 -K O W t a p -K O Z c 3 h 1 3 -K O Multi-mapped reads Unique reads ERV1 ERVK ERVL MMERGLN-int ETnERV2-int ETnERV3-int IAP-d-int IAPEY2_LT R IAPEY3-int IAPEY5_I-int IAPEz-int IAPLTR3 IAPLTR3-int IAPLTR4 IAPLTR4_I MMERVK10C-int MERVL-int LINE1 M e tt l3 _ K O 1 L1MdA_I L1MdA_II L1MdGf_I L1MdGf_II L1MdTf_I
L IN E L T R /E R V K L T R /E R V 1 L T R /E R V L L T R /E R V L -M a L

Article

Extended Data Fig. ;2& E)7 2$49$ 1!no=popo q q q q q q q q q q 34 ( '2 8 ( 5& 5& ' #$ "( )$ ( '#/:C$ 4 4!/$ 2&% 5 &4m'$ #' $ "%r! $ %)(1!$ ""$ "%#$ 9"8 4&7C$ 4 4!* 0! D s$ + :1m X FL MNO[J X YFMQL eX P SL J NMYJ L FYP X NFYJ L YFUL X FP QW FYP X NFYJ L J YeR L FNP X FiL YFKL [QW OXP R L P SYP L eQW QL NVP YX FQUL iX dQL P SQL FYOQL Nh L P SQL X R R TX FiL YTP SNW X P K L P SQL UYP QL Nh L X R R TQ L YFUL YFKL X UQFP X h KX FiL X Fh NW OYP X NF _ QR MW X VQL YFKL UX R P TW VYFMQL MYTR QUL VKL P SQL R P TUKL YFUL SNeL X P L eYR L OXFX OX QU_ q q q q q q q q q bW NdX UQL

W QUQP QW OXFQL R YO[J QL R X QL IL X h L FNL R YO[J Q R X QL MYJ MTJ YP X NFL eYR L [QW h NW OQU L UQR MW X VQL SNeL R YO[J QL R X QR L eQW QL MSNR QFL YFUL [W NdX UQL YL W YP X NFYJ QL h NW L eSKL P SQR QL R YO[J QL R X QR L YW QL R Th h X MX QFP _ QR MW X VQL P SQL UYP YL MNJ J QMP X NFL [W NMQUTW Q L X FMJ
P R L P NL W Q[QYP L P SQL Q\[QW X OQFP L eQW QL R TMMQR R h TJ _ QR MW X VQL SNeL R YO[J QR NW iYFX R OR [YW P X MX [YFP R L eQW QL YJ J NMYP QUL X FP NL iW NT[R _ L h L YJ J NMYP X NFL eYR L FNP L W YFUNOL UQR MW X VQL SNeL MNdYW X YP QR L eQW QL MNFP W NJ J QU_ L h L P SX R L X R L FNP L W QJ QdYFP L P NL KNTW L R P TUK L Q\[J YX FL eSK_ QR MW X VQL P SQL Q\P QFP L Nh L VJ X FUX FiL TR QUL UTW X FiL UYP YL YM]TX R X P X NFL YFUL YFYJ KR X R _ L h L VJ X FUX FiL eYR L FNP L [NR R X VJ Q L UQR MW X VQL eSKL IL Q\[J YX FL eSKL VJ X FUX
W X YJ L W QiX R P W YP X NFL FTOVQW L h W NOLJ X FX MYJ W X YJ R _ iNdL NW L YFL Q]TX dYJ QFP L YiQFMK_ NP QL eSQW QL P SQL h TJ J L P W X YJ L [W NP NMNJ L MYFL VQL YMMQR R QUL IL X h L FNP L YdYX J YVJ Q L Q\[J YX FL eSK_ QR MW X VQL P SQL R QP P X FiR L YFUL J NMYJ QR L Nh L UYP YL MNJ J QMP X NF L FNP X FiL P SQL P X OQL [QW X NUR L Nh L W QMW TX P OQFP L YFUL UYP YL MNJ J QMP X NF_ QR MW X VQL SNeL KNTL [W Q UQh X FQUL [W X OYW KL YFUL R QMNFUYW KL NTP MNOQL OQYR TW QR L YFUL SNeL KNTL YR R QR R QUL P SQR QL OQYR TW QR _ q q
)( ( ! , D D 555: "4.$ : "7 2:"$ ): %6D %D m' /D &44: 4%$ &44k1@zuvoox ¡ 123 45 65 67 625 895 65 3 17425

<$ 7 $ "#&( &.& '.2$ $ "
k"2. 5 $ " * : %:r1+ q q q q -$ 647 7 5 47 7 4( #=5& )#&"# ' !"##$ "Af1. '

/ k &!)&"&7 / $ C'7 ( $ 6& $ &( 2#7 $ "%&"#! #$ 4( $ 6&"&7 / $ [QMX h K¦ L h TFMP X NFYJ L R P W TMP TW YJ L UX h h TR X NF L [QW h TR X NF_ [QMX h KL X FL QR J Y [QMX h KL P SQL [TJ R QL R Q]TQFMQL P K[QL iW YUX QFP L QMSN L R [X FL QMSN L QP M_ L X OYiX FiL P K[QL §b L R [X W YJ L QP M_ L h X QJ UL Nh L dX Qe L OYP W X \L R X Q L R J X MQL P SX MFQR R L NW X QFP YP X NFL YFUL § h J X [L YFiJ Q_ P YP QL eSQP SQW L YL eSNJ QL VW YX FL R MYFL eYR L TR QUL IL UQh X FQL P SQL YW QYL Nh L YM]TX R X P X NF L UQR MW X VX FiL SNeL P SQL W QiX NFL eYR L UQP QW OXFQU_ bW NdX UQL UQP YX J L NFL R Nh P eYW QL dQW R X NFL YFUL W QdX R X NFL FTOVQW L YFUL NFL R [QMX h X ML [YW YOQP QW R L ONUQJ h TFMP X NFR L VW YX FL Q\P W YMP X NF L R QiOQFP YP X NF L R ONNP SX FiL QW FQJ L R X Q L QP M_ _ h L UYP YL eQW QL FNW OYJ X QU R P YFUYW UX QU L UQR MW X VQL P SQL Y[[W NYMS QR ¦ L R [QMX h KL J X FQYW L NW L FNF J X FQYW L YFUL UQh X FQL X OYiQL P K[QR L TR QUL h NW L P W YFR h NW OYP X NFL IL X FUX MYP QL P SYP L UYP YL eQW QL FNP L FNW OYJ X QUL YFUL Q\[J YX FL W YP X NFYJ QL h NW L J YML Nh L FNW OYJ X YP X NF_ QR MW X VQL P SQL P QO[J YP QL TR QUL h NW L FNW OYJ X YP X NF P W YFR h NW OYP X NF L R [QMX h KX FiL R TV¤ QMP L R [YMQL NW L iW NT[L R P YFUYW UX QUL R [YMQL Q_ i_ L NW X iX FYJ L YJ YX W YMS L ^ ¨©ª L ^«ª¬ L IL X FUX MYP QL P SYP L P SQL UYP YL eQW QL FNP L FNW OYJ X QU_ QR MW X VQL KNTW L [W NMQUTW Q R L h NW L YW P X h YMP L YFUL R P W TMP TW QUL FNX R QL W QONdYJ L R [QMX h KX FiL ONP X NFL [YW YOQP QW R L P X R R TQL R X iFYJ R L YFUL [SKR X NJ NiX MYJ L R X iFYJ R L SQYW P L W YP Q L W QR [X W YP X NF _ Qh X FQL KNTW L R Nh P eYW QL YFU NW L OQP SNUL YFUL MW X P QW X YL h NW L dNJ TOQL MQFR NW X Fi L YFUL R P YP QL P SQL Q\P QFP L Nh L R TMSL MQFR NW X Fi_ [QMX h KL P K[QL OYR R L TFX dYW X YP Q L OTJ P X dYW X YP Q L L [W QUX MP X dQ L QP M_ L YFUL UQR MW X VQL QR R QFP X YJ L UQP YX J R L Nh L P SQL ONUQJ L YP L P SQL h X W R P L YFUL R QMNFUL J QdQJ R L Q_ i_ L h X \QU L W YFUNOLNW L OX\QUL Qh h QMP R Z L UW X h P L NW L YTP N MNW W QJ YP X NF _ Qh X FQL [W QMX R QL Qh h
L R [QMX h KX FiL eQX iSP QUL iW Y[SL NW L VX FYW X QUL iW Y[S L R TV¤ QMP L NW L iW NT[ J QdQJ L YFUL P SQL iJ NVYJ L YFU NW L FNUQL R TOOYW X QR L TR QUL Q_ i_ L MJ TR P QW X FiL MNQh h X MX QFP L Qh h X MX QFMK L QP M_ _ [QMX h KL X FUQ[QFUQFP L dYW X YVJ QR L h QYP TW QR L Q\P W YMP X NFL YFUL UX OQFR X NFL W QUTMP X NF L ONUQJ L P W YX FX FiL YFUL QdYJ TYP X NFL OQP W X MR _ ❆♣♣❡♥❞✐① ❆❞❞✐t✐♦♥❛• ♣✉❜•✐❝❛t✐♦♥s ❆✳✷ ❆rt✐❝•❡ ✹✿ ❙P❊◆ ✐s r❡q✉✐r❡❞ ❢♦r ❳✐st ✉♣r❡❣✉•❛✲ t✐♦♥ ❞✉r✐♥❣ ✐♥✐t✐❛t✐♦♥ ♦❢ ❳ ❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛✲ t✐♦♥ ✭❇✐♦|①✐✈✳ ✷✵✷✵✮ |♦❜❡rt✲❋✐♥❡str❛ ❚✱ ❚❛♥ ❇❋✱ ▼✐r❛✲❇♦♥t❡♥❜❛• ❍✱ ❚✐♠♠❡rs ❊✱ •♦♥t❛♥✲P❛r❞♦ ❈✱ ▼❡r✲ ③♦✉❦ ❙✱ •✐❛✐♠♦ ❇❉✱ ❉♦ss✐♥ ❋✱ ✈❛♥ ■•❝❦❡♥ ❲❋❏✱ ▼❛rt❡♥s ❏❲▼✱ ❇♦r❣r❡✛❡ ❚✱ ❍❡❛r❞ ❊✱ •r✐❜♥❛✉ ❏✳ ❙P❊◆ ✐s |❡q✉✐r❡❞ ❢♦r ❳✐st ❯♣r❡❣✉•❛t✐♦♥ ❞✉r✐♥❣ ■♥✐t✐❛t✐♦♥ ♦❢ ❳ ❈❤r♦♠♦s♦♠❡ ■♥❛❝t✐✈❛t✐♦♥✳ ❜✐♦|①✐✈✳ ✷✵✷✵✳ ❆|❚■❈▲❊ ❖◆ ◆❊❳❚ P❆•❊ ✷✺✵

Introduction

To compensate for gene dosage imbalance between females (XX) and males (XY), female placental mammals randomly inactivate one X chromosome early during embryonic development 1 . In mice, random X chromosome inactivation (XCI) takes place in the epiblast in three phases: initiation, establishment and maintenance. During the initiation phase, the long non-coding RNA (lncRNA) Xist is upregulated from the future inactive X (Xi) chromosome [2][3][4] . Xist is located within the X Chromosome Inactivation Centre (Xic), an X-linked region required for XCI that contains different cis-regulatory elements, including Tsix, another lncRNA gene that is transcribed antisense to and completely overlaps Xist 5 . Tsix negatively regulates Xist expression via antisense transcription and chromatin remodelling [6][7][8][9][10][11][12] . Together, Xist and Tsix form a master switch that controls initiation of XCI: in the pluripotent state, Tsix is biallelically expressed, repressing both alleles of Xist, while upon initiation of XCI the balance changes, resulting in downregulation of Tsix and upregulation of Xist on the future Xi. Trans-regulators including pluripotency factors OCT4, NANOG, REX1 as well as XCI activators are key in controlling this switch by regulating Xist and Tsix transcription (reviewed in 13 ). Later, during the establishment phase of XCI, the 17 kb lncRNA Xist spreads in cis along the future Xi and recruits different proteins involved in gene silencing that render the X transcriptionally inactive (reviewed in 14 ). Active histone marks (H3K4me2/me3, H3 and H4 acetylation) are removed and repressive histone marks are instigated, catalysed by Polycomb group complexes and (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted December 30, 2020. ; https://doi.org/10.1101/2020.12.30.424676 doi: bioRxiv preprint 3 other protein complexes (reviewed in 15 ). Finally, in the maintenance phase, the inactive state of the Xi is epigenetically propagated across cell divisions.

Different studies consisting of Xist RNA immunoprecipitations coupled to mass spectrometry [16][17][18] and genetic screens 19,20 identified SPEN (also known as SHARP in human and MINT in mouse) as a crucial factor in the establishment phase of XCI. SPEN is a large protein with four N-terminal RNA Recognition Motifs (RRM) and a highly conserved C-terminal SPOC domain able to recruit different proteins involved in transcriptional silencing 21,22 . SPEN is also involved in the Notch signalling pathway and nuclear receptor signalling, where it acts as a transcriptional corepressor 23,24 .

SPEN is crucial for X-linked gene silencing 16,17,19,20 by binding the Xist Repeat A (RepA) via its RRM domains 17,19 and interacting via its SPOC domain with the corepressors NCoR/SMRT to recruit/activate histone deacetylase 3 (HDAC3), which is responsible for the removal of histone H3 and H4 acetylation at promoters and enhancers of genes located on the future Xi 16,25 . Despite this crucial role for SPEN in establishment of the Xi, these studies did not report defects in Xist upregulation and coating 16,17,19,20,25 (Supplementary Table 1).

Here, we show that SPEN accumulates on the Xi very early during differentiation and is required for Xist upregulation. We show that SPEN has a dual function, required to silence Tsix and facilitate Xist upregulation, while also stabilizing Xist RNA. Together, our results indicate that SPEN is not only necessary for X-linked gene silencing but also plays a crucial earlier role in the regulation of initiation of XCI.

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted December 30, 2020. ; https://doi.org/10.1101/2020.12.30.424676 doi: bioRxiv preprint 4

Results

SPEN is required for Xist upregulation

Previous work has shown how SPEN is crucial for silencing of X-linked genes, but these studies did not investigate the role of SPEN in initiation of Xist expression. Therefore, we generated Spen homozygous (Spen -/-) and heterozygous (Spen +/-) knockout mouse embryonic stem cells (ESCs) by deleting the complete open reading frame (ORF) using the CRISPR/Cas9 technology (Supplementary Fig. 1a). These lines were generated in a hybrid F1 129/Sv:Cast/EiJ (129/Cast) genetic background with a doxycycline-responsive endogenous Xist promoter located on the Cast X chromosome 26 (Fig. 1a). The Spen ORF deletion was verified by PCR on genomic DNA (gDNA) and Western blot analysis (Supplementary Fig. 1b-c). Allelespecific RNA-seq analysis (Supplementary Fig. 1d) of wild type (Wt) undifferentiated ESCs (day 0) containing the doxycycline-responsive Xist promoter treated with and without doxycycline for 4 days (Fig. 1b) showed skewed X-linked gene silencing towards the Cast allele (Fig. 1c top-left, Supplementary Fig. 1e). On the other hand, the same analysis in Spen -/-ESCs showed impaired X-linked gene silencing (Fig. 1c bottom-left), as described before 16,17,19,20 (Supplementary Table 1).

To trigger XCI in the context of differentiation, we forced Xist upregulation by doxycycline treatment followed by monolayer differentiation (Fig. 1b). Allele-specific RNA-seq analysis of Wt and Spen -/-ESCs treated with doxycycline at day 7 of monolayer differentiation (Supplementary Fig. 1d) also revealed a lack of X-linked gene silencing along the entire X-chromosome (Fig. 1c bottom-right, Supplementary Fig. 1e). Similarly, allele-specific RT-qPCR analysis of the X-linked gene Rnf12 revealed impaired silencing (Fig. 1d). Although previous work indicated that in Spen -/-ESCs a group of lowly expressed X-linked genes are susceptible to SPEN-independent gene silencing 27 , our RNA-seq analysis shows no silencing of this specific group of genes (Supplementary Fig. 1f,g). We observed Xist upregulation in Wt and Spen -/-cells, however doxycycline induction resulted in lower Xist expression levels in Spen -/-compared to Wt ESCs (Fig. 1e), an effect that was also reported in a recent study 27 .

Next, we recapitulated physiological XCI by monolayer differentiation in the absence of doxycycline to allow normal Xist upregulation and X-linked gene silencing (Fig. 1b). Allele-specific RNA-seq analysis of Wt and Spen -/-ESCs at day 3 without doxycycline (Supplementary Fig. 1d) shows no significant differences in silencing (Fig. 1c top-right, Supplementary Fig. 1e), making evident the time scale differences between forced Xist upregulation in the undifferentiated ESC stage and physiological XCI. Even so, at the latest time points of differentiation (day 5 and 7) Spen -/-cells clearly lost the capacity to induce Rnf12 silencing (Fig. 1f). Remarkably, in Spen -/-cells, Xist upregulation from the 129 allele was completely abrogated (Fig. 1g), (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted December 30, 2020. ; https://doi.org/10.1101/2020.12.30.424676 doi: bioRxiv preprint contrasting earlier evidence that suggested that SPEN is not required for Xist upregulation and coating 16,17,19,20,25 (Supplementary Table 1). In addition, differentiating Spen -/-ESCs lack Xist clouds, determined by RNA-FISH (Fig. 1h,i), while Tsix was significantly more expressed from the Wt 129 allele in Spen -/-cells compared to Wt cells at day 5 and 7 of monolayer differentiation (Fig. 1j), suggesting that SPEN might be necessary for Tsix silencing. Importantly, RT-qPCR analysis confirmed proper silencing of pluripotency genes Rex1 and Nanog, and upregulation of endoderm marker Gata6 (Supplementary Fig. 1h) in Spen -/- ESCs upon monolayer differentiation, indicating that loss of Xist expression is not related to defective ESCs differentiation.

Furthermore, while Spen +/-cells are able to upregulate Xist and silence Rnf12 upon doxycycline treatment followed by monolayer differentiation (Supplementary Fig. 2a,b), they show reduced Rnf12 silencing upon physiological differentiation without a defect in Xist upregulation (Supplementary Fig. 2c,d). Given that SPEN levels are reduced in Spen +/-cells (Supplementary Fig. 1c), these results demonstrate that SPEN dosage is important in XCI.

Spen rescue leads to normal Xist expression levels

To confirm previous results, we performed a rescue experiment by stably re-expressing Spen through introduction of the full-length Spen cDNA in the ROSA26 locus 25 of Spen -/-ESCs (Supplementary Fig. 3a).

Successful integration and expression of Spen was verified by PCR on gDNA and RT-qPCR, respectively (Supplementary Fig. 3b,c). The generated rescue clones (Clone A, B and C) display two-to three-fold overexpression of Spen RNA at the ESC stage and during monolayer differentiation (Supplementary Fig. 3c). In contrast to the Spen -/-lines, all Spen -/-(cDNA) rescue clones express Xist at similar levels to Wt clones during monolayer differentiation (Fig. 2a). Allele-specific expression analysis of Rnf12 and Tsix (129 allele) indicated that the silencing defect is partially rescued in Spen -/-(cDNA) clones (Supplementary Fig. 3d,e).

Interestingly, in undifferentiated Spen -/-(cDNA) ESCs clones, Xist levels were higher than in Wt controls (Fig. 2a), and Xist RNA-FISH analysis revealed a significant percentage of Spen -/-(cDNA) ESCs having Xist clouds (Fig. 2b,c). This abnormal cloud formation could be related to higher SPEN abundance due to overexpression in the undifferentiated state, possibly stabilizing Xist or silencing Tsix. In addition, these results indicate that the observed defect in Xist expression is SPEN mediated and takes place at the very early initiation steps of XCI.

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted December 30, 2020. ; https://doi.org/10.1101/2020.12.30.424676 doi: bioRxiv preprint 6 SPEN accumulation on the Xi upon ESCs differentiation shows two distinguishable states: early and late SPEN accumulation In light of the previous results, we expect SPEN to accumulate on the Xi at the very early steps of physiological XCI. To investigate this hypothesis and since commercial SPEN antibodies suitable for immunofluorescence (IF) are lacking, we generated ESCs endogenously expressing a C-terminally tagged SPEN-GFP (Supplementary Fig. 4a). Correct GFP integration was confirmed by PCR on gDNA and by FACS analysis (Supplementary Fig. 4b,c). IF detection of GFP at different time points of monolayer differentiation revealed SPEN accumulation as early as day 1 in about 30% of the nuclei (Fig. 3a,b).

Interestingly, we could distinguish two different states: early and late SPEN accumulation on the Xi (Fig. 3a-c). Early accumulation was dispersed and had a lower IF intensity, while SPEN accumulation at later stages was more compact and with a higher IF signal. Early accumulation signals were only detected at the initial days of monolayer differentiation (day 1 to 5), while late accumulation signals appeared at day 2, progressively increased over time and plateaued at day 7 (Fig. 3b).

Previous studies have shown that SPEN and Xist co-localize in doxycycline-inducible Xist undifferentiated ESC lines 20,25 , although the normal timing and relation between SPEN and Xist in differentiating cells remained unexplored. Therefore, we investigated SPEN accumulation in relation to Xist, by performing GFP IF combined with Xist RNA FISH in differentiating cells (Fig. 3d, Supplementary Fig. 4d). This analysis indicated that at day 2 of differentiation, about 55% of the cells with Xist clouds showed SPEN colocalization (Fig. 3e). We also studied the relation of SPEN with other key players in XCI, including the PRC2 catalytic subunit EZH2 and its catalytic product H3K27me3. As found for Xist, we could detect colocalization of EZH2 and its associated histone modification with SPEN (Supplementary Fig. 4e-g). At day 2 about 60% of the nuclei with early SPEN accumulation displayed EZH2 accumulation in the absence of H3K27me3 deposition, whereas at day 5 about 90% of the cells showed H3K27me3 enrichment at SPEN positive Xi (Fig. 3f). Our results show that SPEN accumulation on the Xi can be distinguished in an early state and late state of SPEN accumulation, where the early state is marked by small dispersed accumulation of SPEN co-localizing with EZH2 without detectable H3K27me3 at day 2, whereas the late state SPEN accumulation is compact and co-localizes with EZH2 and H3K27me3.

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted December 30, 2020. ; https://doi.org/10.1101/2020.12.30.424676 doi: bioRxiv preprint Xist RNA stability is compromised in Spen -/-ESCs We have shown that SPEN is required for endogenous Xist upregulation and XCI upon ESCs differentiation.

In addition, we noticed that forced Xist upregulation resulted in reduced Xist RNA levels in Spen -/-cells compared to Wt cells (Fig. 1e, Fig. 4a). Accordingly, the percentage of nuclei with Xist clouds was lower in Spen -/-cells with doxycycline induced Xist versus Wt cells and Xist clouds were in general smaller (Fig. 4b,c). This finding could be explained by a role for SPEN in stabilizing Xist RNA by complex formation. To study the role of SPEN in Xist stability, we determined the half-life of doxycycline-induced Xist RNA in Spen -/-and Wt ESCs treated with actinomycin D to block its transcription. The remaining levels of Xist RNA at different time-points was assessed by RT-qPCR, and the Xist RNA decay rate and half-life were then calculated (Fig. 4d). In Wt ESCs, the Xist RNA half-life was 6h 37min, similar to what was described in a previous study 28 , whereas the Xist half-life was reduced to 3h 52min in Spen -/-cells. These results indicate that SPEN plays a role in promoting Xist RNA stability. However, these results cannot explain why physiological Xist upregulation is lost in Spen -/-ESCs upon differentiation. SPEN, HDAC3 and H3K27ac are enriched at the Tsix regulatory region Our results showed that at late days of monolayer differentiation, Spen -/-cells display higher Tsix levels than control cells (Fig. 1j), suggesting that SPEN might be recruited by Xist to silence Tsix. Hence, we explored SPEN genomic binding at the Xist-Tsix locus using published SPEN CUT&RUN data in undifferentiated ESCs with a doxycycline-responsive Xist promoter 25 . SPEN accumulates on the Xist gene body as well as on the Tsix regulatory region, SPEN accumulation is evident at 24 hours of doxycycline induction, but more prominent after 4 and 8 hours of induction (Fig. 5a,b). This Tsix regulatory region comprises the minor and major Tsix promoters and Xite, an enhancer of Tsix 29 . Xist-mediated recruitment of SPEN is important for recruitment and/or activation of HDAC3, responsible for the removal of H3K27ac from the future Xi 16,30 . Analysis of published HDAC3 and H3K27ac ChIP-seq data from undifferentiated female ESCs upon 24h Xist induction 30 reveals HDAC3 binding and H3K27ac loss at the Tsix regulatory region, where SPEN is recruited (Fig. 5a). Moreover, X-linked Rnf12 (Supplementary Fig. 5a) and Pgk1 (Supplementary Fig. 5b) display SPEN and HDAC3 enrichment at their promoters and H3K27ac loss upon Xist induction. Interestingly, Rnf12, an early silenced gene 31 , shows SPEN and HDAC3 promoter binding in the undifferentiated state without Xist induction, suggesting that in the undifferentiated state Xist might be sufficiently expressed at very low levels to partly silence Rnf12, explaining the Rnf12 allelic ratio difference between Wt and Spen -/-ESCs at day 0 (Fig. 1f, Supplementary Fig. 3d).

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted December 30, 2020. ; https://doi.org/10.1101/2020.12.30.424676 doi: bioRxiv preprint 8 To test whether SPEN recruitment leads to H3K27me3 accumulation, we performed H3K27me3 ChIP-seq on Spen -/-and Wt ESCs prior to and after differentiation (day 3) (Fig. 5a). This analysis revealed H3K27me3 enrichment in the Tsix regulatory region in Wt cells upon differentiation, while Spen -/-cells do not show H3K27me3 enrichment in accordance with the lack of Xist upregulation. Interestingly, the H3K27me3 hotspot, located at the 3' end of Tsix 32,33 is clearly reduced upon differentiation both in Wt and Spen -/- cells, while Wt and Spen -/-cells show no difference in H3K27me3 levels, indicating that SPEN does not play a role in H3K27me3 enrichment at the hotspot (Fig. 5a). Altogether, these data support a model where Xist-mediated SPEN recruitment leads to Tsix promoter silencing.

SPEN is required to silence Tsix to allow Xist upregulation

To further investigate the role of SPEN in silencing Tsix, we generated compound Spen -/-:Tsix defective hybrid ESC lines. If Xist-mediated recruitment of SPEN to the Tsix regulatory region is crucial for Tsix silencing and Xist upregulation, we expect Xist upregulation upon differentiation to be rescued in these double knockout cell lines. We made use of a Tsix-Stop line containing a triple poly(A) signal downstream of the major Tsix promoter that blocks its transcription on the 129 allele 7 , and a Tsix-Cherry line with a mCherry coding sequence introduced downstream of the major Tsix promoter on the Cast allele 34 (Fig. 5c). As controls, we generated Spen -/-ESCs in the same hybrid background (F1:129/Cast) where both Xist alleles are intact 35 , in contrast to the previously studied ESCs that contained one doxycycline-inducible Xist allele. Deletion of the Spen ORF was confirmed in all cell lines by PCR on gDNA and Western blot analysis (Supplementary Fig. 6a,b). For each line we generated two independent knockout clones. All the ESC lines were differentiated in parallel and in biological duplicates, followed by allele-specific Xist RNA expression analysis by RT-qPCR. As expected, the F1:129/Cast control Spen -/-line showed no Xist upregulation (Fig. 5d), similar to the phenotype observed in the Spen knockout clones generated in the heterozygous doxycycline-inducible Xist cell line (Fig. 1g). Remarkably, the introduction of a poly(A) signal in Spen -/-:Tsix-Stop lines fully rescued Xist upregulation upon differentiation (Fig. 5e), indicating that SPEN is required for Xist upregulation via Tsix repression. However, the Spen -/-:Tsix-Cherry lines displayed only mild upregulation of Xist upon differentiation (Fig. 5e). These differences in the rescue phenotype between Spen -/-:Tsix-Cherry and Spen -/-:Tsix-Stop lines can be explained by remaining Tsix transcription in the Tsix-Cherry line, which is fully ablated in the Tsix-Stop lines (Supplementary Fig. 6c). As expected, the Spen -/-: Tsix-Stop lines are not able to silence X-linked Rnf12 despite normal Xist levels in the Tsix-Stop line (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted December 30, 2020. ; https://doi.org/10.1101/2020.12.30.424676 doi: bioRxiv preprint 9 (Supplementary Fig. 6d). Moreover, the Spen -/-:Tsix-Stop lines display Xist clouds that were similar in morphology (Fig. 5f) and number compared to Wt F1:129/Cast cells (Fig. 5g).

Taken together, our results indicate a novel and essential role for SPEN in Xist upregulation, mainly via silencing of Tsix, and to a lesser extent by stabilizing Xist RNA. SPEN is therefore not only crucial in X-linked gene silencing but also in the early initiation steps of XCI, playing a role in the feedforward loop leading to Xist activation (Fig. 6).

Discussion

The role of the silencing factor SPEN in the establishment phase of XCI has been addressed in various studies, but whether SPEN is relevant for the initiation phase, involving Xist upregulation, remains unknown. Here, we show that SPEN-defective ESCs do not upregulate Xist upon differentiation and study the molecular mechanism behind this observation. To explore the role of SPEN in XCI, various studies used Xist-inducible ESC lines to generate SPEN knockdown or knockout cell lines (Supplementary Table 1).

Forcing Xist expression in ESCs is a powerful way to understand X-linked gene silencing, but is not suitable to investigate the initiation phase of XCI. Studies exploring the role of SPEN in cells undergoing physiological XCI involved Spen knockdown strategies 17,20 , therefore, the levels of SPEN might have been sufficient to allow normal Xist upregulation, while showing a defect in X-linked gene silencing. Likewise, our Spen +/-ESC lines show Xist upregulation, but reduced X-linked silencing compared to Wt lines. In agreement with our results, one study reports lower Xist abundance and cloud formation upon forced Xist induction from the endogenous locus in Spen -/-ESCs 27 . Another recent study suggested that Spen -/-ESCs are not able to differentiate upon Leukemia Inhibitory Factor (LIF) removal and differentiation towards neural progenitor cells 36 , while we observe that Spen -/-ESCs display a normal morphology in the undifferentiated state and undergo normal differentiation upon monolayer differentiation. Nevertheless, we observe more cell death of Spen -/-cells compared to Wt cells upon monolayer differentiation, although we consider this might not be XCI-related since X0 Spen -/-cells also die upon differentiation (data not shown). This observation is probably not surprising since SPEN plays a role in various biological processes as a transcriptional repressor 23,24,37 .

Previous studies performed with doxycycline-inducible Xist lines show that SPEN and Xist co-localize soon after Xist induction 20,25 . However, Xist upregulation from a doxycycline-inducible promoter happens at a (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted December 30, 2020. ; https://doi.org/10.1101/2020.12.30.424676 doi: bioRxiv preprint 10 very different timescale than during physiological XCI. In the present study, we observe that upon Spen-GFP ESCs monolayer differentiation we can distinguish early and late SPEN accumulation on the Xi. Cells with SPEN early accumulation are detectable from day 1 of differentiation with their number decreasing overtime, while the number of cells with SPEN late accumulations progressively increases. Other XCI key players, such EZH2 and H3K27me3, also sequentially accumulate on the Xi.

In addition, we provide evidence that SPEN is required to silence Tsix to allow Xist upregulation. (I) In Spen - /-cells, we detect higher levels of Tsix at the latest time points of monolayer differentiation, compared to Wt cells. In line with this, paternal Tsix levels in female E3.5 blastocysts with a mutated Xist RepA, necessary for SPEN recruitment, are higher than in Wt blastocysts 38 , suggesting that higher Tsix levels in Spen -/-cells are due to defective Tsix silencing, rather than a lack of Xist antisense transcription. (II) SPEN overexpression in ESCs leads to higher Xist levels and ectopic Xist cloud formation in the undifferentiated state, suggesting that SPEN overexpression might lead to partial silencing of the Tsix promoter, facilitating Xist expression. (III) SPEN binds to active promoters and enhancers to silence the X chromosome 25 . Spatial proximity to the Xist locus is a strong predictor of X-linked gene silencing efficiency 26,39 . One of the closest actively transcribed promoters to Xist is Tsix and we indeed observe SPEN binding at the Tsix regulatory region. (IV) Spen -/-cell lines cannot upregulate Xist, while compound Spen:Tsix defective cell lines (Spen -/- :Tsix-Stop) display normal Xist levels upon differentiation, indicating that in the absence of SPEN, Tsix, which acts a as brake on Xist transcription, cannot be silenced. Furthermore, our results show that SPEN plays a role in Xist stability, however, we also observe that Xist levels in differentiating Spen -/-:Tsix-Stop cells are comparable to Wt cells, suggesting that Xist stability might not be compromised. This difference may be attributed to the massive Xist overexpression in Xist-inducible systems, where the excess of Xist RNA molecules, in the absence of SPEN, may affect overall Xist RNA stability readings.

During initiation of XCI, SPEN helps remodel the chromatin environment of the Xist-Tsix locus. SPEN and HDAC3 are present at the Tsix regulatory region and H3K27ac levels decrease upon Xist induction.

Accordingly, we propose that during XCI initiation, SPEN binds Xist nascent transcripts 25 , recruits and/or activates HDAC3 16,30 , which removes histone acetylation marks, weakening Tsix promoter activity and facilitating Xist expression (Fig. 6).

The mechanism driving the symmetry breaking event leading to monoallelic upregulation of Xist has been a focus of many studies. Several studies showed that the Xic and more specifically the Xist-Tsix master switch are tightly regulated in a deterministic process, involving monoallelic down-regulation of Tsix or Xpairing mechanisms [40][41][42] . Our studies, and studies of others, indicate that a gene regulatory network (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted December 30, 2020. ; https://doi.org/10.1101/2020.12.30.424676 doi: bioRxiv preprint 11 composed of trans-acting activators and inhibitors of XCI, involving positive and negative feedback loops, instruct the cis-regulatory landscape of the Xic to direct monoallelic upregulation of Xist 12,[43][44][45] . Our present work reveals that Tsix silencing not only involves downregulation of inhibitors of XCI, including pluripotency factors OCT4, SOX2, KLF4, c-MYC and REX1 [START_REF] Donohoe | The pluripotency factor Oct4 interacts with Ctcf and also controls X-chromosome pairing and counting[END_REF][START_REF] Navarro | Molecular coupling of Tsix regulation and pluripotency[END_REF][START_REF] Gontan | RNF12 initiates X-chromosome inactivation by targeting REX1 for degradation[END_REF] , but also involves Xist-mediated recruitment of SPEN. This can happen on either allele, but asynchronous Xist transcription bursts will facilitate Xistmediated monoallelic silencing of Tsix through SPEN, resulting in further upregulation of Xist and concomitant silencing of the XCI activator Rnf12, providing a negative feedback loop to prevent upregulation of Xist on the future active X chromosome.

Main figure legends

RT-qPCR

Total RNA was isolated from cell pellets using the ReliaPrep RNA Cell Miniprep System (Promega, Z6012) and reversed transcribed using Superscript III (Invitrogen, 18080093) and random hexamers (Invitrogen, N8080127), following the manufacturer's instructions. All RT-qPCRs were done using the GoTaq qPCR Master Mix (Promega, A6002) in a CFX384 real-time PCR detection system (Bio-Rad). Hist2h2aa1 was used as a normalization control, except in the RNA stability assay were β-actin was used. All expression primers are listed in Supplementary Table 4. The optimal allele-specific primer pair concentration to amplify the 129 and Cast allele at the same efficiency was optimized using pure 129, Cast and 129-Cast gDNA.

RNA stability assay

Wt and Spen -/-ESC lines with a doxycycline-responsive Xist promoter were cultured in medium supplemented with doxycycline to induce Xist expression for 4 days. While in doxycycline treatment, 5 µg mL -1 actinomycin D (Sigma-Aldrich, A1410) was added for different time (t= 0, 2, 4, 6 and 10 hours). Before collection, ESCs were plated in non-gelatinized plates to remove the feeders and harvested for RNA isolation, cDNA synthesis and RT-qPCR. Total Xist levels were determined and normalized to β-actin and (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted December 30, 2020. ; https://doi.org/10.1101/2020.12.30.424676 doi: bioRxiv preprint 26 the percentage of remaining Xist RNA was calculated by dividing Xist expression levels at the different times of collection relative to t=0. Using linear regression analysis the RNA decay rate constant (k decay) was calculated from the slope of the curve that best fitted our data and the RNA half-life (t1/2) obtained with the following formula t1/2 = ln2/(k decay).

Immunofluorescence (IF)

Undifferentiated ESCs were attached to slides using a cytospin, while differentiating cells were grown on coverslips coated with gelatin or laminin. Cells were fixed with 4% paraformaldehyde (PFA) in PBS (10 min, RT), permeabilized with 0.4% Triton-X100 and 5% goat serum in PBS (15 min, on ice) and blocked with 10% goat serum in PBST, composed of 0.05% Tween-20 in PBS (30 min, RT). Primary antibodies incubation was done in blocking buffer (2h, RT), after three washings with PBST the slides were incubated with the secondary antibody (1h, RT). After three washings, the second washing containing DAPI, the slides were mounted with ProLong Gold antifade mounting medium (Invitrogen, P36930). The used primary antibodies were the following: αGFP (Abcam, ab290, 1:500), H3K27me2/3 (Active motif, 39535, 1:100) and EZH2(BD43 clone, kindly provided by Dr. Kristian Helin, 1:100). Images were taken using a ZEISS Axio Imager M2 including digital microscopy camera AxioCam 503 and analysed with ImageJ.

IF combined with RNA Fluorescent in Situ Hybridization (FISH)

The Xist FISH probe was made from 2 µg of a 5.5 kb DNA fragment of mouse Xist comprising exons 3 to 7. Cells on coverslips or slides were fixed with 4% PFA in PBS (10 min, RT), permebilized with 0.5% Triton-X100 in PBS and blocked in TS-BSA buffer (0.1 M Tris-HCl (pH 7.5), 0.15 M NaCl, 2 mg mL -1 BSA (Jena Bioscience, BU-102) in H2O). The αGFP (Abcam, ab290, 1:500) primary antibody was incubated in blocking buffer (30 min, 37 o C), washed in PBS (3x) and then incubated with the secondary antibody (30 min, 37 o C).

All solutions were supplemented with 10 µM VRC and 0.2 U µL -1 RNAseOUT. Slides were washed in PBS (3x), post-fixed with 4% PFA (10 min, RT) and washed again in PBS (x3). Dehydration was done with (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted December 30, 2020. ; https://doi.org/10.1101/2020.12.30.424676 doi: bioRxiv preprint 27 increasing ethanol concentration (70%, 90% and 100%). The pre-hybridized probe was added on top of the coverslips or slides (20h, 37 o C, humid chamber). Various washings were done with 50% formamide/2x SSC (5 min, 37 o C, 2x), 2x SSC (5 min, 37 o C, 2x) and TS buffers (5 min, RT, 2x). Then the slides were mounted and visualized as explained in the IF section.

FISH

The FISH only protocol was done as described in the IF-FISH section, skipping the IF section. Directly after the permeabilization, the dehydration step was performed.

RNA-seq

Doxycycline treated cells were sorted to isolate dsRed positive cells upregulating Xist from the desired allele. RNA was isolated using the ReliaPrep RNA Cell Miniprep System. A total of 16 DNA libraries were created, according to the Smart-seq2 protocol [START_REF] Picelli | Smart-seq2 for sensitive full-length transcriptome profiling in single cells[END_REF] , using the Nextera DNA Flex library prep kit (Illumina) to create a library from full-length cDNA. Samples were sequenced on a HiSeq2500 sequencer (50 bp singleend reads).

Chromatin immunoprecipitation (ChIP)-seq 50x10^6 cells were trypsinised, resuspended and fixed in 50 mL warm medium and 1% PFA for 10 min at NGS data analysis: allele-specific RNA-seq and ChIP-seq Both the RNA-seq as the ChIP-seq data were processed allele-specifically. The single nucleotide polymorphism (SNPs) in the 129/Sv and Cast/Ei lines were downloaded from the Sanger institute (v.5 SNP142) [START_REF] Keane | Mouse genomic variation and its effect on phenotypes and gene regulation[END_REF] . These were used as input for SNPsplit v0.3.4 [START_REF] Krueger | Allele-specific splitting of alignments between genomes with known SNP genotypes[END_REF] , to construct an N-masked reference genome based on mm10 in which all SNPs between 129/Sv and Cast/Ei were masked. The 50 bp single-end RNAseq and 50 bp paired-end ChIP-seq reads were mapped to this N-masked reference genome using the default settings of hisat2 v2.2.1 and bowtie2 v2.4.1, respectively [START_REF] Kim | A fast spliced aligner with low memory requirements[END_REF][START_REF] Langmead | Fast gapped-read alignment with Bowtie 2[END_REF] . SNPsplit (--paired for the ChIP-seq analysis) was then used to assign the reads to either the 129/Sv or Cast/Ei bam file based on the best alignment or to a common bam file if mapping to a region without allele-specific SNPs. The allele-specific and unassigned bam files were sorted using samtools v1.10 54 .

For the RNA-seq, the number of mapped reads per gene were counted for both alleles separately using HTSeq v0.12.4 (--nonunique=none -m intersection-nonempty) [START_REF] Anders | HTSeq-A Python framework to work with high-throughput sequencing data[END_REF] based on the gene annotation from ensembl v98. For each condition, genes with more than 20 allele-specific reads across both replicates were used to calculate the allelic ratio, defined as Xi/(Xi+Xa). For the day 0 and day 7, Cast/Ei and 129/Sv were used as the Xi and active allele (Xa), respectively, whereas for the day 3, Cast/Ei and 129/Sv were used as Xa and Xi, respectively. The allelic ratios of X-linked genes were visualized as violin plots with boxplots of the same data on top. Allelic ratios of individual genes were plotted along the X chromosome. Different conditions were plotted together showing only the X-linked genes that had at least 20 allelespecific reads in both conditions. We visualized differences in allelic ratios of X-linked genes between conditions by plotting both ratios on the different axes of a scatter plot. Genes were highlighted when they were identified as lowly silenced genes in Spen -/-ESCs from 27 , which were defined as genes showing -0.05 < z < -0.2 where z (gene silencing) = [Xi/(Xi+Xa)]dox -[Xi/(Xi+Xa)]noDox. Significant differences between conditions were tested using a Mann-Whitney test with P-value < 0.05.

The allele-specific ChIP-seq bam files were normalized using the 'callpeak' and 'bdgcmp' functions of MACS2 v2.2.7.1 [START_REF] Zhang | Model-based analysis of ChIP-Seq (MACS)[END_REF] . We called broad peaks (-f BAMPE --broad --bdg) and used the Poisson P-value as method for normalizing the tracks. The input-normalized tracks were visualized using pyGenomeTracks (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted December 30, 2020. ; https://doi.org/10.1101/2020.12.30.424676 doi: bioRxiv preprint 29 v3.4. [START_REF] Lopez-Delisle | pyGenomeTracks: reproducible plots for multivariate genomic data sets[END_REF] . For validation, we downloaded several publicly available datasets. The SPEN CUT&RUN data (SRX5903674, SRX5903675, SRX5903676, SRX5903677, SRX5903678, SRX5903679, SRX5903682, SRX5903683) 25 , was processed similar to our analysis using a C57BL/6NJ-Cast/Ei reference genome.

However, the allele-specific tracks were normalized based on the total number of mapped reads per sample. The scaling factor was calculated as 10^6 / total number of mapped reads and used as parameter --scaleFactor to both allelic tracks using deepTools bamCoverage v3.5.0. 58 . A binsize of 1 was used and paired-end reads were extended. The allele-specific tracks from HDAC3 and H3K27Ac (SRX4384412, SRX4384420, SRX4384476, SRX4384484, SRX4887836, SRX4887839) were downloaded from 30 . For all datasets, replicates for each condition were averaged using deepTools bigwigCompare v.3.5.0 with the settings '--operation mean --binSize 1' [START_REF] Ramírez | deepTools2: a next generation web server for deep-sequencing data analysis[END_REF] . In the genome browser overview showing the allele-specific tracks, the y-axis was scaled for each group of samples separately. Zoom-in Figure 5b Fig. 5 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted December 30, 2020. ; https://doi.org/10.1101/2020.12. 30

■ 4 M

 4 ♥tr♦❞✉❝t✐♦♥ ❳ ❈❤r♦♠♦s♦♠❡ ■♥❛❝t✐✈❛t✐♦♥ s♦♠❡ ✏❜❡♥❡✜❝✐❛•✑ ♠❛•❡✲s♣❡❝✐✜❝ ❣❡♥❡s✱ ✇❤✐•❡ ❢❡♠❛•❡s ✇♦✉•❞ ✐♥❤❡r✐t ✏❤❛r♠❢✉•✑ ❣❡♥❡s ♣r❡✈✐✲ ♦✉s•② ❢♦✉♥❞ ♦♥•② ♦♥ t❤❡ ♣r♦t♦✲( ✭❋✐❣✉r❡ ✶✳✶❉✮✳ ❚❤❡r❡❢♦r❡✱ ♠❡✐♦t✐❝ r❡❝♦♠❜✐♥❛t✐♦♥ ❛r♦✉♥❞ t❤❡ ♠❛•❡✲s♣❡❝✐✜❝ ❣❡♥❡s ✇❛s s✉♣♣r❡ss❡❞ s♦ ❛s t♦ ♣r❡s❡r✈❡ ❛ str✐❝t ❞✐st✐♥❝t✐♦♥ ❜❡t✇❡❡♥ t❤❡ ♣r♦t♦✲❳ ❛♥❞ t❤❡ ♣r♦t♦✲( ✭❋✐❣✉r❡ ✶✳✶❊✮✳ ❯♥❛❜•❡ t♦ r❡❝♦♠❜✐♥❡ ✇✐t❤ t❤❡ ♣r♦t♦✲❳✱ t❤❡ ♣r♦t♦✲( ❛❝❝✉♠✉•❛t❡❞ ❞❡•❡t❡r✐♦✉s ♠✉t❛t✐♦♥s ✇❤✐❝❤ ❝♦✉•❞ ♥❡✈❡r ❜❡ ❜❛•❛♥❝❡❞ ♦✉t ❞✉r✐♥❣ ♠❡✐♦s✐s ✭❋✐❣✉r❡ ✶✳✶❊✮ ❬▼✉••❡r✱ ✶✾✶✽❀ |✐❝❡✱ ✶✾✽✼✱ ✶✾✾✻(✳ ❚❤✐s ♠✉t❛t✐♦♥❛• ❜✉r❞❡♥ •❡❞ t♦ t❤❡ ❞❡❣❡♥❡r❛t✐♦♥ ♦❢ ♠♦st ♦❢ t❤❡ ( ❝❤r♦♠♦s♦♠❡ ❛♥❞ ✐ts ❡♥❝♦❞✐♥❣ ❣❡♥❡s✱ t❤❡r❡❜② ♣r♦✈✐❞✐♥❣ ❛♥ ❡①♣•❛♥❛t✐♦♥ ❢♦r t❤❡ ❞✐✛❡r❡♥❝❡ ❜❡t✇❡❡♥ t❤❡ ♠♦❞❡r♥ ❳ ❛♥❞ ( ❝❤r♦♠♦s♦♠❡s ✐♥ ♠❛♠✲ ♠❛•s ✭❋✐❣✉r❡ ✶✳✶❊✮ ❬❈❤❛r•❡s✇♦rt❤✱ ✶✾✾✻❀ ❈❤❛r•❡s✇♦rt❤ ❡t ❛•✳✱ ✷✵✵✺❀ •r❛✈❡s✱ ✷✵✵✻❀ •r❛✈❡s ❡t ❛•✳✱ ✷✵✵✻❀ ❱✐❝♦s♦ ❛♥❞ ❈❤❛r•❡s✇♦rt❤✱ ✷✵✵✻(✳ fold expression ❋✐❣✉r❡ ✶✳✶ ✕ ▼❛♠♠❛•✐❛♥ s❡① ❝❤r♦♠♦s♦♠❡ ❡✈♦•✉t✐♦♥ ❛♥❞ t❤❡ r❡q✉✐r❡♠❡♥t ❢♦r ❞♦s❛❣❡ ❝♦♠♣❡♥s❛t✐♦♥✳ ✭❆✮ ▼✉t❛t✐♦♥s ✐♥ t❤❡ ❙❖❳✸ ❣❡♥❡ •❡❛❞ t♦ ✭❇✮ t❤❡ ❡♠❡r❣❡♥❝❡ ♦❢ ❛ s❡①✲ ❞❡t❡r♠✐♥✐♥❣ ❣❡♥❡✱ ❙|(✱ ♦♥ ♦♥❡ ♦❢ t❤❡ t✇♦ ♣r♦t♦✲s❡① ❝❤r♦♠♦s♦♠❡s✳ ✭❈✮ ▼❛•❡✲s♣❡❝✐✜❝ ❣❡♥❡s ❛❝❝✉♠✉•❛t❡ ♦♥ t❤❡ ♣r♦t♦✲(✱ •❡❛❞✐♥❣ t♦ ✭❉✮ ❞❡•❡t❡r✐♦✉s ✐♥t❡r✲s❡① ❛rr❛♥❣❡♠❡♥ts ❢♦••♦✇✐♥❣ r❡❝♦♠✲ ❜✐♥❛t✐♦♥ ❡✈❡♥ts ✐♥ ♠❡✐♦s✐s✳ ✭❊✮ ❈♦♥s❡q✉❡♥t✐❛• s✉♣♣r❡ss✐♦♥ ♦❢ ♠❡✐♦t✐❝ r❡❝♦♠❜✐♥❛t✐♦♥ ✐♥ t❤❡ ♠❛•❡ ❣❡r♠•✐♥❡ ❢❛✈♦rs ✜①❛t✐♦♥ ♦❢ ❞❡•❡t❡r✐♦✉s ♠✉t❛t✐♦♥s✱ r❡s✉•t✐♥❣ ✐♥ t❤❡ ♣r♦❣r❡ss✐✈❡ ❞❡❣❡♥❡r❛t✐♦♥ ♦❢ t❤❡ ♣r♦t♦✲( ❝❤r♦♠♦s♦♠❡✳ ❆s ❣❡♥❡s ❛r❡ •♦st ♦♥ t❤❡ ♣r♦t♦✲(✱ s❡•❡❝t✐✈❡ ♣r❡ss✉r❡s ❡♥s✉r❡ t✇♦✲❢♦•❞ ✉♣r❡❣✉•❛t✐♦♥ ♦❢ ❝♦rr❡s♣♦♥❞✐♥❣ ❣❡♥❡s ♦♥ t❤❡ ♣r♦t♦✲❳✳ ✭❋✮ ❚❤❡ r❡s✉•t✐♥❣ ✐♥❝r❡❛s❡ ✐♥ ❳✲•✐♥❦❡❞ ❣❡♥❡ ❞♦s❛❣❡ ✐♥ ❢❡♠❛•❡s ✐s ❝♦♠♣❡♥s❛t❡❞ t❤r♦✉❣❤ ❳ ❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥✳ ♦❞✉❝t✐♦♥ ❳ ❈❤r♦♠♦s♦♠❡ ■♥❛❝t✐✈❛t✐♦♥ ❡①♣r❡ss✐♦♥ ❤❛s ❜❡❡♥ ✐♥✈❡st✐❣❛t❡❞ ✐♥ t❤❡ ❝♦♥t❡①t ♦❢ ❛ ♣r♦•❡❝t t♦ ✇❤✐❝❤ ■ ♣❛rt✐❝✐♣❛t❡❞ ❛t t❤❡ ❜❡❣✐♥♥✐♥❣ ♦❢ ♠② ❞♦❝t♦r❛• st✉❞✐❡s ❬➏②•✐❝③ ❡t ❛•✳✱ ✷✵✶✾(✳ ❚❤✐s st✉❞② ✇✐•• ❜❡ ❢✉rt❤❡r ❞✐s❝✉ss❡❞ ✐♥ ❝❤❛♣t❡r ✺ ♦❢ t❤❡ r❡s✉•ts ♣❛rt ♦❢ ♠② ❞✐ss❡rt❛t✐♦♥✳ marks ❋✐❣✉r❡ ✶✳✽ ✕ ❙❝❤❡♠❡ ❞❡♣✐❝t✐♥❣ t❤❡ ❝❤r♦♠❛t✐♥ ❝❤❛♥❣❡s ♦❝❝✉r✐♥❣ ❞✉r✐♥❣ ❳❈■✳ ✶✳✾ ▼❛✐♥t❡♥❛♥❝❡ ♦❢ ❳✲❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥ |❡♠❛r❦❛❜•②✱ ❤♥|◆P❑ ✇❛s ❢♦✉♥❞ t♦ ✐♥t❡r❛❝t ❞✐r❡❝t•② ✇✐t❤ P❈•❋✸ ❛♥❞ P❈•❋✺✱ t✇♦ ❝♦♠♣♦♥❡♥ts ♦❢ t❤❡ ♥♦♥✲❝❛♥♦♥✐❝❛• P❈•❋✸✴✺✲P|❈✶ ❝♦♠♣•❡① ✭❋✐❣✉r❡ ✶✳✶✶✮✱ ❛♥❞ t❤✐s ✐♥t❡r✲ ❛❝t✐♦♥ ✇❛s ❢♦✉♥❞ t♦ ❜❡ ♥❡❝❡ss❛r② ❢♦r ❳✐st✲♠❡❞✐❛t❡❞ P|❈✶ r❡❝r✉✐t♠❡♥t ❬P✐♥t❛❝✉❞❛ ❡t ❛•✳✱ ✷✵✶✼(✳ ❈♦♥s✐st❡♥t•②✱ |(❇P ✭❛♥♦t❤❡r ❝♦♠♣♦♥❡♥t ♦❢ t❤❡ ♥♦♥✲❝❛♥♦♥✐❝❛• P|❈✶ ❝♦♠♣•❡①✮ ❛♥❞ |■◆•✶❇ ✭t❤❡ ❝❛t❛•②t✐❝ s✉❜✉♥✐t ♦❢ P|❈✶✱ r❡s♣♦♥s✐❜•❡ ❢♦r ❍✷❆❑✶✶✾✉❜✶ ❞❡♣♦s✐t✐♦♥✮ ✇❡r❡ r❡tr✐❡✈❡❞ ✐♥ t✇♦ ♦❢ t❤❡ ❳✐st ♣r♦t❡♦♠✐❝ st✉❞✐❡s ❬❈❤✉ ❡t ❛•✳✱ ✷✵✶✺❀ ▼✐♥❛•✐❣✐ ❡t ❛•✳✱ ✷✵✶✺(✳ ✶✳✶✷ ✕ ▼♦❞❡• ❢♦r ▲❇|✲♠❡❞✐❛t❡❞ r❡❝r✉✐t♠❡♥t ♦❢ ❳✐st ❛♥❞ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡ t♦ t❤❡ ♥✉❝•❡❛r •❛♠✐♥❛✳ ❚❤❡ ▲❇❙ r❡❣✐♦♥s ♦❢ ❳✐st ✕ ✇❤✐❝❤ ❜✐♥❞ t❤❡ |❙ ♠♦t✐❢ ♦❢ ▲❇| ✕ ❛r❡ ❝♦•♦r❡❞ ✐♥ ♣✉r♣•❡✳ ✷✻ ■♥tr♦❞✉❝t✐♦♥ ❳ ❈❤r♦♠♦s♦♠❡ ■♥❛❝t✐✈❛t✐♦♥ ■♠♣♦rt❛♥t•② ✐♥ t❤✐s st✉❞②✱ ♦♥•② ✺ ❳✲•✐♥❦❡❞ ❣❡♥❡s ✇❡r❡ ❛ss❛②❡❞ ❛s ❛ ♣r♦①② ❢♦r ❳❈■✱ ✉s✐♥❣ |◆❆ ❋■❙❍ ❬❈❤❡♥ ❡t ❛•✳✱ ✷✵✶✻❀ ▼❝❍✉❣❤ ❡t ❛•✳✱ ✷✵✶✺(✳ ■♥ ❛ r❡❝❡♥t r❡♣♦rt ❤♦✇❡✈❡r✱ ✐♥ ✇❤✐❝❤ ❳✲•✐♥❦❡❞ tr❛♥s❝r✐♣t✐♦♥ ✇❛s ❛ss❛②❡❞ ❝❤r♦♠♦s♦♠❡✲✇✐❞❡ ✉s✐♥❣ |◆❆s❡q✱ t❤❡ ❇r♦❝❦❞♦r✛ •❛❜ s❤♦✇❡❞ t❤❛t ▲❜r ❦♥♦❝❦♦✉t ✕ ♦r ❞❡•❡t✐♦♥ ♦❢ t❤❡ ▲❇❙✲✶ r❡❣✐♦♥ ♦❢ ❳✐st ✕ r❡s✉•t❡❞ ✐♥ ✈❡r② ♠✐♥♦r ❣❡♥❡ s✐•❡♥❝✐♥❣ ❞❡❢❡❝ts ❬◆❡st❡r♦✈❛ ❡t ❛•✳✱ ✷✵✶✾(✳ ❚❤✐s ♥❡✇ st✉❞② ❤❡♥❝❡ s✉❣❣❡sts t❤❛t ▲❇| ♦♥•② ♣•❛②s ❛ ♥❡❣•✐❣✐❜•❡ r♦•❡ ✐♥ ❳✐st✲♠❡❞✐❛t❡❞ s✐•❡♥❝✐♥❣✱ ❛♥❞ t❤❛t t❤❡ ❛ss♦❝✐❛t✐♦♥ ♦❢ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡ ✇✐t❤ t❤❡ ♥✉❝•❡❛r •❛♠✐♥❛ ✐s ♥♦t ❝r✉❝✐❛• ❢♦r ❳❈■ ❬◆❡st❡r♦✈❛ ❡t ❛•✳✱ ✷✵✶✾(✳ ✶✳✶✸✳✸ |❇▼✶✺ ❛♥❞ ❲❚❆P •✐♥❦ ❳✐st ✇✐t❤ ♠ ✻ ❆ |◆❆ ♠❡t❤②•❛t✐♦♥ ❈❡••✉•❛r |◆❆s ❛r❡ s✉❜•❡❝t t♦ ❛ ✇✐❞❡ ❛rr❛② ♦❢ ❝❤❡♠✐❝❛• ♠♦❞✐✜❝❛t✐♦♥s✱ ❛♠♦♥❣ ✇❤✐❝❤ ◆ ✻ ✲ ♠❡t❤②•❛❞❡♥♦s✐♥❡ ✭♠ ✻ ❆✮ ❤❛s r❡❝❡♥t•② ❡♠❡r❣❡❞ ❛s ❛♥ ✐♠♣♦rt❛♥t ♣•❛②❡r ✐♥ ❣❡♥❡ ❡①♣r❡ss✐♦♥ r❡❣✉•❛t✐♦♥ ❜② ❛✛❡❝t✐♥❣ ♠|◆❆ ❤♦♠❡♦st❛s✐s ❛t s❡✈❡r❛• •❡✈❡•s ✐♥❝•✉❞✐♥❣ s♣•✐❝✐♥❣✱ st❛❜✐•✐t②✱ •♦❝❛•✐③❛t✐♦♥ ❛♥❞ tr❛♥s•❛t✐♦♥ ❬|♦✉♥❞tr❡❡ ❡t ❛•✳✱ ✷✵✶✼(✳ ♠ ✻ ❆ ♠❡t❤②•❛t✐♦♥ ✐s ❝❛t❛•②③❡❞ ❜② ❛ ♥✉❝•❡❛r ✧✇r✐t❡r✧ ❝♦♠♣•❡① ❝♦♠♣♦s❡❞ ♦❢ t❤❡ ▼❊❚❚▲✸✴▼❊❚❚▲✶✹ ♠❡t❤②•tr❛♥s❢❡r❛s❡s ❛♥❞ t❤❡ ❛❞❛♣t♦r ♣r♦t❡✐♥s ❲❚❆P✱ |❇▼✶✺✱ )❈✸❍✶✸✱ ❱■|▼❆ ✭❑■❆❆✶✹✷✾✮ ❛♥❞ ❈❇▲▲✶ ✭❍❆❑❆■✮ ❬)❛❝❝❛r❛ ❡t ❛•✳✱ ✷✵✶✾(✳ ❖♥❝❡ ❞❡✲ ♣♦s✐t❡❞✱ ♠ ✻ ❆ r❡❝r✉✐ts ♠ ✻ ❆ ✧r❡❛❞❡r✧ ♣r♦t❡✐♥s✱ ❛♠♦♥❣ ✇❤✐❝❤ t❤❡ (❚❍ ❞♦♠❛✐♥✲❝♦♥t❛✐♥✐♥❣ ♣r♦t❡✐♥s (❚❍❉❈✶ ❛♥❞ (❚❍❉❋✶✴✷✴✸ ✕ •♦❝❛•✐③❡❞ ✐♥ t❤❡ ♥✉❝•❡✉s ❛♥❞ t❤❡ ❝②t♦s♦• r❡s♣❡❝✲ t✐✈❡•② ✕ ❤❛✈❡ ❡①t❡♥s✐✈❡•② ❜❡❡♥ s❤♦✇♥ t♦ ❛✛❡❝t ♠|◆❆ ❢❛t❡ ✐♥ t❤❡✐r r❡s♣❡❝t✐✈❡ s✉❜❝❡••✉•❛r ❝♦♠♣❛rt♠❡♥ts ❬)❛❝❝❛r❛ ❡t ❛•✳✱ ✷✵✶✾(✳ ■♥ ✷✵✶✺✱ ❛ r♦•❡ ❢♦r t❤❡ ♠ ✻ ❆ |◆❆ ♠❡t❤②•❛t✐♦♥ ♣❛t❤✇❛② ❞✉r✐♥❣ ❳❈■ ✇❛s ❜r♦✉❣❤t t♦ •✐❣❤t ✭❋✐❣✉r❡ ✶✳✶✸✮✳ ■♥❞❡❡❞✱ |❇▼✶✺ ✭|◆❆✲❜✐♥❞✐♥❣ ♠♦t✐❢ ♣r♦t❡✐♥ ✶✺✮ ❛♥❞ ❲❚❆P ✭❲ ✐•♠s t✉♠♦r ✶ ❛ss♦❝✐❛t❡❞ ♣r♦t❡✐♥✮ ✇❡r❡ ❢♦✉♥❞ t♦ ✐♥t❡r❛❝t ✇✐t❤ ❳✐st ✕ s❡❡♠✐♥❣•② t❤r♦✉❣❤ ✐ts ❆✲r❡♣❡❛t ✕ ✐♥ t✇♦ ♦❢ t❤❡ ♣r♦t❡♦♠✐❝ st✉❞✐❡s ✭❋✐❣✉r❡ ✶✳✶✸✮ ❬▼❝❍✉❣❤ ❡t ❛•✳✱ ✷✵✶✺❀ ❈❤✉ ❡t ❛•✳✱ ✷✵✶✺(✳ ❋✉rt❤❡r♠♦r❡✱ |❇▼✶✺ ❛♥❞ ❲❚❆P ✇❡r❡ ❜♦t❤ ✐❞❡♥t✐✜❡❞ ❛s ❝❛♥❞✐❞❛t❡s ✐♥ ♦♥❡ ♦❢ t❤❡ t✇♦ ❣❡♥❡t✐❝ s❝r❡❡♥s✱ ❛♥❞ t❤❡✐r ❦♥♦❝❦❞♦✇♥ ✇❛s ❛ss♦❝✐❛t❡❞ ✇✐t❤ ❞❡❢❡❝ts ✐♥ ❳✐st✲♠❡❞✐❛t❡❞ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❬▼♦✐♥❞r♦t ❡t ❛•✳✱ ✷✵✶✺(✳ ❋♦••♦✇ ✉♣ st✉❞✐❡s s❤♦✇❡❞ t❤❛t ❳✐st✴❳■❙❚ ✐s ❤✐❣❤•② ♠ ✻ ❆✲♠❡t❤②•❛t❡❞ ✐♥ ♠♦✉s❡ ❛♥❞ ❤✉♠❛♥s r❡s♣❡❝t✐✈❡•② ❬◆❡st❡r♦✈❛ ❡t ❛•✳✱ ✷✵✶✾❀ P❛t✐• ❡t ❛•✳✱ ✷✵✶✻(✱ ❛♥❞ t❤❛t ❜✐♥❞✐♥❣ ♦❢ |❇▼✶✺ t♦ ❳✐st |◆❆ ✐s r❡q✉✐r❡❞ ❢♦r ♠ ✻ ❆✲♠❡t❤②•❛t✐♦♥ ♦❢ ❳✐st ✭❋✐❣✉r❡ ✶✳✶✸✮ ❬P❛t✐• ❡t ❛•✳✱ ✷✵✶✻(✳ P❛t✐• ❡t ❛•✳ ❢✉rt❤❡r s❤♦✇❡❞ t❤❛t |❇▼✶✺ ✭❛♥❞ ✐ts ♣❛r❛•♦❣ |❇▼✶✺❇✮ r❡❝r✉✐ts t❤❡ ♠ ✻ ❆✲ ♠❡t❤②•❛t✐♦♥ ❝♦♠♣•❡① t♦ ❳✐st ❜② ✐♥t❡r❛❝t✐♥❣ ❞✐r❡❝t•② ✇✐t❤ ❲❚❆P ✭❋✐❣✉r❡ ✶✳✶✸✮✳ ■♠♣♦rt❛♥t•②✱ ❦♥♦❝❦❞♦✇♥s ♦❢ ▼❡tt•✸✱ ❲t❛♣ ♦r |❜♠✶✺✴|❜♠✶✺❜ ✇❡r❡ s❤♦✇♥ t♦ r❡s✉•t ✐♥ ❞❡✜❝✐❡♥t ❳✐st✲♠❡❞✐❛t❡❞ ❣❡♥❡ s✐•❡♥❝✐♥❣✱ s✉❣❣❡st✐♥❣ t❤❛t ❳✐st |◆❆ ♠❡t❤②•❛t✐♦♥ ✐s r❡✲ q✉✐r❡❞ ❢♦r ❣❡♥❡ s✐•❡♥❝✐♥❣ ❞✉r✐♥❣ ❳❈■ ❬P❛t✐• ❡t ❛•✳✱ ✷✵✶✻(✳ ❚❤✐s ❡✛❡❝t ✇❛s s❤♦✇♥ t♦ ❜❡ ✷✼ ■♥tr♦❞✉❝t✐♦♥ ❳ ❈❤r♦♠♦s♦♠❡ ■♥❛❝t✐✈❛t✐♦♥ ♠❡❞✐❛t❡❞ ❜② t❤❡ ❜✐♥❞✐♥❣ ♦❢ t❤❡ (❚❍❉❈✶ r❡❛❞❡r ♣r♦t❡✐♥ t♦ ♠❡t❤②•❛t❡❞ ❳✐st ❬P❛t✐• ❡t ❛•✳✱ ✷✵✶✻(✱ ❛•t❤♦✉❣❤ t❤❡ ♠❡❝❤❛♥✐st✐❝ ❜❛s✐s ❢♦r (❚❍❉❈✶✲♠❡❞✐❛t❡❞ ❳✲•✐♥❦❡❞ ❣❡♥❡ s✐•❡♥❝✐♥❣ ✐s ❝♦♠♣•❡t❡•② ✉♥❦♥♦✇♥ ✭❋✐❣✉r❡ ✶✳✶✸✮✳ M E T T L 1 ▼♦❞❡• ❢♦r ❆✲r❡♣❡❛t ❞❡♣❡♥❞❡♥t r❡❝r✉✐t♠❡♥t ♦❢ t❤❡ ♠ ✻ ❆ |◆❆ ♠❡t❤②✲ •❛t✐♦♥ ♠❛❝❤✐♥❡r②✳ |❇▼✶✺ ❜✐♥❞✐♥❣ t♦ ❳✐st ❆✲r❡♣❡❛t •❡❛❞s t♦ r❡❝r✉✐t♠❡♥t ♦❢ t❤❡ ♠ ✻ ❆ ✇r✐t✐♥❣ ♠❛❝❤✐♥❡r② ❛♥❞ s✉❜s❡q✉❡♥t ❳✐st |◆❆ ♠❡t❤②•❛t✐♦♥✳ ❇✐♥❞✐♥❣ ♦❢ t❤❡ ♠ ✻ ❆ r❡❛❞❡r (❚❍❉❈✶ ❛•✲ •❡❣❡❞•② ♣•❛②s ❛ r♦•❡ ✐♥ ❳✲•✐♥❦❡❞ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❞✉r✐♥❣ ❳❈■✳ ❙✐♠✐•❛r t♦ ✇❤❛t ✇❛s ❞✐s❝✉ss❡❞ ♣r❡✈✐♦✉s•② ✐♥ s✉❜s❡❝t✐♦♥ ✶✳✶✸✳✷ ❛❜♦✉t ▲❇|✱ t❤❡ ❡✈✲ ✐❞❡♥❝❡ ❢♦r ❛ ♠❛•♦r r♦•❡ ♦❢ ❳✐st |◆❆ ♠❡t❤②•❛t✐♦♥ ✐♥ ❣❡♥❡ s✐•❡♥❝✐♥❣ ✇❛s s♦•❡•② ❜❛s❡❞ ♦♥ |◆❆ ❋■❙❍ ❛ss❛②s ♦❢ ✷ ❳✲•✐♥❦❡❞ ❣❡♥❡s✳ |❡❝❡♥t•② ❤♦✇❡✈❡r✱ t❤❡ ❇r♦❝❦❞♦r✛ •❛❜ ✉s❡❞ |◆❆s❡q t♦ s❤♦✇ t❤❛t ❣❡♥❡ s✐•❡♥❝✐♥❣ ❝❤r♦♠♦s♦♠❡ ✇✐❞❡ ✐s ♦♥•② ♠✐♥♦r•② ❛✛❡❝t❡❞ ✉♣♦♥ ❦♥♦❝❦♦✉ts ♦❢ |❜♠✶✺✱ ❲t❛♣ ♦r ▼❡tt•✸ ❬◆❡st❡r♦✈❛ ❡t ❛•✳✱ ✷✵✶✾(✳ ❈♦♥s✐st❡♥t•②✱ t❤❡② ❛•s♦ r❡♣♦rt❡❞ ♥♦ ❞r❛♠❛t✐❝ s✐•❡♥❝✐♥❣ ❞❡❢❡❝ts ✉♣♦♥ ❞❡•❡t✐♦♥ ♦❢ ♠❛•♦r ♠ ✻ ❆ s✐t❡s ✇✐t❤✐♥ ❳✐st ❬◆❡st❡r♦✈❛ ❡t ❛•✳✱ ✷✵✶✾❀ ❈♦❦❡r ❡t ❛•✳✱ ✷✵✷✵(✳ ❚❛❦❡♥ t♦❣❡t❤❡r✱ t❤❡s❡ r❡s✉•ts str♦♥❣•② s✉❣❣❡st t❤❛t ❳✐st |◆❆ ♠❡t❤②•❛t✐♦♥ ✐s •❛r❣❡•② ❞✐s♣❡♥s❛❜•❡ ❢♦r ❣❡♥❡ s✐•❡♥❝✐♥❣ ❞✉r✐♥❣ ❳❈■✳ ✷✽ ■♥tr♦❞✉❝t✐♦♥ ❙P❊◆✿ ❉✐s❝♦✈❡r② ❛♥❞ ❢✉♥❝t✐♦♥ ❈❤❛♣t❡r ✷ ❙P❊◆✿ ❉✐s❝♦✈❡r② ❛♥❞ ❢✉♥❝t✐♦♥ ❆•t❤♦✉❣❤ ✐❞❡♥t✐✜❡❞ ♠♦r❡ t❤❛♥ ✷✵ ②❡❛rs ❛❣♦✱ t❤❡ ❙P❊◆ ♣r♦t❡✐♥ r❡♠❛✐♥s ✈❡r② ♣♦♦r•② ❝❤❛r❛❝t❡r✐③❡❞✳ ❆ ❢❡✇ st✉❞✐❡s✱ ♣❡r❢♦r♠❡❞ ❛❝r♦ss ❞✐✛❡r❡♥t ♠♦❞❡• ♦r❣❛♥✐s♠s✱ ❤❛✈❡ r❡✈❡❛•❡❞ s♦♠❡ ♦❢ ✐ts ❢✉♥❝t✐♦♥s ❛♥❞ ♣r♦✈✐❞❡❞ ❤✐♥ts ✐♥t♦ ✐ts ♠❡❝❤❛♥✐s♠ ♦❢ ❛❝t✐♦♥✳ ■♥ t❤✐s ❝❤❛♣t❡r✱ ■ ✐♥t❡♥❞ t♦ ♣r❡s❡♥t t❤❡s❡ st✉❞✐❡s s✉❝❝✐♥❝t•② ❛♥❞ ❤✐❣❤•✐❣❤t t❤❡ st❛t❡ ♦❢ ❦♥♦✇•❡❞❣❡ ♦♥ ❙P❊◆ ❛t t❤❡ t✐♠❡ ■ ✐♥✐t✐❛t❡❞ ♠② P❤❉ ✇♦r❦ ✐♥ ✷✵✶✼✳✷✳✶ ❋✐rst ❞✐s❝♦✈❡r② ♦❢ ❙P❊◆ ✐♥ ❉r♦s♦♣❤✐•❛ ❚❤❡ ❣❡♥❡ ❙♣❡♥ ✭❙♣•✐t✲❡♥❞s✮ ✇❛s ♦r✐❣✐♥❛••② ✐❞❡♥t✐✜❡❞ ✐♥ ❉r♦s♦♣❤✐•❛ ❡♠❜r②♦s✱ ✇❤❡r❡ ✐ts ♠✉t❛t✐♦♥ ✇❛s ❛ss♦❝✐❛t❡❞ ✇✐t❤ s❡♥s♦r② ❛①♦♥ ❣r♦✇t❤ ❞❡❢❡❝ts ❬❑♦•♦❞③✐❡• ❡t ❛•✳✱ ✶✾✾✺(✳ ❉r♦s♦♣❤✐•❛ ❙♣❡♥ ✇❛s s❤♦✇♥ t♦ ❡♥❝♦❞❡ ❛ ✻✵✵❦❉❛ ♥✉❝•❡❛r ♣r♦t❡✐♥✱ ❝♦♥t❛✐♥✐♥❣ t❤r❡❡ |◆❆ r ❡❝♦❣♥✐t✐♦♥ ♠ ♦t✐❢s ✭||▼s✮ ❛t ✐ts ◆✲t❡r♠✐♥✉s ❛♥❞ ✜✈❡ ♥✉❝•❡❛r •♦❝❛•✐③❛t✐♦♥ s✐❣♥❛•s ❞✐s✲ tr✐❜✉t❡❞ ✇✐t❤✐♥ ✐ts ❝❡♥t❡r r❡❣✐♦♥ ❬❲✐❡••❡tt❡ ❡t ❛•✳✱ ✶✾✾✾(✳ ❋✉rt❤❡r ❣❡♥❡t✐❝ st✉❞✐❡s ✐♥ ❉r♦s♦♣❤✐•❛ s❤♦✇❡❞ t❤❛t ❙♣❡♥ r❡❣✉•❛t❡s s✐❣♥❛•✐♥❣ ❜② t❤❡ ❲♥t✴❲✐♥❣•❡ss ❬▲✐♥ ❡t ❛•✳✱ ✷✵✵✸(✱ ❊•❋| ❬❑✉❛♥❣ ❡t ❛•✳✱ ✷✵✵✵❀ |❡❜❛② ❡t ❛•✳✱ ✷✵✵✵( ❛♥❞ ◆♦t❝❤ ❬❉♦r♦q✉❡③ ❡t ❛•✳✱ ✷✵✵✼( ♣❛t❤✇❛②s✱ t❤r♦✉❣❤ ✇❤✐❝❤ ✐t ✐s ✐

  ❋✐❣✉r❡ ✷✳✶ ✕ ❈♦♥s❡r✈❛t✐♦♥ ❛♥❛•②s✐s ♦❢ ❙P❊◆ ❛❝r♦ss ❛♥✐♠❛•s✳ ❊❛❝❤ ❙P❊◆ ♣r♦t❡✐♥ ✐s ❞r❛✇♥ t♦ s❝❛•❡✱ ❛♥❞ ❛♥♥♦t❛t❡❞ ❞♦♠❛✐♥s ❛r❡ s❝❛•❡❞ ❛♥❞ ♣♦s✐t✐♦♥❡❞ ❛❝❝♦r❞✐♥❣•②✳ ❙♣❡❝✐❡s ♥❛♠❡s ❛r❡ ✐♥❞✐❝❛t❡❞ ♦♥ t❤❡ r✐❣❤t✱ ❛♥❞ ❝♦rr❡s♣♦♥❞✐♥❣ ♣r♦t❡✐♥ ♥❛♠❡s ❛♥❞ s✐③❡s ♦♥ t❤❡ •❡❢t✳ ❚❤❡ ♣❡r❝❡♥t ✐❞❡♥t✐t② ♦❢ ❛♠✐♥♦ ❛❝✐❞ s❡q✉❡♥❝❡s ✭❝♦♠♣❛r❡❞ t♦ ❍✳ s❛♣✐❡♥s✮ ✐s ✐♥❞✐❝❛t❡❞ ❢♦r ❡❛❝❤ ❞♦♠❛✐♥✳ ||▼✿|◆❆ r❡❝♦❣♥✐t✐♦♥ ♠♦t✐❢✱ |■❉✿ ♥✉❝•❡❛r r❡❝❡♣t♦r ✐♥t❡r❛❝t✐♥❣ ❞♦♠❛✐♥✱ |❇P■❉✿ |❇P❏✲✐♥t❡r❛❝t✐♥❣ ❞♦♠❛✐♥✱ ❙P❖❈✿ ❙♣❡♥ ♣❛r❛•♦❣ ❛♥❞ ♦rt❤♦•♦❣ ❈✲t❡r♠✐♥❛• ❞♦♠❛✐♥✳ ❍✳ s❛♣✐❡♥s ✭❤✉♠❛♥✮✱ ▼✳ ♠✉s❝✉✲ •✉s ✭♠♦✉s❡✮✱ •✳ ❣❛••✉s ✭❝❤✐❝❦❡♥✮✱ ❉✳ r❡r✐♦ ✭③❡❜r❛✜s❤✮✱ ❇✳ ✢♦r✐❞❛❡ ✭❛♠♣❤✐♦①✉s✮✱ ❉✳ ♠❡•❛♥♦❣❛st❡r ✭❢r✉✐t ✢②✮✱ ❈✳ ❡•❡❣❛♥s ✭♥❡♠❛t♦❞❡✮✳ ✷✳✸ ■❞❡♥t✐✜❝❛t✐♦♥ ♦❢ ❙P❊◆✴❙❍❆|P✴▼■◆❚ ✐♥ ♠❛♠✲ ♠❛•s ❚❤❡ ✜rst ❡✈✐❞❡♥❝❡ ❢♦r ❙P❊◆ ❢✉♥❝t✐♦♥ ✐♥ ♠❛♠♠❛•s ❝❛♠❡ ❢r♦♠ ✐ts ✐❞❡♥t✐✜❝❛t✐♦♥✱ ✐♥ r❛ts✱ ❛s ❛♥ ✐♥t❡r❛❝t♦r ♦❢ ▼❙❳✷✱ ❛ tr❛♥s❝r✐♣t✐♦♥❛• r❡♣r❡ss♦r ✐♠♣♦rt❛♥t ❢♦r ❜♦♥❡ ❞❡✈❡•♦♣♠❡♥t ❬◆❡✇❜❡rr② ❡t ❛•✳✱ ✶✾✾✾(✳ ❚❤r♦✉❣❤ ✐ts ||▼ ♠♦t✐❢s✱ ❙P❊◆ ✇❛s s❤♦✇♥ t♦ ❜✐♥❞ t❤❡ r❛t ♦st❡♦❝❛•❝✐♥ ♣r♦♠♦t❡r ❞✐r❡❝t•②✱ s✉❜s❡q✉❡♥t•② r❡❝r✉✐t✐♥❣ ▼❙❳✷ t♦ r❡♣r❡ss tr❛♥s❝r✐♣t✐♦♥ ♦❢ t❤❡ ♦st❡♦❝❛•❝✐♥ ❣❡♥❡ ❬◆❡✇❜❡rr② ❡t ❛•✳✱ ✶✾✾✾(✳ ■♥ t❤✐s ❡❛r•② r❡♣♦rt✱ ❙P❊◆ ✇❛s r❡♥❛♠❡❞ ✸✵ ■♥tr♦❞✉❝t✐♦♥ ❙P❊◆✿ ❉✐s❝♦✈❡r② ❛♥❞ ❢✉♥❝t✐♦♥ ▼■◆❚✱ ❢♦r ▼ ❙❳✷✲✐♥t❡r❛❝t✐♥❣ ♥✉❝•❡❛r t❛r❣❡t✳ ■♥ ✷✵✵✶✱ t❤❡ ❤✉♠❛♥ ❤♦♠♦•♦❣ ♦❢ ▼■◆❚ ✇❛s ✐❞❡♥t✐✜❡❞ ✐♥ ❛ ②❡❛st t✇♦✲❤②❜r✐❞ s❝r❡❡♥ ❛s ❛♥ ✐♥t❡r❛❝t♦r ♦❢ t❤❡ ♥✉❝•❡❛r r❡❝❡♣t♦r ❝♦r❡♣r❡ss♦r ❙▼|❚✱ ❛♥❞ ❝r✉❝✐❛• ❝•✉❡s ✐♥t♦ t❤❡ ❢✉♥❝t✐♦♥ ♦❢ ✐ts ||▼s ❛♥❞ ❙P❖❈ ❞♦♠❛✐♥s✱ ❞✐s❝✉ss❡❞ ❜❡•♦✇✱ ✇❡r❡ ♣r♦✈✐❞❡❞ ❬❙❤✐ ❡t ❛•✳✱ ✷✵✵✶(✳ ❍✉♠❛♥ ▼■◆❚ ✇❛s ♥❛♠❡❞ ❙❍❆|P✱ ❢♦r ❙ ▼|❚✴❍ ❉❆❈✶ ❛ss♦❝✐❛t❡❞ r❡♣r❡ss♦r ♣r♦t❡✐♥✱ ❤♦✇❡✈❡r✱ ❢♦r ❝•❛r✐t② ✐ss✉❡s✱ ▼■◆❚✴❙❍❆|P✴❙P❊◆ ✇✐•• ❤❡r❡❛❢t❡r ❜❡ r❡❢❡rr❡❞ t♦ ❛s ❙P❊◆✳ ✷✳✹ ❋✉♥❝t✐♦♥s ♦❢ ❙P❊◆✬s ||▼s ❙P❊◆✬s ||▼s ✭✷✱ ✸ ❛♥❞ ✹✱ ❋✐❣✉r❡ ✷✳✶✮ ✇❡r❡ ✐♥✐t✐❛••② r❡♣♦rt❡❞ t♦ ✐♥t❡r❛❝t ❞✐r❡❝t•② ✇✐t❤ ❛ ♥♦♥✲❝♦❞✐♥❣ |◆❆ ❝❛••❡❞ ❙|❆ ✭st❡r♦✐❞✲r❡❝❡♣t♦r |◆❆ ❛❝t✐✈❛t♦r✱ ❋✐❣✉r❡ ✷✳✷✮✱ ✇❤✐❝❤ ♣•❛②s ❛♥ ✐♠♣♦rt❛♥t r♦•❡ ✐♥ t❤❡ r❡❣✉•❛t✐♦♥ ♦❢ ♥✉❝•❡❛r✲r❡❝❡♣t♦r s✐❣♥❛•✐♥❣ ❬❙❤✐ ❡t ❛•✳✱ ✷✵✵✶(✳ ❆ str✉❝t✉r❛• st✉❞② •❛t❡r r❡✈❡❛•❡❞ t❤❛t t❤❡s❡ t❤r❡❡ ||▼s ❜✐♥❞ ❜♦t❤ s✐♥❣•❡ ❛♥❞ ❞♦✉❜•❡✲ str❛♥❞❡❞ |◆❆ s❡q✉❡♥❝❡s ✇✐t❤✐♥ ❙|❆✱ ❛♥❞ ❝♦♦♣❡r❛t❡ t♦ ❡♥s✉r❡ ❜✐♥❞✐♥❣ s♣❡❝✐✜❝✐t② ❬❆r✐❡t✐ ❡t ❛•✳✱ ✷✵✶✹(✳ ❚❤❡ ||▼✶ ❞♦♠❛✐♥✱ •♦❝❛t❡❞ ♦✉ts✐❞❡ ♦❢ t❤❡ ||▼✷✲✹ ❜•♦❝❦ ✭❋✐❣✉r❡ ✷✳✶✮✱ s❡❡♠s t♦ ❜❡ ❞✐s♣❡♥s❛❜•❡ ❢♦r ❜✐♥❞✐♥❣ ❙|❆ ✐♥ ✈✐tr♦✳ ❲❤❡t❤❡r ||▼✶ ❝❛rr✐❡s ❜♦♥❛✲✜❞❡ |◆❆✲❜✐♥❞✐♥❣ ❝❛♣❛❝✐t② ✐s ✉♥❦♥♦✇♥✱ ❜✉t t❤❡ ❢❛❝t t❤❛t ||▼✶ ❛♣♣❡❛r❡❞ s♣❡❝✐✜❝❛••② ✐♥ ❝❤♦r❞❛t❡s✱ ✇❤❡r❡ ✐t ✐s ❤✐❣❤•② ❝♦♥s❡r✈❡❞ ✭❋✐❣✉r❡ ✷✳✶✮✱ s✉❣❣❡sts t❤❛t ✐t ✐s ✐♠♣♦rt❛♥t ❢♦r s♦♠❡ ❛s♣❡❝t ♦❢ ❙P❊◆ ❢✉♥❝t✐♦♥ ✐♥ ♠❛♠♠❛•s✳ ■t ✐s ✇♦rt❤② t♦ ♥♦t❡ t❤❛t ❛•t❤♦✉❣❤ ❙P❊◆✬s ||▼s ✇❡r❡ ♦r✐❣✐♥❛••② s❤♦✇♥ t♦ ❞✐r❡❝t•② ❜✐♥❞ ❉◆❆ ❛t t❤❡ •❡✈❡• ♦❢ t❤❡ r❛t ♦st❡♦❝❛•❝✐♥ ♣r♦♠♦t❡r ✐♥ ✈✐tr♦ ❬◆❡✇❜❡rr② ❡t ❛•✳✱ ✶✾✾✾(✱ s✉❝❤ ❜✐♥❞✐♥❣ ✇❛s •❛t❡r ❢♦✉♥❞ t♦ ❜❡ ❢❛✐r•② ✇❡❛❦✱ ❛♥❞ ✉♥❛❜•❡ t♦ ❝♦♠♣❡t❡ ✇✐t❤ |◆❆✲❜✐♥❞✐♥❣ ❬❆r✐❡t✐ ❡t ❛•✳✱ ✷✵✶✹(✳ ❍❡♥❝❡✱ ✐t ✐s ❜❡•✐❡✈❡❞ t❤❛t |◆❆ ✐s t❤❡ ♣r❡❞♦♠✐♥❛♥t ♥✉❝•❡✐❝ ❛❝✐❞ s✉❜str❛t❡ ♦❢ ❙P❊◆✬s ||▼s✳ ✷✳✺ ❋✉♥❝t✐♦♥s ♦❢ ❙P❊◆✬s ❙P❖❈ ❞♦♠❛✐♥ ❛♥❞ •✐♥❦s ✇✐t❤ ♥✉❝•❡❛r r❡❝❡♣t♦r s✐❣♥❛•✐♥❣ ❆•t❤♦✉❣❤ ♣r❡✈✐♦✉s r❡♣♦rts ❤❛❞ ✐❞❡♥t✐✜❡❞ ❛ str♦♥❣ ❝♦♥s❡r✈❛t✐♦♥ ♦❢ t❤❡ ❙P❖❈ ❞♦♠❛✐♥ ❛❝r♦ss ❛♥✐♠❛•s ❬❲✐❡••❡tt❡ ❡t ❛•✳✱ ✶✾✾✾❀ ❑✉❛♥❣ ❡t ❛•✳✱ ✷✵✵✵(✱ ✐ts ❢✉♥❝t✐♦♥ ✇❛s ❜r♦✉❣❤t t♦ •✐❣❤t t✇♦ ②❡❛rs •❛t❡r✱ ✇❤❡♥ t❤❡ ❙P❖❈ ❞♦♠❛✐♥ ♦❢ ♠❛♠♠❛•✐❛♥ ❙P❊◆ ✇❛s s❤♦✇♥ t♦ ✐♥t❡r❛❝t ❞✐r❡❝t•② ✇✐t❤ ◆❈♦| ✭◆❈❖|✶✮ ❛♥❞ ❙▼|❚ ✭◆❈❖|✷✱ ❋✐❣✉r❡ ✷✳✷✮ ❬❙❤✐ ❡t ❛•✳✱ ✷✵✵✶(✳ ❚❤❡ str✉❝t✉r❛• ❜❛s✐s ♦❢ t❤✐s ✐♥t❡r❛❝t✐♦♥ ❤❛s ❜❡❡♥ r❡s♦•✈❡❞ ❬❆r✐②♦s❤✐ ❛♥❞ ❙❝❤✇❛❜❡✱ ✷✵✵✸(✱ ❛♥❞ ✸✶ ■♥tr♦❞✉❝t✐♦♥ ❙P❊◆✿ ❉✐s❝♦✈❡r② ❛♥❞ ❢✉♥❝t✐♦♥ ✇✐•• ❜❡ ❢✉rt❤❡r ❞✐s❝✉ss❡❞ ✐♥ t❤❡ ✜♥❛• ❝❤❛♣t❡r ♦❢ t❤❡ r❡s✉•ts s❡❝t✐♦♥ ✭❝❤❛♣t❡r ✼✮ ✐♥ t❤❡ ❝♦♥t❡①t ♦❢ ❛ ♥♦✈❡• ✐♥t❡r❛❝t✐♦♥ ❜❡t✇❡❡♥ ❙P❖❈ ❛♥❞ |◆❆ ♣♦•②♠❡r❛s❡ ✷ ■ ❞✐s❝♦✈❡r❡❞ ❞✉r✐♥❣ ♠② P❤❉✳ ◆❈♦| ❛♥❞ ❙▼|❚ ❛r❡ t✇♦ str✉❝t✉r❛••② r❡•❛t❡❞ ❢♦✉♥❞✐♥❣ ♠❡♠❜❡rs ♦❢ t❤❡ ♥✉❝•❡❛r r❡❝❡♣✲ t♦r ❝♦r❡♣r❡ss♦r ❢❛♠✐•②✳ ❚❤❡s❡ ♣r♦t❡✐♥s ❜✐♥❞ ✉♥•✐❣❛♥❞❡❞ ♥✉❝•❡❛r r❡❝❡♣t♦rs ✭◆|s✮ ❬▲❛③❛r✱ ✷✵✵✸(✱ ❛♥❞ ♠❡❞✐❛t❡ tr❛♥s❝r✐♣t✐♦♥❛• r❡♣r❡ss✐♦♥ ❛t ◆|✲t❛r❣❡t ❣❡♥❡s ❜② r❡❝r✉✐t✐♥❣ ❞✐st✐♥❝t ❢❛♠✐•✐❡s ♦❢ ❤✐st♦♥❡ ❞❡❛❝❡t②•❛s❡s s✉❝❤ ❛s ❍❉❆❈s ✶✴✷✴✸ ✭❝•❛ss ■✮ ❛♥❞ ❍❉❆❈s ✹✴✺✴✼ ✭❝•❛ss ■■✮ ❬)❤❛♥❣ ❡t ❛•✳✱ ✶✾✾✼❀ ◆❛❣② ❡t ❛•✳✱ ✶✾✾✼❀ •✉❡♥t❤❡r ❡t ❛•✳✱ ✷✵✵✶❀ ❍✉❛♥❣ ❡t ❛•✳✱ ✷✵✵✵❀ ❑❛♦ ❡t ❛•✳✱ ✷✵✵✵(✳ |❡♠❛r❦❛❜•②✱ ❙❤✐ ❡t ❛•✳ ❢♦✉♥❞ t❤❛t ❙❍❆|P ❛•s♦ ❜✐♥❞s ✉♥•✐❣❛♥❞❡❞ ♥✉❝•❡❛r r❡❝❡♣t♦rs t❤r♦✉❣❤ ✐ts r❡❝❡♣t♦r ✐♥t❡r❛❝t✐♦♥ ❞♦♠❛✐♥ ✭|■❉✱ ❋✐❣✉r❡ ✷✳✷✮✱ ❛♥❞ ❤❡♥❝❡ ♣r♦♣♦s❡❞ t❤❛t ❙❍❆|P ❛♥❞ ◆❈♦|✴❙▼|❚ ❝♦✉•❞ ❡①❡rt ❝♦♦r❞✐♥❛t❡❞ r❡♣r❡ss✐♦♥ t❤r♦✉❣❤ t❤❡✐r ♠✉t✉❛• r❡✲ ❝r✉✐t♠❡♥t ❛t ❣❡♥❡s ❜♦✉♥❞ ❜② ✉♥•✐❣❛♥❞❡❞ ◆|s ❬❙❤✐ ❡t ❛•✳✱ ✷✵✵✶(✳

[

  Arieti et al., 2014] [Monfort et al., 2015; Lu et al., 2016]❋✐❣✉r❡ ✷✳✷ ✕ ❙❝❤❡♠❡ s✉♠♠❛r✐③✐♥❣ t❤❡ ♣r❡✈✐♦✉s•② r❡♣♦rt❡❞ r♦•❡s ♦❢ ❙P❊◆✬s ❢✉♥❝t✐♦♥❛• ❞♦♠❛✐♥s✳ ❙P❊◆ ❞♦♠❛✐♥s ❛r❡ ♥❛♠❡❞ ❛s ✐♥ ❋✐❣✉r❡ ✷✳✶✳ ◆|✿ ♥✉❝•❡❛r r❡❝❡♣t♦r ✷✳✻ ❙P❊◆ ❛♥❞ ◆♦t❝❤ s✐❣♥❛•✐♥❣ ■♥ ♠✐❝❡✱ ❙P❊◆ ✇❛s ❢✉rt❤❡r s❤♦✇♥ t♦ ♣•❛② ❛ r♦•❡ ✐♥ t❤②♠♦❝②t❡ ❛♥❞ s♣•❡♥✐❝ ❇✲❝❡•• ❞✐✛❡r❡♥t✐❛t✐♦♥✱ t❤r♦✉❣❤ s✉♣r❡ss✐♦♥ ♦❢ ◆♦t❝❤ s✐❣♥❛•✐♥❣ ❬❑✉r♦❞❛ ❡t ❛•✳✱ ✷✵✵✸❀ ❚s✉•✐ ❡t ❛•✳✱ ✷✵✵✼(✳ ❆❝❝♦r❞✐♥❣•②✱ ❙♣❡♥ ❦♥♦❝❦✲♦✉t ♠✐❝❡ ❞✐❡ ❞✉r✐♥❣ ❡♠❜r②♦♥✐❝ ❞❡✈❡•♦♣♠❡♥t ❜❡t✇❡❡♥P❛rt ■■■ |❡s✉•ts |❡s✉•ts ❈❤r♦♠❛t✐♥ ❝❤❛♥❣❡s ✐♥ ❳❈■ ❈❤❛♣t❡r ✺ ❈❤r♦♠❛t✐♥ ❝❤❛♥❣❡s ✐♥ ❳❈■

  s ❞✉r✐♥❣ ❳❈■ ❛♥❞ ✉♥❞❡rst❛♥❞ t❤❡✐r ❢✉♥❝t✐♦♥❛• •✐♥❦ ✇✐t❤ ❳✲•✐♥❦❡❞ ❣❡♥❡ s✐•❡♥❝✐♥❣✳ P✉❜•✐s❤❡❞ ❡❛r•② ✷✵✶✾ ✐♥ ❈❡••✱ ♠② s❝✐❡♥t✐✜❝ ❝♦♥tr✐❜✉t✐♦♥ t♦ t❤✐s ♣r♦•❡❝t r❡s✉•t❡❞ ✐♥ ❛ ❝♦✲❛✉t❤♦rs❤✐♣✳ ❚❤❡ r❡s✉•ts ❛r❡ t♦ ❜❡ ❢♦✉♥❞ ✐♥ t❤❡ ♠❛✐♥ t❡①t✴✜❣✉r❡s ❛♥❞ s✉♣♣•❡♠❡♥t❛r② ✜❣✉r❡s ♦❢ t❤❡ ❛rt✐❝•❡ ❛♣♣❡♥❞❡❞ ❛t t❤❡ ❡♥❞ ♦❢ t❤✐s ❝❤❛♣t❡r ✭s❡❝t✐♦♥ ✺✳✹ ❛♥❞ ❬➏②•✐❝③ ❡t ❛•✳✱ ✷✵✶✾(✮✳ ■♥ t❤✐s ❝❤❛♣t❡r✱ ■ ✇✐•• ❜r✐❡✢② ❞❡s❝r✐❜❡ t❤❡ ❦❡② r❡s✉•ts ❞❡r✐✈❡❞ ❢r♦♠ t❤✐s st✉❞②✱ ♦✉t•✐♥❡ ✇❤❛t ♠② ❝♦♥tr✐❜✉t✐♦♥ ❝♦♥s✐st❡❞ ✐♥ ❛♥❞ ❤✐❣❤•✐❣❤t ✇❤✐❝❤ ✐♥s✐❣❤ts ✇❡r❡ ❣❛✐♥❡❞ t❤❛♥❦s t♦ ✐t✳ ■♠♣♦rt❛♥t•②✱ s❡✈❡r❛• r❡s✉•ts ♦❜t❛✐♥❡❞ t❤r♦✉❣❤♦✉t t❤✐s st✉❞② ❛r❡ t♦ ❜❡ ✐♥t❡❣r❛t❡❞ ✇✐t❤ t❤❡ q✉❡st✐♦♥s ■ r❛✐s❡❞ ❛♥❞ ❛❞❞r❡ss❡❞ ✐♥ t❤❡ ❝♦♥t❡①t ♦❢ ♠② ♠❛✐♥ P❤❉ ♣r♦•❡❝t ❝❡♥t❡r❡❞ ❛r♦✉♥❞ ❙P❊◆ ❢✉♥❝t✐♦♥ ✐♥ ❳❈■ ✭s❡❡ ❝❤❛♣t❡r ✻✮✳ ✺✳✶ •❡♥❡r❛• ♠❡t❤♦❞♦•♦❣② ❜❡❤✐♥❞ )②•✐❝③ ❡t ❛•✳✱ ✷✵✶✾ ✺✳✶✳✶ ❚❤❡ ❚❳✶✵✼✷ ❝❡••✉•❛r s②st❡♠ ❚❤✐s ♣r♦•❡❝t ♠❛❞❡ ✉s❡ ♦❢ ❛♥ ❡•❡❣❛♥t ❢❡♠❛•❡ ♠❊❙❈ •✐♥❡ ❝❛••❡❞ ❚❳✶✵✼✷ ❬❙❝❤✉•③ ❡t ❛•✳✱ ✷✵✶✹(✱ ✐♥ ✇❤✐❝❤ ❛ ❞♦①②❝②❝•✐♥❡ ✐♥❞✉❝✐❜•❡ ♣r♦♠♦t❡r ❤❛s ❜❡❡♥ ✐♥t❡❣r❛t❡❞ ♦♥ ♦♥❡ ❛••❡•❡ ♦❢ t❤❡ ✺✼ |❡s✉•ts ❈❤r♦♠❛t✐♥ ❝❤❛♥❣❡s ✐♥ ❳❈■ ❡♥❞♦❣❡♥♦✉s ❳✐st ❣❡♥❡✱ ❛••♦✇✐♥❣ ❢♦r ❝♦♥❞✐t✐♦♥❛• ♠♦♥♦❛••❡•✐❝ ✉♣r❡❣✉•❛t✐♦♥ ♦❢ ❳✐st |◆❆ ❛♥❞ s✉❜s❡q✉❡♥t ❣❡♥❡ s✐•❡♥❝✐♥❣ ✭❋✐❣✉r❡ ✺✳✶❆✮✳ ❚❤✐s ♦✛❡rs t✇♦ ❜❡♥❡✜ts✿ ❋✐rst✱ ❳❈■ ✐s ✐♥❞✉❝❡❞ s②♥❝❤r♦♥♦✉s•② ✐♥ ❛•• ❝❡••s✱ ♦✈❡rr✐❞✐♥❣ t❤❡ ❤❡t❡r♦❣❡♥❡✐t② ♦❜s❡r✈❡❞ ✇✐t❤ ✏✉♥❞✐♥❞✉❝❡❞✑ ❳❈■✱ ❛••♦✇✐♥❣ t♦ ❢♦••♦✇ t❤❡ ♣r♦❝❡ss ✉s✐♥❣ ♣♦♣✉•❛t✐♦♥✲❜❛s❡❞ ❛ss❛②s✳ ❙❡❝♦♥❞✱ ✐t ❡♥❛❜•❡s ✉♥❝♦✉♣•✐♥❣ ❳✐st ✉♣r❡❣✉•❛t✐♦♥✴❳❈■ ❢r♦♠ ❝❡••✉•❛r ❞✐✛❡r❡♥t✐❛t✐♦♥ ✭s❡❡ s❡❝t✐♦♥ ✶✳✻✮✱ t❤❡r❡❜② r❡♠♦✈✐♥❣ ❛♥② ❝♦♥❢♦✉♥❞✐♥❣ ❢❛❝t♦rs •✐♥❦❡❞ t♦ ❞✐✛❡r❡♥t✐❛t✐♦♥ t❤❛t ✇♦✉•❞ ❝♦♠♣•✐❝❛t❡ ❞❛t❛ ✐♥t❡r♣r❡t❛t✐♦♥✳ ■t ✐s ❛•s♦ ✇♦rt❤ ♥♦t✐♥❣ t❤❛t ❛•t❤♦✉❣❤ ❞♦①✲✐♥❞✉❝t✐♦♥ ♦❢ ❳✐st r❡s✉•ts ✐♥ ❢❛st❡r ❳❈■✱ t❤❡ ♦r❞❡r ✐♥ ✇❤✐❝❤ ❣❡♥❡s ❛r❡ s✐•❡♥❝❡❞ ✐s s✐♠✐•❛r t♦ t❤❛t ♦❢ ❡♥❞♦❣❡♥♦✉s ❳❈■ ✭❍❡❛r❞✱ ✉♥♣✉❜•✐s❤❡❞✮✳ ❋✉rt❤❡r♠♦r❡✱ ❚❳✶✵✼✷ ❛r❡ ❞❡r✐✈❡❞ ❢r♦♠ ❛ ❝r♦ss ❜❡t✇❡❡♥ t✇♦ ❤✐❣❤•② ♣♦•②♠♦r♣❤✐❝ ♠♦✉s❡ str❛✐♥s ✭❈✺✼❇▲✻✴❏ ① ❈❛st✴❊✐❏ ✮✱ ❤❡r❡❛❢t❡r r❡❢❡rr❡❞ t♦ ❛s ❇✻ ❛♥❞ ❈❛st✱ ✇✐t❤ ❙◆Ps ♦❝✲ ❝✉rr✐♥❣ ❡✈❡r② ✷✵✵✲✸✵✵ ❜❛s❡ ♣❛✐rs ✭❋✐❣✉r❡ ✺✳✶❇✮✳ ❚❤✐s ❤✐❣❤ ❙◆P ❞❡♥s✐t② ❛••♦✇s ❞✐s✲ ❝r✐♠✐♥❛t✐♦♥ ❜❡t✇❡❡♥ ❛••❡•❡s ♦❢ t❤❡ t✇♦ ❳ ❝❤r♦♠♦s♦♠❡s✳ ❍❡♥❝❡✱ ❛••❡•❡✲s♣❡❝✐✜❝ ✐♥❢♦r♠❛✲ t✐♦♥ ❝❛♥ ❜❡ r❡tr✐❡✈❡❞ ❛❝r♦ss ❛ ❞✐✈❡rs❡ r❛♥❣❡ ♦❢ ❣❡♥♦♠✐❝ ❛♣♣r♦❛❝❤❡s✱ ✐♥❝•✉❞✐♥❣ tr❛♥✲ s❝r✐♣t♦♠✐❝s✱ ❝❤r♦♠❛t✐♥✲❢r❛❣♠❡♥t s❡q✉❡♥❝✐♥❣ ❛s ✇❡•• ❛s ❝❤r♦♠♦s♦♠❡ ❝♦♥❢♦r♠❛t✐♦♥ ❝❛♣t✉r❡ ✭❋✐❣✉r❡ ✺✳✶❇✮✳transcript allelic ratio: and differentiation-independent XCI allele specificity: discrimination between Xa and Xi transcripts/chromatin ❙❝❤❡♠❡ ❞❡♣✐❝t✐♥❣ t❤❡ ❚❳✶✵✼✷ ♠♦✉s❡ ❡♠❜r②♦♥✐❝ st❡♠ ❝❡•• s②st❡♠✳ ✭❆✮ ❆ ❞♦①②❝②❝•✐♥❡✲✐♥❞✉❝✐❜•❡ ♣r♦♠♦t❡r ✭❚❡t❖✮ ✉♣str❡❛♠ ♦❢ t❤❡ ❡♥❞♦❣❡♥♦✉s ❇✻ ❳✐st ❛••❡•❡ ❛••♦✇s ✐♥❞✉❝✐❜•❡✱ s②♥❝❤r♦♥♦✉s ❛♥❞ ❞✐✛❡r❡♥t✐❛t✐♦♥✲✐♥❞❡♣❡♥❞❡♥t ❳❈■✳ ✭❇✮ ❇❡✐♥❣ ❞❡r✐✈❡❞ ❢r♦♠ ❛ ❝r♦ss ❜❡t✇❡❡♥ ❇✻ ❛♥❞ ❈❛st ♠♦✉s❡ str❛✐♥s✱ t❤❡ ❚❳✶✵✼✷ ❝❡•• •✐♥❡ ❛••♦✇s ❞✐s❝r✐♠✐♥❛t✐♦♥ ❜❡t✇❡❡♥ t❤❡ ❳❛ ❛♥❞ t❤❡ ❳✐ ✉s✐♥❣ ❛••❡•❡✲s♣❡❝✐✜❝ s❡q✉❡♥❝✐♥❣ ❛♣♣r♦❛❝❤❡s✳ ❆••❡•✐❝ r❛t✐♦s ❝❛♥ t❤❡♥ ❜❡ ❞❡r✐✈❡❞✱ ❞✐r❡❝t•② ✐♥❢♦r♠✐♥❣ ❛❜♦✉t t❤❡ tr❛♥s❝r✐♣t✐♦♥❛• st❛t✉s ✭s❤♦✇♥ ❛s ❤♦r✐③♦♥t❛• •✐♥❡s ❛♥❞ t❤❡ ❜•✉❡ ❜❛r♣•♦t✮ ❛s ✇❡•• ❛s t❤❡ ❝❤r♦♠❛t✐♥ ❡♥✈✐r♦♥♠❡♥t ✭s❤♦✇♥ ❛s tr✐❛♥❣•❡s ❛♥❞ t❤❡ ❣r❡❡♥ ❜❛r♣•♦t✮ ♦❢ •♦❝✐ ♦♥ t❤❡ ❳✐✳ |♦✉t✐♥❡•②✱ ✐♥ ❛ ❣✐✈❡♥ ❡①♣❡r✐♠❡♥t ✐♥✈♦•✈✐♥❣ ❣❡♥♦♠✐❝s✱ ❡❛❝❤ •♦❝✉s ✇✐•• ❜❡ ❛ttr✐❜✉t❡❞ ❛♥ ❛••❡•✐❝ r❛t✐♦✱ ✇❤✐❝❤ ✐s ❞❡✜♥❡❞ ❛s B6 reads B6 reads +Cast reads ✳ ❲❤❡♥ •♦♦❦✐♥❣ ❛t ❛ t②♣✐❝❛• ❳✲•✐♥❦❡❞ ❣❡♥❡ ✭✐✳❡✳ ♦♥❡ t❤❛t ❣❡ts s✐•❡♥❝❡❞✮✱ ✐ts ❛ss♦❝✐❛t❡❞ tr❛♥s❝r✐♣t ❛••❡•✐❝ r❛t✐♦ ✭❞❡r✐✈❡❞ ❢r♦♠ ❡✳❣✳ ✺✽ |❡s✉•ts ❈❤r♦♠❛t✐♥ ❝❤❛♥❣❡s ✐♥ ❳❈■ |◆❆s❡q✮ ✇✐•• ❜❡ ≈✵✳✺ ♣r✐♦r t♦ ❳✐st ✐♥❞✉❝t✐♦♥ ✭❋✐❣✉r❡ ✺✳✶❇✮ ✕ ❛s ❜♦t❤ ❇✻ ❛♥❞ ❈❛st ❛••❡•❡s ❛r❡ ❡①♣r❡ss❡❞ ❛t s✐♠✐•❛r •❡✈❡•s✳ ❙✐♠✐•❛r•②✱ t❤❡ ❍✸❑✷✼♠❡✸✲♠❛r❦❡❞ ❝❤r♦♠❛t✐♥ ❛••❡•✐❝ r❛t✐♦ ✭❞❡r✐✈❡❞ ❢r♦♠ ❡✳❣✳ ❈❤■Ps❡q✮ ❛t t❤❛t s❛♠❡ ❣❡♥❡ ✇✐•• ❛•s♦ ❜❡ ≈✵✳✺ ✭❋✐❣✉r❡ ✺✳✶❇✮ ✕ ❛s ❝❤r♦♠❛t✐♥ ✐s ❡q✉❛••② ♠❛r❦❡❞ ✭❛•❜❡✐t ❛t •♦✇ •❡✈❡•s✮ ❜② ❍✸❑✷✼♠❡✸ ♦♥ ❜♦t❤ ❛••❡•❡s ♦❢ t❤❛t ❣❡♥❡✳ ❍♦✇❡✈❡r✱ s✐♥❝❡ t❤❡ ✐♥❞✉❝✐❜•❡ ❳✐st ❛••❡•❡ ✐s ♦♥ t❤❡ ❇✻ ❝❤r♦♠♦s♦♠❡ ✭❋✐❣✉r❡ ✺✳✶❆✮✱ t❤❡ tr❛♥s❝r✐♣t ❛••❡•✐❝ r❛t✐♦ ✇✐•• ❞❡❝r❡❛s❡ ❜❡•♦✇ ✵✳✺ ❢♦••♦✇✐♥❣ ❳✐st ✐♥❞✉❝t✐♦♥ ✭❋✐❣✉r❡ ✺✳✶❇✮ ✕ ❛s t❤❡ ❇✻ ❛••❡•❡ ❣❡ts tr❛♥s❝r✐♣t✐♦♥❛••② s✐•❡♥❝❡❞✱ ❛♥❞ t❤❡ ❢r❛❝t✐♦♥ ♦❢ ❇✻ r❡❛❞s ❞✐♠✐♥✲ ✐s❤❡s✳ ❈♦♥✈❡rs❡•②✱ t❤❡ ❍✸❑✷✼♠❡✸✲♠❛r❦❡❞ ❝❤r♦♠❛t✐♥ ❛••❡•✐❝ r❛t✐♦ ✇✐•• ✐♥❝r❡❛s❡ ❛❜♦✈❡ ✵✳✺ ✭❋✐❣✉r❡ ✺✳✶❇✮ ✕ ❛s P|❈✷ ✭❛♥❞ ❝♦♥s❡q✉❡♥t ❍✸❑✷✼♠❡✸✮ ❛❝❝✉♠✉•❛t❡s ♦♥ t❤❡ ❇✻ ❳ ❝❤r♦✲ ♠♦s♦♠❡ ❢♦••♦✇✐♥❣ ❳✐st ✐♥❞✉❝t✐♦♥ ✭s❡❡ s❡❝t✐♦♥ ✶✳✽✮✱ ❛♥❞ t❤❡ ❢r❛❝t✐♦♥ ♦❢ ❇✻ r❡❛❞s ✐♥❝r❡❛s❡s✳ ✺✳✶✳✷ ❊①♣❡r✐♠❡♥t❛• ❛♣♣r♦❛❝❤ ❚♦ ❝❤❛r❛❝t❡r✐③❡ t❤❡ ❞②♥❛♠✐❝s ♦❢ ❝❤r♦♠❛t✐♥ ♠♦❞✐✜❝❛t✐♦♥s ❞✉r✐♥❣ ❳❈■✱ ❏❛♥ )②•✐❝③ ❛♥❞ ❆✉ré•✐❡ ❇♦✉s❛r❞ ❣❡♥❡r❛t❡❞ ❛♥❞ ❛♥❛•②③❡❞ ❛••❡•❡✲s♣❡❝✐✜❝ ❈❤■P✲s❡q ❞❛t❛s❡ts ❢♦r ✼ ❞✐✛❡r❡♥t ❛❝t✐✈❡ ❛♥❞ r❡♣r❡ss✐✈❡ ❤✐st♦♥❡ ♠❛r❦s ✭❚❛❜•❡ ✺✳✶✮ ❞✉r✐♥❣ ❛ t✐♠❡❝♦✉rs❡ ✭✵✱ ✹✱ ✽✱ ✶✷ ❛♥❞ ✷✹ ❤♦✉rs✮ ♦❢ ❳✐st ✐♥❞✉❝t✐♦♥ ✐♥ ❚❳✶✵✼✷ ♠❊❙❈s ✭s❡❡ )②•✐❝③ ❡t ❛•✳✱ ❋✐❣✉r❡ ✶❆✮✳ ❣❡♥♦♠✐❝ ❢❡❛t✉r❡ ❛ss♦❝✐❛t❡❞ ❤✐st♦♥❡ ♠❛r❦s ❛❝t✐✈❡ ♣r♦♠♦t❡r ❍✸❑✹♠❡✸✱ ❍✸❑✾❛❝✱ ❍✹❛❝✱ ❛♥❞ ❍✸❑✷✼❛❝ ❛❝t✐✈❡ ❡♥❤❛♥❝❡r ❍✸❑✹♠❡✶ ❛♥❞ ❍✸❑✷✼❛❝ P♦•②❝♦♠❜✲♠❛r❦❡❞ ❍✷❆❑✶✶✾✉❜✶ ✭P|❈✶✮✱ ❍✸❑✷✼♠❡✸ ✭P|❈✷✮ ❚❛❜•❡ ✺✳✶ ✕ ▲✐st ♦❢ ❤✐st♦♥❡ ♠♦❞✐✜❝❛t✐♦♥s ♣r♦✜•❡❞ ✐♥ )②•✐❝③ ❡t ❛•✳✱ ❈❡••✳ ✷✵✶✾ ❚♦ ❢✉rt❤❡r ✉♥❞❡rst❛♥❞ t❤❡ •✐♥❦ ❜❡t✇❡❡♥ t❤❡ ❞②♥❛♠✐❝s ♦❢ t❤❡s❡ ❤✐st♦♥❡ ♠♦❞✐✜❝❛t✐♦♥s ❛♥❞ tr❛♥s❝r✐♣t✐♦♥❛• s✐•❡♥❝✐♥❣ ♦❢ ❳✲•✐♥❦❡❞ ❣❡♥❡s✱ ❛••❡•❡✲s♣❡❝✐✜❝ tr❛♥s✐❡♥t tr❛♥s❝r✐♣t♦♠❡ s❡✲ q✉❡♥❝✐♥❣ ✭❚❚✲s❡q✮ ❬❙❝❤✇❛•❜ ❡t ❛•✳✱ ✷✵✶✻( ✕ ✇❤✐❝❤ s❡♥s✐t✐✈❡•② ♠♦♥✐t♦rs tr❛♥s❝r✐♣t✐♦♥❛• ❛❝t✐✈✐t② t❤r♦✉❣❤ s♣❡❝✐✜❝ s❡q✉❡♥❝✐♥❣ ♦❢ ♥❡✇•② s②♥t❤❡s✐③❡❞ ✭✇✐t❤✐♥ •❡ss t❤❛♥ ✜✈❡ ♠✐♥✉t❡s✮ |◆❆s ✕ ✇❛s ♣❡r❢♦r♠❡❞ ❛t ❡❛❝❤ t✐♠❡♣♦✐♥t ♦❢ t❤❡ ❛❢♦r❡♠❡♥t✐♦♥❡❞ ❳✐st ✐♥❞✉❝t✐♦♥ t✐♠❡✲ ❝♦✉rs❡✳ ✺✾ |❡s✉•ts ❈❤r♦♠❛t✐♥ ❝❤❛♥❣❡s ✐♥ ❳❈■ ✺✳✷ ❑❡② r❡s✉•ts ✐♥❞❡♣❡♥❞❡♥t ♦❢ ♠② ❝♦♥tr✐❜✉t✐♦♥ ✺✳✷✳✶ ❙♣r❡❛❞✐♥❣ ♦❢ P♦•②❝♦♠❜ ♠❛r❦s ❞✉r✐♥❣ ❳❈■ r❡q✉✐r❡s ❣❡♥❡ s✐•❡♥❝✐♥❣ ❆s ❡①♣❡❝t❡❞✱ ✐t ✇❛s ❢♦✉♥❞ t❤❛t t❤❡ P♦•②❝♦♠❜ r❡♣r❡ss✐✈❡ ♠❛r❦s ✭❍✷❆❑✶✶✾✉❜✶ ❛♥❞ ❍✸❑✷✼♠❡✸✮ r❛♣✐❞•② ❛❝❝✉♠✉•❛t❡ ❛❝r♦ss t❤❡ ❳ ❝❤r♦♠♦s♦♠❡ ✉♣♦♥ ❳✐st ❝♦❛t✐♥❣ ✭s❡❡ )②•✐❝③ ❡t ❛•✳✱ ❋✐❣✉r❡ ✻✮✳ ❚❤❡s❡ ♠❛r❦s ✜rst ❛❝❝✉♠✉•❛t❡ ✐♥t❡r❣❡♥✐❝❛••②✱ ❛♥❞ ♦♥•② s♣r❡❛❞ ✇✐t❤✐♥ ❣❡♥✐❝ r❡❣✐♦♥s ✉♣♦♥ ❣❡♥❡ s✐•❡♥❝✐♥❣ ✭s❡❡ )②•✐❝③ ❡t ❛•✳✱ ❋✐❣✉r❡ ✻ ❛♥❞ ❋✐❣✉r❡ ❙✻❋✮✳ ❈♦♥s✐st❡♥t•②✱ ✐♥ ❆✲ r❡♣❡❛t ♠✉t❛♥t ❝❡••s ✭✐♥ ✇❤✐❝❤ ❳✐st ❢❛✐•s t♦ s✐•❡♥❝❡ ❣❡♥❡s✮✱ P♦•②❝♦♠❜ ♠❛r❦s st✐•• ❛❝❝✉♠✉•❛t❡ str♦♥❣•② ✐♥t❡r❣❡♥✐❝❛••②✱ ❜✉t ❢❛✐• t♦ s♣r❡❛❞ ✐♥t♦ ❛❝t✐✈❡ ❣❡♥❡s ✭s❡❡ )②•✐❝③ ❡t ❛•✳✱ ❋✐❣✉r❡ ✼ ❛♥❞ ❋✐❣✉r❡ ❙✼✮✳ ❈♦••❡❝t✐✈❡•②✱ t❤❡s❡ r❡s✉•ts ❛r❡ ❝♦♥s✐st❡♥t ✇✐t❤ ♣r❡✈✐♦✉s st✉❞✐❡s ❝♦♥❞✉❝t❡❞ ✐♥ ♦t❤❡r s②st❡♠s✱ ✇❤✐❝❤ ❢♦✉♥❞ t❤❛t P♦•②❝♦♠❜ ❛❝t✐✈✐t② ❝❛♥ ♦♥•② ❜❡ ♠❛r❦❡❞ ❛t tr❛♥s❝r✐♣t✐♦♥❛••② ✐♥✲ ❛❝t✐✈❡ s✐t❡s ❬|✐✐s✐♥❣ ❡t ❛•✳✱ ✷✵✶✹❀ ❑❛♥❡❦♦ ❡t ❛•✳✱ ✷✵✶✹❀ ❍♦s♦❣❛♥❡ ❡t ❛•✳✱ ✷✵✶✻(✳ ❋✉rt❤❡r♠♦r❡✱ t❤❡ ♦❜s❡r✈❛t✐♦♥ t❤❛t P♦•②❝♦♠❜ ♠❛r❦s st✐•• str♦♥❣•② ❛❝❝✉♠✉•❛t❡ ✐♥ ❳✐st ❆✲r❡♣❡❛t ❞❡•❡t✐♦♥ ❝❡••s ✐s ❝♦♥s✐st❡♥t ✇✐t❤ t❤❡ ❢❛❝t t❤❛t P♦•②❝♦♠❜ r❡❝r✉✐t♠❡♥t ♣r❡❞♦♠✐♥❛♥t•② ♦❝❝✉rs t❤r♦✉❣❤ t❤❡ ❇✲r❡♣❡❛t ♦❢ ❳✐st ✭s❡❡ s✉❜s❡❝t✐♦♥ ✶✳✶✸✳✶✮✳ ✺✳✷✳✷ P|❈✶ r❡❝r✉✐t♠❡♥t ♣r❡❝❡❞❡s P|❈✷ ❞✉r✐♥❣ ❳❈■ ❚❤❡ P|❈✶✲❛ss♦❝✐❛t❡❞ ❍✷❆❑✶✶✾✉❜✶ ♠❛r❦ ✇❛s ❢♦✉♥❞ t♦ ❛❝❝✉♠✉•❛t❡ ❜❡❢♦r❡ t❤❡ P|❈✷✲ ❛ss♦❝✐❛t❡❞ ❍✸❑✷✼♠❡✸ ♠❛r❦ ✭s❡❡ )②•✐❝③ ❡t ❛•✳✱ ❋✐❣✉r❡ ✻✮✱ s✉❣❣❡st✐♥❣ t❤❛t P|❈✶ r❡❝r✉✐t✲ ♠❡♥t ❞✉r✐♥❣ ❳❈■ ♣r❡❝❡❞❡s P|❈✷ r❡❝r✉✐t♠❡♥t✳ ❙✉❝❤ ❣❡♥♦♠✐❝s✲❜❛s❡❞ ❡✈✐❞❡♥❝❡ ❝♦♥✜r♠s ❛ P|❈✶ ✜rst✱ P|❈✷ s❡❝♦♥❞ ♠♦❞❡• ✇❤✐❝❤ ✇❛s ♣r❡✈✐♦✉s•② ♣✉t ❢♦rt❤ t❤r♦✉❣❤ ♠✐❝r♦s❝♦♣②✲❜❛s❡❞ ♦❜s❡r✈❛t✐♦♥s ✭s❡❡ s✉❜s❡❝t✐♦♥ ✶✳✶✸✳✶✮✳ ■♠♣♦rt❛♥t•②✱ t❤✐s ✐s ✐♥ s❤❛r♣ ❝♦♥tr❛st ✇✐t❤ ✇❤❛t ✐s ❝•❛ss✐❝❛••② ♦❜s❡r✈❡❞ ✇✐t❤ ♣♦•②❝♦♠❜✲♠❛r❦❡❞ •♦❝✐ ✐♥ ♥♦♥✲❳❈■ ❝♦♥t❡①ts✱ ✇❤❡r❡ P|❈✷ r❡✲ ❝r✉✐t♠❡♥t ❛♥❞ ❍✸❑✷✼♠❡✸ ❞❡♣♦s✐t✐♦♥ ✐s r❡q✉✐r❡❞ ❢♦r t❤❡ s✉❜s❡q✉❡♥t r❡❝r✉✐t♠❡♥t ♦❢ P|❈✶ ❬❉✐ ❈r♦❝❡ ❛♥❞ ❍❡•✐♥✱ ✷✵✶✸(✳ ✺✳✷✳✸ ❍❉❆❈✸ ✐s ✐♠♣♦rt❛♥t ❢♦r ❳✐st✲❞❡♣❡♥❞❡♥t ❣❡♥❡ s✐•❡♥❝✐♥❣ ❆♠♦♥❣ ❛•• ❛❝t✐✈❡ ❤✐st♦♥❡ ♠❛r❦s ♣r♦✜•❡❞✱ •♦ss ♦❢ ❤✐st♦♥❡ ❛❝❡t②•❛t✐♦♥ ❛t ❍✸❑✷✼ ♦❝✲ ❝✉r❡❞ ♠♦st r❛♣✐❞•② ✉♣♦♥ ❳✐st ✉♣r❡❣✉•❛t✐♦♥ ✭s❡❡ )②•✐❝③ ❡t ❛•✳✱ ❋✐❣✉r❡ ✶❇✮✳ ◆♦t❛❜•②✱ •♦ss ♦❢ ❍✸❑✷✼❛❝ ❛t ❡♥❤❛♥❝❡rs ❛♥❞ ♣r♦♠♦t❡rs ♦❝❝✉rr❡❞ ✇✐t❤ ❢❛st❡r ❦✐♥❡t✐❝s t❤❛♥ •♦ss ♦❢ ❍✸❑✹♠❡✶ ❛♥❞ ❍✸❑✹♠❡✸ r❡s♣❡❝t✐✈❡•② ✭s❡❡ )②•✐❝③ ❡t ❛•✳✱ ❋✐❣✉r❡ ✶❈✲❊✮✳ ❋✉rt❤❡r♠♦r❡✱ ✇❤❡♥ t❤❡ ♣r♦✜•❡s ✻✵ |❡s✉•ts ❈❤r♦♠❛t✐♥ ❝❤❛♥❣❡s ✐♥ ❳❈■ ♦❢ ❛❝t✐✈❡ ♠❛r❦ ❞②♥❛♠✐❝s ❛t ❳✲•✐♥❦❡❞ ♣r♦♠♦t❡rs ✇❡r❡ ❝♦♠♣❛r❡❞ t♦ t❤❡ tr❛♥s❝r✐♣t✐♦♥❛• s✐✲ •❡♥❝✐♥❣ ❞②♥❛♠✐❝s ♦❢ ❝♦rr❡s♣♦♥❞✐♥❣ ❣❡♥❡s ✭♦❜t❛✐♥❡❞ ❜② ❚❚✲s❡q✮✱ ❛ ❤✐❣❤❡st ❝♦rr❡•❛t✐♦♥ ✇❛s ♦❜s❡r✈❡❞ ✇✐t❤ •♦ss ♦❢ ❍✸❑✷✼ ❛❝❡t②•❛t✐♦♥ ✭s❡❡ )②•✐❝③ ❡t ❛•✳✱ ❋✐❣✉r❡ ✷❆✲❈✮✳ ❈♦••❡❝t✐✈❡•②✱ t❤❡s❡ ❞❛t❛ s✉❣❣❡st❡❞ t❤❛t t❤❡r❡ ❝♦✉•❞ ❜❡ ❛ ❢✉♥❝t✐♦♥❛• •✐♥❦ ❜❡t✇❡❡♥ ❤✐st♦♥❡ ❞❡❛❝❡t②•❛t✐♦♥ ❛♥❞ ❳✐st✲✐♥❞✉❝❡❞ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❞✉r✐♥❣ ❳❈■✳ ❚♦ t❡st t❤✐s ❤②♣♦t❤❡s✐s✱ t❤❡ ✐♠♣❛❝t ♣❡rt✉r❜✐♥❣ ❤✐st♦♥❡ ❞ ❡❛❝❡t②•❛s❡s ✭❍❉❆❈s✮ ❤❛s ♦♥ ❳✲•✐♥❦❡❞ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❞✉r✐♥❣ ❳❈■ ✇❛s ✐♥✈❡st✐❣❛t❡❞✳ ❯s✐♥❣ ❛ ❝♦♠❜✐♥❛t✐♦♥ ♦❢ ❍❉❆❈ ✐♥✲ ❤✐❜✐t♦rs✱ s❡•❡❝t✐✈❡•② ✐♥❤✐❜✐t✐♥❣ ❍❉❆❈s ❡①♣r❡ss❡❞ ✐♥ ❊❙ ❝❡••s ✭❍❉❆❈✶✴✷✱ ❍❉❆❈✸✱ ❍❉❆❈✹✱ ❍❉❆❈✺✱ ❍❉❆❈✻ ❛♥❞ ❍❉❆❈✽✮✱ ✐t ✇❛s ❢♦✉♥❞ t❤❛t ✐♥❤✐❜✐t✐♦♥ ♦❢ ❍❉❆❈✸ ✭❜✉t ♥♦t ♦t❤❡r ❍❉❆❈s✮ r❡s✉•t❡❞ ✐♥ ❞❡✜❝✐❡♥t s✐•❡♥❝✐♥❣ ♦❢ s❡✈❡r❛• ❳✲•✐♥❦❡❞ ❣❡♥❡s ✭s❡❡ )②•✐❝③ ❡t ❛•✳✱ ❋✐❣✉r❡ ✸❆✲❇✮✳ ❚❤✐s ♦❜s❡r✈❛t✐♦♥ ✇❛s ❢✉rt❤❡r ❝♦♥✜r♠❡❞ ❜② ♠♦♥✐t♦r✐♥❣ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❝❤r♦♠♦s♦♠❡✲ ✇✐❞❡ ✉s✐♥❣ |◆❆s❡q ✐♥ ❍❞❛❝✸ ✲❑❖ ❚❳✶✵✼✷ ❝❡••s ✭s❡❡ )②•✐❝③ ❡t ❛•✳✱ ❋✐❣✉r❡ ✸❈✮✱ ✇❤✐❝❤ s❤♦✇❡❞ s✐❣♥✐✜❝❛♥t ❞❡❢❡❝ts ✐♥ ❣❡♥❡ s✐•❡♥❝✐♥❣ ❛❢t❡r ✷✹ ❤♦✉rs ♦❢ ❳✐st ✐♥❞✉❝t✐♦♥ ✭s❡❡ )②•✐❝③ ❡t ❛•✳✱ ❋✐❣✉r❡ ✸❉✲•✮✳ ❋✉rt❤❡r♠♦r❡✱ ❈❤■P✲s❡q r❡✈❡❛•❡❞ t❤❛t •♦ss ♦❢ ❍✸❑✷✼❛❝ ♦♥ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡ ✐s s❡✈❡r❡•② ❤✐♥❞❡r❡❞ ✐♥ ❍❞❛❝✸ ❑❖ ❝❡••s ✭s❡❡ )②•✐❝③ ❡t ❛•✳✱ ❋✐❣✉r❡ ✹✮✱ ❛♥❞ t❤❛t ❣❡♥❡s ✇❤♦s❡ s✐•❡♥❝✐♥❣ ✐s ♠♦st ❛✛❡❝t❡❞ ❜② ❍❉❆❈✸ •♦ss ❛•s♦ s❤♦✇ ♠♦st ❞r❛st✐❝ ❞❡❢❡❝ts ✐♥ ❍✸❑✷✼ ❞❡❛❝❡t②•❛t✐♦♥ ❛t t❤❡✐r ♣r♦♠♦t❡rs ✭s❡❡ )②•✐❝③ ❡t ❛•✳✱ ❋✐❣✉r❡ ✹❈✲❉✮✳ ❚❛❦❡♥ t♦❣❡t❤❡r✱ t❤❡s❡ r❡s✉•ts ❤✐❣❤•✐❣❤t t❤❛t ❍❉❆❈✸✲♠❡❞✐❛t❡❞ ❞❡❛❝❡t②•❛t✐♦♥ ✐s ✐♠♣♦r✲ t❛♥t ❢♦r ❡✣❝✐❡♥t ❣❡♥❡ s✐•❡♥❝✐♥❣ ❞✉r✐♥❣ ❳ ❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥✳ ❍♦✇❡✈❡r✱ ✇❤❡t❤❡r ❛♥❞ ❤♦✇ ❍❉❆❈✸ ✐s r❡❝r✉✐t❡❞ t♦ ✐ts ❳✲•✐♥❦❡❞ t❛r❣❡ts✱ ❛♥❞ ✇❤✐❝❤ ♣❛rt♥❡rs ❞♦❡s ✐t ✐♥t❡r❛❝t ✇✐t❤ ✐♥ t❤❡ ❝♦♥t❡①t ♦❢ ❳❈■ r❡♠❛✐♥❡❞ ✉♥❝•❡❛r ❛t t❤❛t st❛❣❡ ♦❢ t❤❡ ♣r♦•❡❝t✳ ❲✐t❤ ♥♦ ❣♦♦❞ ❍❉❆❈✸ ❛♥t✐❜♦❞✐❡s t♦ ♣❡r❢♦r♠ ■P✲❜❛s❡❞ ❡①♣❡r✐♠❡♥ts ♥❡❝❡ss❛r② t♦ ❜r✐♥❣ s♦♠❡ ✐♥s✐❣❤ts t♦ t❤❡s❡ q✉❡st✐♦♥s✱ ■ ❤❛❞ t❤❡ ♦♣♣♦rt✉♥✐t② t♦ ❝♦♥tr✐❜✉t❡ t♦ t❤✐s ♣r♦•❡❝t ✇✐t❤ ❡①♣❡r✐♠❡♥t❛• t♦♦•s ■ ❤❛❞ ✐♥ ♣❛r❛••❡• ❞❡✈❡•♦♣❡❞ ✐♥ t❤❡ ❝♦♥t❡①t ♦❢ ♠② ♠❛✐♥ ♣r♦•❡❝t ❝❡♥t❡r❡❞ ♦♥ ❙P❊◆ ✭❝❤❛♣t❡r ✻✮✳ ✺✳✸ ❑❡② r❡s✉•ts t♦ ✇❤✐❝❤ ■ ❝♦♥tr✐❜✉t❡❞ ✺✳✸✳✶ •❡♥❡r❛t✐♦♥ ❛♥❞ ❝❤❛r❛❝t❡r✐③❛t✐♦♥ ♦❢ ❡♥❞♦❣❡♥♦✉s ❋▲❆•✲t❛❣❣❡❞ ❍❉❆❈✸ ♠❊❙❈s ❚♦ ❝✐r❝✉♠✈❡♥t t❤❡ •❛❝❦ ♦❢ ❣♦♦❞ ❛♥t✐❜♦❞✐❡s ❛❣❛✐♥st ❍❉❆❈✸✱ ■ ♦♣t❡❞ t♦ ❣❡♥❡r❛t❡ ❛ ❝❡•• •✐♥❡ ✐♥ ✇❤✐❝❤ t❤❡ ❡♥❞♦❣❡♥♦✉s ❍❉❆❈✸ ♣r♦t❡✐♥ ✇♦✉•❞ ❜❡ ❢✉s❡❞ t♦ ❛ t❛❣ t♦ ❛••♦✇ s✉❜s❡q✉❡♥t ❛✣♥✐t② ♣✉r✐✜❝❛t✐♦♥ ❛♥❞ ✐❞❡♥t✐❢② ❍❉❆❈✸ ❜✐♥❞✐♥❣ ❞②♥❛♠✐❝s ❛t ❣❡♥♦♠✐❝ t❛r❣❡ts ❞✉r✐♥❣ ❳❈■ ✻✶ |❡s✉•ts ❈❤r♦♠❛t✐♥ ❝❤❛♥❣❡s ✐♥ ❳❈■ ✭❈❤■P✲s❡q✮ ❛s ✇❡•• ❛s ✐ts ❛ss♦❝✐❛t❡❞ ♣r♦t❡✐♥ ♣❛rt♥❡rs ✭❈♦✲■P✲▼❙✮✳ ❚❛❦✐♥❣ ❛❞✈❛♥t❛❣❡ ♦❢ ❈|■❙P|✴❈❛s✾✲♠❡❞✐❛t❡❞ ❤♦♠♦•♦❣② ❞✐r❡❝t❡❞ r❡♣❛✐r✱ ■ ♣❡r❢♦r♠❡❞ ❛ ❤♦♠♦③②❣♦✉s ❦♥♦❝❦✲✐♥ ♦❢ ❛ ✸①❋▲❆• ❝❛ss❡tt❡ ✐♠♠❡❞✐❛t❡•② ✉♣str❡❛♠ ♦❢ t❤❡ ❙❚❖P ❝♦❞♦♥ ♦❢ ❍❞❛❝✸ ✭❋✐❣✉r❡ ✺✳✷❆✮✳ ❍♦♠♦③②❣♦✉s ❦♥♦❝❦✲✐♥ ❝♦✉•❞ ❜❡ ❝♦♥✜r♠❡❞ ✉s✐♥❣ ❙◆P✲❣❡♥♦t②♣✐♥❣ ❢♦••♦✇✐♥❣ P❈| ♦♥ ❣❡♥♦♠✐❝ ❉◆❆ ✭❋✐❣✉r❡ ✺✳✷❆✮✳ ❚❤❡ ❡♥❞♦❣❡♥♦✉s•② t❛❣❣❡❞ ❍❉❆❈✸ ♣r♦✲ t❡✐♥ ✇❛s ❡①♣r❡ss❡❞ ❛t •❡✈❡•s s✐♠✐•❛r t♦ ✐ts ✉♥t❛❣❣❡❞ ❝♦✉♥t❡r♣❛rt ✭❋✐❣✉r❡ ✺✳✷❇✮✱ ❛♥❞ ✐ts ❢✉♥❝t✐♦♥ r❡♠❛✐♥❡❞ ♣r❡s❡r✈❡❞ ❣✐✈❡♥ t❤❛t ❣❡♥❡ s✐•❡♥❝✐♥❣ ❞✉r✐♥❣ ❳❈■ ✇❛s ♥♦t ❛✛❡❝t❡❞ ✭❋✐❣✉r❡ ✺✳✷❈✮✳

  WT ❋✐❣✉r❡ ✺✳✷ ✕ •❡♥❡r❛t✐♦♥ ❛♥❞ ❝❤❛r❛❝t❡r✐③❛t✐♦♥ ♦❢ ❍❉❆❈✸✲❋▲❆• ❚❳✶✵✼✷ ♠❊❙❈s✳ ✭❆✮ ❙❝❤❡♠❡ ❞❡♣✐❝t✐♥❣ ❈|■❙P|✲♠❡❞✐❛t❡❞ t❛r❣❡t✐♥❣ ♦❢ ❛ ✸①❋▲❆• ❝❛ss❡tt❡ ❛t ❜♦t❤ ❛••❡•❡s ♦❢ t❤❡ ❡♥❞♦❣❡♥♦✉s ❍❞❛❝✸ ❣❡♥❡✳ ❚❤❡ ✐♥s❡rt✐♦♥ ✇❛s t❛r❣❡t❡❞ ✐♠♠❡❞✐❛t❡•② ✉♣str❡❛♠ ♦❢ t❤❡ st♦♣ ❝♦❞♦♥✳ ❚❤❛♥❦s t♦ t❤❡ ♣♦•②♠♦r♣❤✐❝ ❚❳✶✵✼✷ ❜❛❝❦❣r♦✉♥❞✱ ❤♦♠♦③②❣♦✉s ❦♥♦❝❦✲✐♥s ❝❛♥ ❜❡ ✐❞❡♥t✐✜❡❞ t❤r♦✉❣❤ ❙❛♥❣❡r s❡q✉❡♥❝✐♥❣ ♦❢ ❛ P❈| ♣r♦❞✉❝t s♣❛♥♥✐♥❣ ❛❝r♦ss t❤❡ ✐♥s❡rt✐♦♥ s✐t❡✱ ❛♥❞ ❝♦✈❡r✐♥❣ ❛ ❙◆P ✇✐t❤✐♥ t❤❡ ❍❞❛❝✸ •♦❝✉s ✭rs✷✷✹✸✻✻✼✼✶✮✳ ✭❇✮ ❲❡st❡r♥ ❜•♦t ❞❡t❡❝t✐♦♥ ♦❢ ❍❉❆❈✸ ❢r♦♠ ✇✐•❞✲t②♣❡ ❛♥❞ ❍❉❆❈✸✲❋▲❆• ❦♥♦❝❦✲✐♥ ✇❤♦•❡ ❝❡•• ❡①tr❛❝ts✳ ❇•♦ts ✇❡r❡ ♣r♦❜❡❞ ✇✐t❤ ❛♥t✐✲❍❉❆❈✸ ✭•❡❢t✮ ♦r ❛♥t✐✲❋▲❆• ✭r✐❣❤t✮ ❛♥t✐❜♦❞✐❡s✳ ✭❈✮ ❇❛r♣•♦ts s❤♦✇✐♥❣ tr❛♥s❝r✐♣t ❛••❡•✐❝ r❛t✐♦ ✭❞❡r✐✈❡❞ ❢r♦♠ ♣②r♦s❡q✉❡♥❝✐♥❣ ❛ss❛②s✮ ❢♦r t❤r❡❡ ❳✲•✐♥❦❡❞ ❣❡♥❡s ❜❡❢♦r❡ ❛♥❞ ❛❢t❡r ❞♦①②❝②❝•✐♥❡✲♠❡❞✐❛t❡❞ ✐♥❞✉❝t✐♦♥ ♦❢ ❳✐st ❢♦r ✷✹ ❤♦✉rs✱ ✐♥ ❲❚ ❛♥❞ ❍❉❆❈✸✲❋▲❆• ❦♥♦❝❦✲✐♥ ❝❡••s✳ ✭❉✮ ❲❡st❡r♥ ❜•♦t ❞❡t❡❝t✐♦♥ ♦❢ ❍❉❆❈✸ ❢♦••♦✇✐♥❣ ❛♥t✐✲❋▲❆• ❜❡❛❞✲❜❛s❡❞ ✐♠♠✉♥♦♣r❡❝✐♣✐t❛t✐♦♥ ✐♥ ✇✐•❞✲t②♣❡ ❛♥❞ ❍❉❆❈✸✲❋▲❆• ♥✉❝•❡❛r ❡①tr❛❝ts✳ ❚❤r❡❡ ❢r❛❝t✐♦♥s ✭✐♥♣✉t✱ ✢♦✇t❤r♦✉❣❤✱ ❛♥❞ ❜❡❛❞ ❡•✉❛t❡✮ ✇❡r❡ s❛♠♣•❡❞✳ ✯ ✿ s✐❣♥❛• ❝♦rr❡s♣♦♥❞✐♥❣ t♦ ❤❡❛✈② ❛♥❞ •✐❣❤t ❝❤❛✐♥s ❢r♦♠ ❜❡❛❞✲❜♦✉♥❞ ❋▲❆• ■❣•✳ Two Active XChromosomes X-Chromosome Inactivation (XCI) EPIGENOMIC ROADMAP OF XCI ROLE OF CHROMATIN MARKS IN XCI i n ter g eni c in di re ct vi a e .g . S P E N in d ir e c t INTRODUCTION Successful development requires the establishment and maintenance of euchromatin and heterochromatin in different parts of the genome. Progressively stable silencing of some genic regions occurs as epigenetic memory continues to accrue, finally leading to facultative heterochromatin formation (Trojer and Reinberg, 2007). Multiple layers of chromatin modifications are believed to enable stable transcriptional silencing. However, little is known about how facultative heterochromatin is dynamically formed and to what extent chromatin changes are involved in the establishment of gene silencing. A powerful model for developmentally induced gene silencing and formation of facultative heterochromatin is X chromosome inactivation (XCI) in female mammals. Although the role for chromatin changes in maintenance of the inactive state has been extensively studied (Disteche and Berletch, 2015), almost nothing is known about their role in the initiation of gene silencing.

Figure 1 .

 1 Figure 1. Histone Deacetylation Is among the First Events of XCI (A) Schematic representation of the experimental design. The hybrid TX1072 mouse ESC line was used, in which Xist can be induced from the endogenous B6 allele. (B) H3K27ac (blue) deacetylation dynamics in comparison with the loss of other active histone marks. Shown are average d-scores (shading is the interquartile range) of all peaks at the X chromosome. (C) Paired comparison of the dynamics in H3K27ac and H3K4 methylation loss. Plotted are IC35 parameters of peaks at active promoters (left) and putative active enhancers (right) and within the quantitative range of the experiment (IC35 < 24 hr). The p value is from a paired Wilcoxon rank-sum test. (D and E) Genome browser tracks showing H3K27ac and H3K4me3 at the Pdk3 promoter (D) or H3K27ac and H3K4me1 at a putative Pdk3 enhancer (E). Allelespecific tracks are overlaid (Cast reads in blue; B6 reads in red). The plots show the quantification of the inactivation dynamics with fitted sigmoidal curves. See also Figure S1.

Figure 2 .

 2 Figure 2. Histone Deacetylation Is Tightly Correlated with Transcriptional Silencing (A) Comparison of IC35 parameters for gene silencing (TT-seq) and histone modifications at associated promoters. The p values are from a paired Wilcoxon rank-sum test. (B) Pairwise comparison of IC35 from gene silencing and histone mark dynamics as in (A). Linear regression fitting (red) and perfect correlation with slope = 1 (black) is shown. Pearson's correlation (r) and p value are shown for each set.

( C )

 C Heatmap showing gene silencing and associated promoter H3K27 deacetylation dynamics. Rows were ordered by their genomic position from centromere to telomere (the green triangle indicates the Xist location). The averages from biological duplicates are shown. (D) Quantification of features associated with early and late deacetylated promoters (H3K27ac). Shown are gene density, LINE density, proximity to LADs, and distance from the Xist locus. The p values are from a Wilcoxon rank-sum test with Benjamini Hochberg correction. See also Figure S2. next page) Cell 176, 182-197, January 10, 2019 187

Figure 3 .

 3 Figure 3. HDAC3 Mediates Efficient Transcriptional Silencing during XCI (A) Screening for HDAC activity involved in XCI using selective inhibitors and pyrosequencing for G6pdX and AtrX. The red line represents silencing in the DMSO control after 24 hr of DOX treatment. Shown are averages (± SD) from 2 experiments. *p < 0.05, Student's t test. (B) RNA FISH for Xist (red) and the X-linked AtrX gene (green) on ESCs treated as in (A). Percentages of biallelic and monoallelic AtrX expression in Xist-expressing cells are shown (>100 nuclei quantified, scale bar, 10 mm). The quantification of Xist cloud formation is shown below.(C) Immunoblot analysis of whole-cell lysates from TX1072 (Hdac3 +/+ ) and two Hdac3 À/À clones probed with antibodies specific for the N terminus of HDAC3 and Lamin B1. (D) Heatmap showing gene silencing after 0 and 24 hr of DOX treatment in TX1072 (Hdac3 +/+ ) and two Hdac3 À/À clones. Rows were ordered by their genomic position from centromere to telomere (the green triangle indicates the Xist location). (E) RNA-seq validation using pyrosequencing on ESCs (TX1072: Hdac3 +/+ and Hdac3 À/À ) differentiated to EpiLCs with DOX. Shown are averages (± SEM) from 3 independent experiments and two Hdac3 À/À clones for 3 genes sensitive to HDAC3 loss (top) and 3 genes less dependent on its activity (bottom). *p < 0.05, Student's t test. (F and G) Validation of Hdac3 À/À phenotype in day 3 EpiLCs differentiated without DOX. (F) FISH was performed with probes for Xist (p510, red) and an X-linked gene, Huwe1 (green). Scale bar, 5 mm. (G) Also shown is quantification of Huwe1 silencing status in more than 100 nuclei with Xist clouds. c 2 test, *p < 0.05. (H) Screening for HDAC activity compensating for HDAC3 loss using pyrosequencing for AtrX, G6pdX, and Rnf12. The experiment was performed like in (A) but in Hdac3 À/À cells. Shown are averages (± SD) from two independent clones. *p < 0.05, Student's t test. See also FigureS3and TableS1.

Figure 4 .

 4 Figure 4. HDAC3 Promotes Silencing by Rapidly Deacetylating Histones(A) IF/RNA FISH on TX1072 (Hdac3 +/+ ) and two Hdac3 À/À ESC clones induced with DOX for 24 hr. Cells were probed using anti-H3K27ac (red) antibodies and a Xist intronic probe (green). The white dotted line encircles the Xist domain. Because of technical variability, contrast settings were individually adjusted to reflect relative enrichment. Scale bar, 10 mm. (B) Quantification of (A) over at least 100 Xist clouds. Shown is the average signal for Xist and H3K27ac when profiles were aligned to the Xist cloud boundary (for more details, refer to FigureS4A and D).(C) Relationship between gene repression (RNA-seq) and promoter deacetylation (H3K27ac) at 24 hr in TX1072 (Hdac3 +/+ , blue) and Hdac3 À/À (red). On the scatterplot, the black dotted line represents fitted linear regression. r = 0.860. Density plots show the distribution of the normalized d-score for H3K27ac (horizontal) and RNA-seq (vertical); note that d-scores in mutant cells are shifted toward biallelic levels (0.5, dotted lines on density plots). Each dot represents the average normalized d-score from biological duplicates. (D) Comparison of promoter deacetylation dynamics and transcriptional silencing after 24 hr of DOX treatment in TX1072 (Hdac3 +/+ ) and Hdac3 À/À ESCs. Examples of genes sensitive (top) and more resistant (bottom) to HDAC3 loss are shown. For RNA-seq, shown is the average from biological duplicates (± SD). See also FigureS4.

Figure 5 .

 5 Figure 5. HDAC3 Is Pre-bound on the X Chromosome, Likely to Promote Efficient Histone Deacetylation (A) HDAC3 sub-nuclear localization in TX1072 (Hdac3 +/+ ) and Hdac3 À/À ESCs induced with DOX for 24 hr. Xi was detected by H4K20me1 enrichment (dotted area) and shows no enrichment for HDAC3. Scale bar, 10 mm. (B) Schematic representation of the Hdac3 FLAG/FLAG ESC line, which expresses endogenous HDAC3 with a 3xFLAG tag. (C) Volcano plot analysis identifying interactors of HDAC3 in ESCs. Binding partners were obtained by using quantitative label-free mass spectrometry analysis performed from five replicates. Shown are the fold changes (Hdac3 FLAG/FLAG versus Hdac3 +/+ ), quantified with an absolute fold change of 2 or more with an adjusted p value of ratio significance of 0.05 or less and with 3 or more peptides. Known HDAC3 (red) and Xist (green) interactors are shown. External plots show proteins with peptides identified only in one sample type (left in Hdac3 +/+ and right in Hdac3 FLAG/FLAG ). (D) Representation of complexes identified as HDAC3 interactors, with pink proteins representing partners, which did not reach significance thresholds. (E) Immunoprecipitation; western blot validating HDAC3 interaction with both NCOR1 and NCOR2. (F) Heatmap of HDAC3 enrichment at all H3K27ac and H4ac peaks across the genome. Metaplots show average enrichment of HDAC3, H3K27ac, and H4ac at promoters and putative enhancers. For HDAC3, the signal was normalized to the no-FLAG ChIP-seq control.

XFigure 6 .

 6 Figure 6. PRC1-Dependent H2AK119Ub Accumulation Precedes H3K27me3 Deposition (A) Quantification of average H2AK119Ub (yellow) and H3K27me3 (red) enrichment at the Xi (B6 allele) compared with t = 0 hr in 10-kb windows spanning the whole chromosome. Shading is the interquartile range. (B) Pairwise comparison of H2AK119Ub and H3K27me3 accumulation dynamics (ED 50 ) at the X chromosome. All 10-kb windows with ED 50 < 24 hr are plotted. The p value is from a paired Wilcoxon rank sum test. (C) Correlation between H2AK119Ub (left) or H3K27me3 (right) pre-marking (t = 0 hr) and de novo enrichment of those marks shortly after Xist RNA induction (t = 4 hr for H2AK119Ub and t = 8 hr for H3K27me3). Plotted are all 10-kb windows spanning the X chromosome. All scales are logarithmic. The p values are from Pearson's correlation test with r = 0.538 (H2AK119Ub) and 0.441 (H3K27me3). (D) H2AK119Ub (yellow) and H3K27me3 (red) accumulation dynamics across the Xi after 4 and 8 hr of DOX treatment. The black line is a locally estimated scatterplot smoothing (LOESS) regression on all 10-kb windows (dots). Shown is the Xist locus (green) and Xist entry sites (blue). (E) Dynamics of transcriptional silencing (top) and H2AK119Ub (yellow) and H3K27me3 (red) accumulation at gene bodies. Genes were separated based on transcriptional silencing dynamics. Shading is the interquartile range. (F) Genome browser tracks showing H2AK119Ub (top) and H3K27me3 (right) accumulation at genes silenced rapidly (Rnf12), slowly (Huwe1, Alg13), or not at all (Kdm6a). Allele-specific tracks were overlaid (B6 in red and Cast in blue). (G) Quantification of PcG mark accumulation at gene bodies (from F) with sigmoidal curves fitted. See also Figure S6.

Figure 7 .

 7 Figure 7. PcG Spreading along the X Chromosome Requires Transcriptional Silencing (A) Schematic representation of the experimental design. By DOX addition, male TXY mouse ESC lines allow expression of Xist or Xist:DA. (B) H3K27me3 (red) and H2AK119Ub (yellow) accumulation across the Xi after 48 hr of DOX compared with no DOX in WT (top) and Xist:DA (bottom) cells. Each dot represents a single 10-kb window. The black line indicates a LOESS regression on all windows. Shown are the Xist locus (green line), active genes (light blue), and examples of gene-dense regions (dotted boxes). (C) Correlation between the distance from an active gene and the H3K27me3 (top) or H2AK119Ub (bottom) accumulation in Xist:DA cells compared with WT cells.(legend continued on next page)

  C (anti-mouse Dynabeads: H3K9ac[1ug/IP], H3K4me3[2.5ug/IP], H3K27ac[1ug/IP]; Protein-A dynabeads: H3K4me1[0.4ug/IP], H3K27me3 [1ug/IP], H2AK119Ub[0.4ug/IP], H4ac[2ug/IP]). Once coated beads were magnet-separated and resuspended in 1mL of concentration-adjusted lysate. Samples were left rotating overnight at 4 C.

(

  Figure S1. Allele-Specific Native ChIP-Seq Monitors Changes in Active Histone Modifications during XCI, Related to Figure 1

(

  A) nChIP enrichment validation using qPCR. Shown is the enrichment of nChIP signal at positive (color) and negative (black) control regions for each mark individually. Shown is a representative result for one biological replicate. Values were normalized to the input sample. (B) Average enrichment plots for active histone modifications across all transcriptional start sites. (C) RNA FISH quantification of Xist induction (p510 probe) during both replicates of nChIP-seq time course. At least 70 nuclei were quantified for each sample. (D) Bar plots showing the percentage of B6(Xi) reads mapping to the X chromosome in the nChIP-seq time course. (E) Genome browser plots of active histone modification at the Gda3/Dppa3/Nanog cluster. Shown are all mapped reads at all time points in biological duplicates. Note highly reproducible and stable pattern of enrichment. (F) Example showcasing the differences between IC35 and ED 50 parameters.

(

  Figure S5. HDAC3 Is Pre-bound on the X Chromosome and Only Weakly Accumulates during XCI, Related to Figure 5

( 6 (

 6 Figure S6. Allele-Specific Natic ChIP-Seq Monitors Changes in Representative Histone Modifications during XCI, Related to Figure6(A) Genome browser plots of H3K27me3 (red) and H2AK119Ub (yellow) enrichment at the Hoxc cluster. Shown are all mapping reads at all time points and biological duplicates. Note highly reproducible and stable pattern of enrichment. (B) Bar plot showing the percentage of B6 reads mapping to the X chromosome in the nChIP-seq time course. *p value < 0.05 calculated using Student's t test.(C) Plot showing the distribution of differential ED 50 (H3K27me3-H2K119Ub) for all 10kb windows along the X chromosome. Each bar represents a single window, these were ordered accordingly to their differential ED 50 . (D) IF/RNA FISH for Xist (gray), H2AK119Ub (red) and H3K27me3 (green) in day 1 or 2 EpiLCs. White dotted lines outline Xist domains. Bar plot quantifies PcG-mark enrichment at 100 Xist clouds. Scale bar = 10 mm. (E) Density plots correlating H2AK119Ub gain (4 versus 0hrs) with H3K27me3 gain (8 versus 4hrs). Shown is the behavior of all 10kb windows spanning the X chromosome. All scales are logarithmic. p value was calculated using Pearson correlation test. The correlation coefficient is r = 0.622 (F) Violin plots showing the distribution of ED 50 for H2AK119Ub (left) and H3K27me3 (right) at windows mapping to the active gene bodies, active enhancers, intergenic regions and active promoters. p values calculated using Wilcoxon rank sum test. (G) ED 50 analysis of H2AK119Ub (yellow), H3K27me3 (red) accumulation at gene bodies in comparison to gene silencing (black, left) and promoter H3K27ac loss (blue, right). p values calculated using paired Wilcoxon rank sum test. (H) Analysis of the point when sigmoid's second derivative reaches max/min. This is a proxy for when H2AK119Ub (yellow) and H3K27me3 (red) start to accumulate at gene bodies in comparison to the beginning of gene silencing (black, left) and promoter H3K27ac loss (blue, right). p values calculated using paired Wilcoxon rank sum test. (I) Dynamics of H2AK119Ub (yellow), H3K27me3 (red) accumulation, promoter deacetylation (H3K27ac, blue) and transcriptional silencing (black) of an X-linked gene: Txlng. Empty dots present ED 50 of sigmoid fittings, while perpendicular lines max/min of second derivative (2ndD). Left axis of normalized d-score is for H3K27ac and TT-seq dynamics, right axis of normalized B6-read accumulation is for H2AK119Ub and H3K27me3. (J-K) Violin plots showing H2AK119Ub (J) and H3K27me3 (K) accumulation at gene bodies of constitutive and facultative escapees when compared to remaining genes. Shown is the normalized enrichment of B6-reads after 24hrs of DOX treatment when normalized to t = 0hr. p values calculated using Wilcoxon rank sum test. (L) Violin plot showing H3K27 deacetylation dynamics (IC35) at promoters of constitutive and facultative escapees when compared to remaining genes. p values calculated using Wilcoxon rank sum test.

  :Xist∆A vs TXY:Xist WT H3K27me3: TXY:Xist∆A vs TXY:

  Figure S7. Dependence of PcG Spreading on Gene Silencing; Proposed Models, Related to Figure7

Goal 1 :Goal 2 :

 12 SPEN function in XCI? SPEN mechanism of action in XCI? (i) SPEN and XCI initiation: How impaired is initiation of XCI, chromosome wide, upon SPEN loss? mRNAseq, RNA FISH and pyrosequencing in vitro (mESCs) and in vivo (blastocysts) (ii) SPEN and XCI maintenance: How impaired is somatic maintenance of XCI, chromosome wide, upon SPEN loss? Hi-C and mRNAseq in NPCs (iii) SPEN localization: What is SPEN's localization relative to the X, before and throughout XCI? Fixed and live cell imaging in mESCs & NPCs (v) SPEN domains: Which domains of SPEN mediate XCI? Complementation strategy in mESCs What do these domains do/interact with? IP/MS in mESCs (iv) SPEN and chromatin: What are the genomic targets of SPEN during XCI? CUT&RUN in mESCs

  t❛r❣❡ts ♦❢ t❤❡ ❝❤r♦♠❛t✐♥✲❛ss♦❝✐❛t❡❞ ❢❛❝t♦r ♦❢ ✐♥t❡r❡st ✭❋✐❣✉r❡ ✻✳✼❆✮✳

  https://doi.org/10.1038/s41586-020-1974-9 Received: 4 June 2019 Accepted: 10 January 2020 Published online: 5 February 2020 1 European Molecular Biology Laboratory, Director's Unit, Heidelberg, Germany. 2 Institut Curie, PSL Research University, CNRS UMR3215, INSERM U934, UPMC Paris-Sorbonne, Paris, France. 3 Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK.

Fig. 1 |

 1 Fig. 1 | SPEN mediates gene silencing across the entire X chromosome in vitro and in vivo. a, Schematic of SPEN-degron Xist-inducible mouse ES cells. Xa, active X chromosome; Xi, inactive X chromosome. b, Western blot showing auxin-induced degradation of endogenous HaloTagged SPEN. This experiment was repeated at least twice with similar results. c, d, Heat map (c) and violin plots (d) showing X-chromosomal transcript allelic ratios after 0 h, 24 h DOX or 24 h DOX + auxin treatment in SPEN-degron mouse ES cells (n = 434 genes, twosided Student's t-test). e, Box plot representation of gene-silencing defect upon SPEN loss in three groups of genes differing by their level of dependence on SPEN for Xist-mediated silencing. The pie chart shows the relative number of genes in each group. f, Pyrosequencing assay of seven X-linked transcripts in mouse ES cells after 0 h, 24 h DOX or 24 h DOX + auxin treatment. Data in c-f are averages of two independent clones; in f, individual data points are shown.g, The mouse crossbreeding scheme for the Spen-knockout experiment. KO, knockout; WT, wild type; mat., maternal; zyg., zygotic; Xm, maternal X chromosome; Xp, paternal X chromosome. h, X-chromosomal transcript allelic ratio distribution (n = 256 genes) in wild-type (n = 2), maternal-only (M) Spenknockout (n = 3), maternal-zygotic (Z) Spen-knockout (n = 5), and Xist-knockout E3.5 embryos (n = 30 single cells, two-sided Wilcoxon rank-sum test. For * refer to ref.10 ). In d, e, h, horizontal lines denote the median, box limits correspond to the upper and lower quartiles.

Fig. 2 |

 2 Fig. 2 | SPEN localizes to the X chromosome immediately upon Xist upregulation and throughout the stages of XCI, but is dispensable for the maintenance of X-linked gene silencing. a, b, Images from combined HaloTag labelling of SPEN (green) and FISH for Xist RNA (red) in mouse ES cells during a time course of Xist induction (a) and in NPCs (b). Scale bars, 5 μm. c, Schematic of the SPEN-degron experiment in NPCs. d, Cumulative distribution of transcript allelic ratios across the X chromosome (n = 387 genes) after SPEN depletion in NPCs. e, f, Cumulative distribution (e) and violin plot representation (f) of the transcript allelic ratio of escapees after SPEN depletion in NPCs (n = 65, two-sided Wilcoxon signed-rank test. NS, not significant. Horizontal lines denote the median, box limits correspond to upper and lower quartiles). Data in d-f are the average of two independent NPC clones. The experiments in a, b were repeated at least twice with similar results.

Fig. 3 |

 3 Fig. 3 | The SPOC domain of SPEN mediates gene silencing and interacts with multiple molecular pathways. a, Spen cDNA fragments used for the rescue experiment. b, Heat map representation of four X-linked transcript allelic ratios (obtained by pyrosequencing) in control, 24 h DOX-and 24 h DOX + 36 h auxin-treated SPEN-degron mouse ES cells overexpressing each cDNA construct. Data represent averages of two to three independent clones. c, Immunofluorescence detection of Flag-tagged SPEN truncations (green) and H2AK119ub1 (red), a marker of the inactive X chromosome, in SPEN-degron mouse ES cells treated with DOX and auxin. Scale bar, 5 μm. d, Schematic showing the tethering of BglG-SPOC to Xist. e, Distribution of gene-repression scores observed across the X chromosome upon the depletion of endogenous SPEN and the tethering of BglG-GFP (green) or BglG-GFP-SPOC (orange) toXist. f, Bar graphs showing the fraction of X-linked genes within four windows of repression score. g, Transcript allelic ratio (obtained by pyrosequencing) for four X-linked genes upon the depletion of endogenous SPEN and the tethering of BglG-GFP or BglG-GFP-SPOC to Xist (*P < 0.01, two-sided Student's t-test). h, Western blot showing co-immunoprecipitated proteins in BglG-GFP and BglG-GFP-SPOC immunoprecipitation experiments. One per cent of the input was loaded (0.1% for RNAPII), and 10% of the pull-down. The experiments in c, h were repeated at least twice with similar results. The data in e-g are the average of four independent clones.

Fig. 4 |

 4 Fig. 4 | SPEN is recruited by Xist to active gene promoters and enhancers, where it silences transcription and subsequently disengages from chromatin. a, SPEN allele-specific accumulation (obtained from CUT&RUN experiments) on peaks at autosomes (grey, n = 948) and on the X chromosome (green, n = 635) after 0 h, 4 h, 8 h and 24 h of Xist induction in mouse ES cells. Shown are average allelic-ratios (shading is the interquartile range) of all peaks. b, UCSC Genome Browser allele-specific track showing SPEN binding around Xist. c, Annotation of SPEN peaks on the X chromosome. d, UCSC Genome Browser allele-specific track showing SPEN binding around Eda2r, an X-linked gene. In b, d, blue denotes Cast-Xa; red denotes B6-Xi; tracks are scaled identically. e, Box plot showing SPEN enrichment at 4 h in peaks outside or within Xist entry sites. f, Violin plot showing gene expression (reads per kilobase per million reads, RPKM) of genes accumulating SPEN (n = 289) or not

Extended Data Fig. 1 |Extended Data Fig. 1 |Extended Data Fig. 2 |Extended Data Fig. 2 |. 3 |

 11223 See next page for caption. SPEN mediates gene silencing across the entire X chromosome in vitro and in vivo. a, Schematic representation of the SPEN-degron genotype with AID-HaloTag insertions in frame with the C terminus of endogenous SPEN. Targeted homozygous insertion of V5-tagged OsTIR1 at the TIGRE locus (top left) results in its constitutive protein expression as assessed by western blot (bottom left). Right, Sanger sequencing results for a PCR amplicon specific to AID-HaloTag insertions and covering a SNP outside of the recombined left homology arm. Detection of both alleles in the amplicon confirms homozygous AID knock-in. b, Fixed-cell imaging of HaloTag in wildtype cells (left), in SPEN-degron mouse ES cells (middle) and in SPEN-degron mouse ES cells exposed to auxin for 4 h (right). Cells were labelled with Halo-JF646 before fixation. SPEN-Halo is properly localized to the nucleus, and is depleted upon auxin treatment. This experiment was repeated at least twice with similar results. c, Bar graph showing the proportion of cells displaying Xist RNA clouds (quantified using RNA FISH) before and after degradation of SPEN (n, number of cells counted; χ 2 test). d, Violin plot showing the distribution of X-chromosomal transcript allelic ratios (obtained by RNA-seq) after 0 h DOX, 24 h DOX or 24 h DOX + auxin treatment in wild-type SPEN-degron mouse ES cells. Horizontal lines denote the median, box limits correspond to upper and lower quartiles, averages of two independent clones shown, n = 434 genes, two-sided Student's t-test. e, RNA FISH experiments for Xist (red) and two X-linked genes: Atrx (grey) and Huwe1 (green), in SPEN-degron mouse ES cells treated with DOX only, or DOX in combination with auxin for 24 h. The proportion of Atrx/Huwe1 monoallelic and biallelic expression among Xistexpressing cells is shown (n, number of cells counted; χ 2 test). f, Illustration of the control hybrid mouse crossbreeding scheme for the experiment shown in Fig.1g, h. g, Quantitative PCR (qPCR) analysis of Spen and Xist transcripts in wild-type (n = 7) and maternal-zygotic Spen-knockout (n = 5) E3.5 embryos. h, Pyrosequencing assay of three X-linked transcripts in maternal-zygotic Spenknockout (n = 5) and wild-type (n = 7) E3.5 embryos (two-sided Student's t-test). In g, h, bars show the mean value and individual data points are shown as dots. See next page for caption. SPEN localizes to the X chromosome immediately upon Xist upregulation and throughout the stages of XCI, but is dispensable for maintenance of X-linked gene silencing. a, Scheme of the strategy for livecell imaging of SPEN protein and Xist RNA. b, Live-cell snapshot after 16 h of Xist induction in the cell line shown in a. This experiment was repeated at least twice with similar results. c, d, Kinetics of total intensity (c) and area (d) of Xist (red) and SPEN (green) domains over time during Xist induction. The data in c, d are the averages of 27 tracked cells. Error bars indicate standard deviation. Images were acquired every 10 min. Time point 1 is defined as the earliest time at which a SPEN or Xist domain is detected in each cell. Intensity and area values were respectively normalized to the maximum value reached for each signal (SPEN and Xist). e, Hi-C map of the inactive (top) and active (bottom) X chromosomes (resolution, 1.024 Mb) in NPCs after 0 h or 48 h of auxinmediated SPEN depletion. f, Heat map of the average contact enrichment on scaled topologically associating domains containing escapees in NPCs after 0 h or 48 h of auxin-mediated SPEN depletion. g, Quantification of the allelic ratio (inactive/active X chromosome) of the Hi-C signal within topologically associating domains (n = 37) shown in f, after 0 h or 48 h of auxin-mediated SPEN depletion. Horizontal lines denote the median, box limits correspond to upper and lower quartiles, two-sided Wilcoxon rank-sum test. In e, f, averages of two independent clones are shown. See next page for caption. Extended Data Fig. 3 | The SPOC domain of SPEN mediates gene silencing and interacts with multiple molecular pathways. a, Scheme of complementation strategy. b, Western blot detection of overexpressed 3×Flagtagged SPEN protein rescue fragments. c, Scheme showing endogenous deletion of SPOC. d, Sub-nuclear localization of endogenous SPEN lacking its SPOC domain upon Xist RNA induction. The inactive X chromosome is identified using immunofluorescence detection of H2AK119ub1. e, Bar graph showing the proportion of cells with Xist RNA clouds (assayed by RNA FISH) in wild-type cells and three independent SPOC-deletion clones after induction of

. 4 |

 4 See next page for caption. Extended Data Fig. 4 | SPEN is recruited by Xist to active gene promoters and enhancers where it silences transcription and subsequently disengages from chromatin. a, Bar graph showing the number of SPEN peaks on each chromosome after 0 h, 4 h, 8 h and 24 h of Xist induction in mouse ES cells. b, Annotation of SPEN peaks on autosomes. c, Heat map showing allelic ratios at SPEN peaks during XCI among different X-linked genomic features. d, Violin plot showing expression (RPKM) of genes accumulating SPEN (n = 259) or not accumulating SPEN (n = 689) at their promoters. Genes showing 0 RPKM were excluded from this plot. e, Box plots showing SPEN enrichment after 4 h of Xist induction within promoter windows of genes grouped on the basis of their level of dependency on SPEN for gene silencing (see Fig.

. 5 |

 5 UCSC Genome Browser allelic tracks of SPEN binding and transcript expression at X-linked genes. a-n, Top, Genome Browser allelic tracks of SPEN binding (from CUT&RUN) at silenced genes (a-g) and nonsilenced genes (h-n) during a time course of Xist induction in mouse ES cells (blue, Cast-Xa; red, B6-Xi; scaled identically within each panel). Bottom, allelic tracks of transcript expression (from RNA-seq) at 0 h and 24 h of Xist induction in mouse ES cells (light grey, Cast-Xa; black, B6-Xi; scaled identically within each panel). The relative position of each gene along the X chromosome is shown at the top of the figure. nature research | reporting summary October 2018 V5-tagged Tir1. The Flag antibody is validated for immunofluorescence in Fig. 3c, with specific nuclear signal being observed only in cells expressing Flag-tagged SPEN truncations. The GFP antibody is validated for CUT&RUN by showing specific genomic signal for GFP-tagged SPEN, which disappears upon treatment of cells with auxin. The H2AK119ub1 has been validated for immunofluorescence in Zylicz et al., 2019 The Lamin B1 antibody have been KO validated by abcam. The PCNA antibody is cited 361 times according to CiteAb. The Ncor1/Ncor2 antibodies are validated for western blot in Fig3. h, as showing increased signal upon immunoprecipitation of SPOC. The Mta1 antibody has been cited 12 times previously according to CiteAb. The Wtap antibody have been KO validated by ProteinTech. The Mettl3 antibody have been KO validated by Abcam. The Hdac3 antibody has been cited 9 times according to CiteAb. The Rpb1 antibody has been cited 280 times previously according to CiteAb. Eukaryotic cell lines Policy information about cell lines Cell line source(s) All cell lines were derived from the TX1072 female mouse embryonic stem cell line (Schulz et al., 2014) Authentication None of the cell lines were authenticated Mycoplasma contamination All cell lines tested negative for mycoplasma contamination Commonly misidentified lines (See ICLAC register) cells used are not in the ICLAC database Animals and other organisms Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research

p

  YSPTSPA YEPRSPGG YTPQSPS YSPTSPS YSPTSPS YSPTSPN YSPTSPS YSPTSPS YSPTSPS YSPTSPS YSPTSPS YSPTSPS YSPTSPS YSPTSPS YSPTSPS YSPTSPS YSPTSPS YSPTSPS YSPTSPS YSPTSPS YSPTSPS YSPTSPN YSPTSPN YTPTSPS YSPTSPS YSPTSPN YTPTSPN YSPTSPS YSPTSPS YSPTSPS YSPSSPR YTPQSPT YTPSSPS YSPSSPS YSPTSPK YTPTSPS YSPSSPE YTPTSPKYSPTSPK YSPTSPK YSPTSPT YSPTTPK YSPTSPT YSPTSPV YTPTSPK YSPTSPT YSPTSPK YSPTSPT YSPTSPKGST YSPTSPG structures of SPOC bound to the SMRT phosphopeptide (Mikami et al., Structure. 2014) ❋✐❣✉r❡ ✼✳✶ ✕ ❊✈✐❞❡♥❝❡ t♦✇❛r❞s ❛ ♣♦ss✐❜•❡ ✐♥t❡r❛❝t✐♦♥ ❜❡t✇❡❡♥ ❙P❖❈ ❛♥❞ t❤❡ ♣❤♦s✲ ♣❤♦r②•❛t❡❞ ❈✲t❡r♠✐♥❛• ❞♦♠❛✐♥ ♦❢ |◆❆ ♣♦•②♠❡r❛s❡ ■■✳ ✭❆✮ ❙❝❤❡♠❡ s❤♦✇✐♥❣ t❤❡ ❛♠✐♥♦ ❛❝✐❞ s❡q✉❡♥❝❡ ♦❢ t❤❡ |◆❆P■■✲❈❚❉✱ ❝♦♠♣r✐s❡❞ ♦❢ ✺✷ ❤❡♣t❛♣❡♣t✐❞❡ r❡♣❡❛ts✳ ❚❤❡ ❝♦♥s❡♥s✉s s❡✲ q✉❡♥❝❡ ♦❢ t❤❡ r❡♣❡❛t❡❞ ❤❡♣t❛♣❡♣t✐❞❡ ✐s s❤♦✇♥ ❛t t❤❡ ❜♦tt♦♠ •❡❢t ♦❢ t❤❡ ♣❛♥❡•✳ ❚❤❡ ❙❡r✐♥❡s ❛t ♣♦s✐t✐♦♥ ✷ ✭❜♦•❞ ❜•✉❡✮✱ ✺ ✭❜♦•❞ ❣r❡❡♥✮ ❛♥❞ ✼ ✭❜♦•❞ ❜•❛❝❦✮ ❛r❡ r❡❛❞✐•② ♣❤♦s♣❤♦r②•❛t❡❞ ✐♥ ✈✐✈♦✳ ✭❇✮ ❚②♣✐❝❛• ❛✈❡r❛❣❡ ❈❤■P ♣r♦✜•❡s ♦❢ |◆❆P■■ ♣❤♦s♣❤♦r②•❛t❡❞ ❛t ❙❡r✷ ✭❜•✉❡✮✱ ❙❡r✺ ✭❣r❡❡♥✮ ❛♥❞ ❙❡r✼ ✭❜•❛❝❦✮ ♦❢ ✐ts ❈❚❉ s❤♦✇s t❤❡ ✐♥tr✐❝❛t❡ •✐♥❦ ❜❡t✇❡❡♥ t❤❡ tr❛♥s❝r✐♣t✐♦

  P✉r✐✜❝❛t✐♦♥ ♦❢ ♠♦✉s❡ ❙P❊◆✬s ❙P❖❈ ❞♦♠❛✐♥ ❡①♣r❡ss❡❞ ✐♥ ❜❛❝t❡r✐❛• ❝❡••s✳ ✭❆✮ ❚❤❡ ❞✐✛❡r❡♥t ❙P❖❈ ❝♦♥str✉❝ts ✭s❡❡ ❚❛❜•❡ ✼✳✶✮ ✇❡r❡ tr❛♥s❢♦r♠❡❞ ✐♥ t❤❡ |♦s❡tt❛✷ ❜❛❝t❡r✐❛• str❛✐♥✱ ❡♥❛❜•✐♥❣ ❡♥❤❛♥❝❡❞ ❡①♣r❡ss✐♦♥ ♦❢ ❡✉❦❛r②♦t✐❝ ♣r♦t❡✐♥s ✇❤✐❝❤ ❤❛✈❡ ♥♦t ❜❡❡♥ ❝♦❞♦♥✲♦♣t✐♠✐③❡❞ ❢♦r ❜❛❝t❡r✐❛✳ ▲✐q✉✐❞ ❜❛❝t❡r✐❛• ❝✉•t✉r❡s ✇❡r❡ ❣r♦✇♥ ❛t ✸✼➦❈✱ ✐♥ t❡rr✐✜❝ ❜r♦t❤✱ ✉♥t✐• •♦❣✲♣❤❛s❡ ✇❛s r❡❛❝❤❡❞ ✭❖❉ ✻✵✵ ❂✵✳✻✮✱ ❛❢t❡r ✇❤✐❝❤ ❙P❖❈ ❡①♣r❡ss✐♦♥ ✇❛s ✐♥❞✉❝❡❞ ♦✈❡r♥✐❣❤t ❛t ✶✽➦❈ ✇✐t❤ ✵✳✺♠▼ ■P❚•✳ ❙P❖❈ ✇❛s ♣✉r✐✜❡❞ ❢r♦♠ ❜❛❝t❡r✐❛• •②s❛t❡s ✉s✐♥❣ ◆✐✲◆❚❆ ❛✣♥✐t②✲r❡s✐♥✳ ❙P❖❈ ✇❛s t❤❡♥ ❝•❡❛✈❡❞✲♦✛ ♦❢ ✐ts ✻①❍✐s✲❙❯▼❖✸ t❛❣ ✇✐t❤ ❛ ❙❯▼❖ ♣r♦t❡❛s❡✳ ❖♥❡ st❡♣ ♦❢ s✐③❡ ❡①❝•✉s✐♦♥ ❝❤r♦✲ ♠❛t♦❣r❛♣❤② ✇❛s s✉✣❝✐❡♥t t♦ ❝♦••❡❝t ❣r❡❛t ②✐❡•❞s ♦❢ ❤✐❣❤•②✲♣✉r✐✜❡❞ ❙P❖❈✳ ✭❇✮ ❈❤r♦♠❛tr♦❣r❛♠ ✭t

  human SPOC(Ariyoshi et al., Genes&Dev. 2003) mouse SPOC (this study) ❈r②st❛••✐③❛t✐♦♥ ❛♥❞ str✉❝t✉r❛• ❞❡t❡r♠✐♥❛t✐♦♥ ♦❢ ♠♦✉s❡ ❙P❖❈✳ ✭❆✮ ❆♠✐♥♦ ❛❝✐❞ s❡q✉❡♥❝❡s ♦❢ ♠♦✉s❡ ✭❜•✉❡✮ ❛♥❞ ❤✉♠❛♥ ✭❣r❡②✮ ❙P❖❈ ❞♦♠❛✐♥s✳ ❆♠✐♥♦ ❛❝✐❞s ❛r❡ ♥✉♠❜❡r❡❞ r❡•❛t✐✈❡ t♦ t❤❡ ❡♥t✐r❡ ❙P❊◆ ♣r♦t❡✐♥ s❡q✉❡♥❝❡✳ ❇♦t❤ ❙P❖❈ s❡q✉❡♥❝❡s s❤❛r❡ ✾✾✪ ❤♦♠♦•♦❣②✱ ✇✐t❤ ❛ ❙❡r✐♥❡ ❛t ♣♦s✐t✐♦♥ ✸✺✶✼ ✐♥ ♠♦✉s❡ ❜❡✐♥❣ s✉❜st✐t✉t❡❞ ❜② ❛ ❚❤r❡♦♥✐♥❡ ❛t ♣♦s✐t✐♦♥ ✸✺✸✼ ✐♥ ❤✉♠❛♥✳ ❚❤❡ ♣♦s✐t✐♦♥ ♦❢ α✲❤❡•✐❝❡s ❛♥❞β✲s❤❡❡ts ✐❞❡♥t✐✜❡❞ ✐♥ ♦✉r ❙P❖❈ str✉❝t✉r❡ ✐s ✐♥❞✐❝❛t❡❞ ❛❜♦✈❡ t❤❡ s❡q✉❡♥❝❡✳ ✭❇✮ P✐❝t✉r❡s s❤♦✇✐♥❣ t②♣✐❝❛• ❙P❖❈ ❝r②st❛•s✳ ✭❈✮ ❈❛rt♦♦♥ r❡♣r❡s❡♥t❛t✐♦♥ ♦❢ ❛ str✉❝t✉r❛• ❛•✐❣♥♠❡♥t ❜❡t✇❡❡♥ ♠♦✉s❡ ❙P❖❈ ✭❜•✉❡✱ r❡s♦•✈❡❞ ✐♥ t❤✐s st✉❞②✮✱ ❛♥❞ ❤✉♠❛♥ ❙P❖❈ ✭❣r❡②✱ r❡s♦•✈❡❞ ✐♥ ❬❆r✐②♦s❤✐ ❛♥❞ ❙❝❤✇❛❜❡✱ ✷✵✵✸(✮✳

  t✐❞❡s ✭❙❡r✷P✴❙❡r✼P✱ ❙❡r✷P✴❙❡r✺P✱ ❙❡r✺P✴❙❡r✼P✮ ❛♥❞ ❙P❖❈ ✇❛s ❛ss❛②❡❞ ✉s✐♥❣ ■❚❈✳ DP (µcal/s) ∆H (kcal/mol)

  ❚❛❜•❡ ✼✳✹ ✕ ❈r②st❛••✐③❛t✐♦♥ ❝♦♥❞✐t✐♦♥s ❢♦r ❝r②st❛•s s❤♦✇♥ ✐♥ ❋✐❣✉r❡ ✼✳✼✳ ❊❛❝❤ ❝r②st❛• ✐s ❞❡s✐❣♥❛t❡❞ ✇✐t❤ ✐ts ❛ss♦❝✐❛t❡❞ ♥✉♠❜❡r ❛s ✐♥ ❋✐❣✉r❡ ✼✳✼✳ ❚❤❡ r❛t✐♦ ❝♦•✉♠♥ ✐♥❞✐❝❛t❡s t❤❡ ❙❡r✺P✲❈❚❉ ♣❡♣t✐❞❡ t♦ ❙P❖❈ ♠♦•❛r r❛t✐♦✳ ❉❛t❛ ❝♦••❡❝t❡❞ ❢r♦♠ ❳✲r❛② ❞✐✛r❛❝t✐♦♥ ♦❢ ❝r②st❛• ✹✵ ✭s❤♦✇♥ ✐♥ ❜♦•❞✮ ✇❛s ✉s❡❞ t♦ r❡s♦•✈❡ t❤❡ str✉❝t✉r❡ ♦❢ t❤❡ ❙P❖❈✴❙❡r✺P✲❈❚❉ ❝♦♠♣•❡①✳ ✕ P✐❝t✉r❡s ♦❢ ❙P❖❈✴❙❡r✺P✲❈❚❉ ♣❡♣t✐❞❡ ❝♦❝r②st❛•s ❣r♦✇♥ ❛❝r♦ss s❡✈❡r❛• ❝♦♥❞✐t✐♦♥s✳ ▼❛❣♥✐✜❝❛t✐♦♥ ✐s ✐❞❡♥t✐❝❛• ❛❝r♦ss ❛•• ♣✐❝t✉r❡s✳ ❈♦♥❞✐t✐♦♥s ❛ss♦❝✐❛t❡❞ ✇✐t❤ ❡❛❝❤ ❝r②st❛• ❛r❡ t♦ ❜❡ ❢♦✉♥❞ ✐♥ ❚❛❜•❡ ✼✳✹✳ ❉❛t❛ ❝♦••❡❝t❡❞ ❢r♦♠ ❳✲r❛② ❞✐✛r❛❝t✐♦♥ ♦❢ ❝r②st❛• ✹✵ ✭r❡❞✲ ❜♦r❞❡r❡❞ ✐♠❛❣❡✮ ✇❛s ✉s❡❞ t♦ r❡s♦•✈❡ t❤❡ str✉❝t✉r❡ ♦❢ t❤❡ ❙P❖❈✴❙❡r✺P✲❈❚❉ ❝♦♠♣•❡①✳ ✶✻✹ |❡s✉•ts ❙P❖❈✴❙❡r✺P✲❈❚❉

P 6 S 7 Y 1 S 2 P 3 T 4

 671234

  ❊•❡❝tr♦st❛t✐❝ ♠❛♣ ♦❢ t❤❡ ❙❡r✺P✲❈❚❉ ❜✐♥❞✐♥❣ s✐t❡ ♦❢ t❤❡ ❙P❖❈ ❞♦♠❛✐♥✳ ■♥t❡r♠♦•❡❝✉•❛r ❤②❞r♦❣❡♥ ❜♦♥❞s ❛♣♣❡❛r ❛s ❞♦tt❡❞ ②❡••♦✇ •✐♥❡s✱ ❛♥❞ ❛r❡ ♥✉♠❜❡r❡❞ ✭❜•❛❝❦ ❝✐r❝•❡s✮ ❛s ✐♥ ❚❛❜•❡ ✼✳✻✳ ❡•❡❝tr♦st❛t✐❝ ♣♦t❡♥t✐❛• ♠❛♣✴❣r❡❡♥ ❝❛rt♦♦♥ r❡♣r❡s❡♥t❛t✐♦♥✿ ❙P❖❈✱ ♦r❛♥❣❡ •✐❝♦r✐❝❡ r❡♣r❡s❡♥t❛t✐♦♥✿ ❙❡r✺P✲❈❚❉ ♣❡♣t✐❞❡ ✶✻✼ P❛rt ■❱ ❉✐s❝✉ss✐♦♥ ❉✐s❝✉ss✐♦♥ ❙P❊◆ ❢✉♥❝t✐♦♥ ❞✉r✐♥❣ ❳❈■ ❈❤❛♣t❡r ✽ ❙P❊◆ ✐s t❤❡ ♠❛st❡r ❡✛❡❝t

  ✉s ◆❈♦| ❛♥❞ ❙▼|❚ ✕ ✇❤✐❝❤ ♠❡❞✐❛t❡s t❤❡✐r ✐♥t❡r❛❝t✐♦♥ ✇✐t❤ ❙P❖❈✱ ❛♥❞ ✐s •♦❝❛t❡❞ ❛t t❤❡✐r ✈❡r② ❈✲t❡r♠✐♥✉s ✭s❡❡ s✉❜s❡❝t✐♦♥ ✼✳✶✳✷✮ ✕ ❤❛s ❜❡❡♥ ❞❡•❡t❡❞ ✭❋✐❣✉r❡ ✾✳✶❇✮✳ Sensitized background: removing NCOR/SMRT-HDAC3 contribution to XCI Direct comparison of SPOC WT and SPOC mutated informs whether SPOC binding to RNAPII is functional in XCI homozygous deletion of the LSDS motif at Ncor1 and Smrt (Ncor2)

  ■■ ❈❚❉✳ ❈❤❛♣✉②✱ ❇✳✱ ❙t❡✇❛rt✱ ❈✳✱ ❉✉♥❢♦r❞✱ ❆✳ ❏✳✱ ❑✐♠✱ ❏✳✱ ❑❛♠❜✉r♦✈✱ ❆✳✱ |❡❞❞✱ |✳ ❆✳✱ ▲❛✇r❡♥❝❡✱ ▼✳ ❙✳✱ |♦❡♠❡r✱ ▼✳ •✳✱ ▲✐✱ ❆✳ ❏✳✱ )✐❡♣❡rt✱ ▼✳✱ ❙t❛✐❣❡r✱ ❆✳ ▼✳✱ ❲❛•❛✱ ❏✳ ❆✳✱ ❉✉❝❛r✱ ▼✳ ❉✳✱ ▲❡s❤❝❤✐♥❡r✱ ■✳✱ |❤❡✐♥❜❛②✱ ❊✳✱ ❚❛②•♦r✲❲❡✐♥❡r✱ ❆✳✱ ❈♦✉❣❤•✐♥✱ ❈✳ ❆✳✱ ❍❡ss✱ ❏✳ ▼✳✱ P❡❞❛♠❛••✉✱ ❈✳ ❙✳✱ ▲✐✈✐t③✱ ❉✳✱ |♦s❡❜r♦❝❦✱ ❉✳✱ |♦s❡♥❜❡r❣✱ ▼✳✱ ❚r❛❝②✱ ❆✳ ❆✳✱ ❍♦r♥✱ ❍✳✱ ❱❛

m 6 A

 6 ✉s r❡tr♦✈✐r✉s❡s✳ ◆❛t✉r❡✳ ✷✵✷✶❀ ✺✾✶✭✼✽✺✾✮✿✸✶✷✕✸✶✻✳ ❆|❚■❈▲❊ ❖◆ ◆❊❳❚ P❆•❊ ✷✶✼ Article RNA methylation regulates the fate of endogenous retroviruses Tomasz Chelmicki 1 ✉ , Emeline Roger 1 , Aurélie Teissandier 1 , Mathilde Dura 1 , Lorraine Bonneville 1 , Sofia Rucli 1 , François Dossin 2 , Camille Fouassier 3 , Sonia Lameiras 4 & Deborah Bourc'his 1 ✉

1 Received 1 InstitutFig. 1 |

 111 Fig. 1 | CRISPR screen for IAP suppressors in mouse ES cells. a, Schematic of the IAPEz reporter and experimental rationale. b, Workflow and timelines for the CRISPR-Cas9 knockout screen (screen I). c, Ranked P values (permutation test by MAGeCK 11 ; Methods) for enriched genes after 5 (left), 7 (middle) and 9 (right) days of blasticidin treatment. Discontinued lines indicate genes ranked in top 100 according to P value (top) and P = 0.05 (bottom). Known IAP regulators (black, when P < 0.05) and Mettl3, Mettl14, Wtap and Zc3h13 (red) are reported. d, Violin plots for all sgRNA read counts represented as the logarithm of reads per million (RPM) after library introduction (1), without selection (2) and after blasticidin selection (3-5); sgRNAs targeting Mettl14 (sgMettl14; red, n = 10), Mettl3 (sgMettl4; green, n = 10), Wtap (sgWtap; blue, n = 10), Zc3h13 (shZc3h13; orange, n = 9) and intergenic sequences (sgRNA_intergenic; grey, n = 199).

Fig. 2 |

 2 Fig. 2 | Depletion of the m 6 A methyltransferase complex increases IAP mRNA levels. a, Immunoblot showing Mettl3, Mettl14, Wtap and Zc3h13 knockout (KO) in ES cells. PCNA and lamin B1 served as loading controls; asterisk indicates unspecific band. Immunoblotting was repeated at least twice with similar results. b, RT-qPCR showing normalized IAP mRNA levels using primers against Δ1, LTR and gag sequences in the indicated knockouts relative to wild-type (WT) control (set to 1). Data are mean ± s.d. of three independent experiments. c, Volcano plot showing log 2 -transformed fold change (log 2 FC) in retrotransposon expression in Mettl3 knockout versus wild-type control using random assignment of multi-mapped reads. Red, blue, orange and green denote significantly deregulated RepeatMasker annotations belonging to ERVK, ERV1, ERVL and L1 families, respectively. Grey denotes non-differentially expressed (non-DE) retrotransposons. P values were computed using limma and adjusted with the Benjamini-Hochberg correction (Methods). log 2 FC > 0.75; false discovery rate (FDR) < 0.05. d, Box plots showing expression (log 10 CPM; in which CPM denotes counts per million) of individual retrotransposon copies in wild-type and knockout ES cells. RNA-seq mapping allowed only unique hits in the reference genome and only copies with at least ten reads in at least one sample were conserved. Horizontal lines denote median, box limits correspond to upper and lower quartiles. P values determined by two-sided Student's t-test.

Fig. 4 |

 4 Fig. 4 | IAP chromatin status and RNA stability upon inducible degron of the m 6 A methyltransferase complex. a, Schematic of METTL3 and METTL14 degron engineering. b, Immunoblot showing auxin-induced degradation of endogenous 3×Flag-AID-METTL3, -METTL14 and -METTL3;METTL4 double degron (dd). PCNA served as a loading control. c, RT-qPCR showing normalized IAP mRNA levels relative to 0h using LTR-specific primers after degron of METTL14 (light blue), METTL3 (blue) and METTL3;METTL14 dd (dark blue). TIR1-only (black) ES cells served as control. Error bars indicate mean ± s.d. of three independent auxin inductions. d, Immunofluorescence staining for IAP Gag in METTL3;METTL14 double degron after 96 h of treatment with DMSO (top) or auxin (bottom). Scale bar, 10 μm. e, Violin plots and box plots (inside) showing H3K4me3, H3K27ac and H3K9me3 levels at IAPEz copies (n = 426) determined by CUT&RUN in METTL3;METTL14 dd after 96 h of DMSO (grey) or auxin (red). Only copies with at least 10 reads in at least one sample were conserved. Box plots as in Fig. 2d. P values were determined by two-sided Student's t-test. f, Composite profiles showing H3K4me3 (top), H3K27ac (middle) and H3K9me3 (bottom) coverage along full-length (greater than 5 kb) IAPEz copies (n = 1,331) in METTL3;METTL14 double degron after 96 h of DMSO (grey) or auxin (red). g, Top, scheme for evaluating IAP mRNA degradation rates in METTL3;METTL14 double degron ES cells exposed to DMSO or auxin and actinomycin D (actD) for 2, 4 and 8 h before (i-ii) and after (iii) auxin wash-off. Bottom, relative IAP mRNA levels and half-life measurements in ES cells exposed to DMSO (light grey), auxin (red) and after auxin wash-off (dark grey) using Δ1-specific primers (n = 3 independent experiments). h, Left, YTHDF2 RIP-qPCR for ERVKs and Nanog in METTL3;METTL14 double degron cells after treatment for 96 h with DMSO (red) or auxin (pink). Rabbit IgG served as control. Data are mean ± s.d. of three independent experiments. P values were determined by two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli. Right, immunoblot showing METTL3 and METTL14 (Flag), and YTHDF2 in METTL3;METTL14 double degron cells after treatment with DMSO or auxin for 96 h. PCNA served as loading control. i, Left, immunoblots showing YTHDF1-YTHDF3 protein levels in mutYthdf1-2-3 and mutYthfd2-3 ES cells. PCNA served as loading control. Right, RT-qPCR showing normalized IAP mRNA levels (geometric mean between Actb and Gapdh) in mutant ES cells relative to wild-type cells using Δ1-, LTR-or gag-specific primers. Data are mean ± s.d. from three independent experiments. Immunoblots in b, h and i and the experiment in d were repeated at least twice with similar results. Results in e and f represent averages of two independent CUT&RUN.

  R package.

. 3 |Extended Data Fig. 3 |

 33 See next page for caption. Generation and validation of Mettl3-, Mettl14-, Wtapand Zc3h13-knockout mouse ES cells. a-d, Mettl3-knockout (a), Mettl14knockout (b), Wtap-knockout (c), and Zc3h13-knockout (d), mediated by CRISPR-Cas9. Schematic representation showing sgRNAs targeting of indicated loci (top) and immunoblots (bottom) confirming depletion of indicated proteins. PCNA or lamin B1 served as loading controls. In red, clones selected for downstream analyses presented in Figs.

10 -. 4 |

 104 Depletion of m

. 5 |

 5 Family-based analysis of retrotransposon expression using random versus unique mapping. a, RNA-seq heat maps showing log 2 FC in expression of indicated retrotransposon families in Mettl3-, Mettl14-, Wtapand Zc3h13-knockout versus wild-type ES cells allowing either multiple mapping with random allocation (left) or unique mapping only (right). In case of unique mappers with intragenic position, only elements transcribed in reverse orientation to the gene were included in the analysis. Note that although ERVK annotations show general upregulation in all knockout ES cells, L1s tended to be downregulated, with the exception of oldest L1MdA_IV, L1ME3Cz and L1MdV_I subfamilies. b, Heat maps of published nuclear RNA-seq data19 showing log 2 FC in expression for indicated retrotransposon families in two Mettl3-knockout versus control ES cells, allowing multiple mapping with random allocation. The two knockout lines show divergent levels of retrotransposon reactivation but consistently show greater reactivation of ERVK compared to ERVL and LINE1 families. Asterisks indicate retrotransposon families with log 2 FC > 0.75 and FDR < 0.05. Data were taken from accession GSE133585.

. 7 |

 7 -----AGAAGATTCTGGTCTGTGGTGTTCTTCCTGGCCGGGCGTGAGAACGCGTCTAAT--AACAATTGGTGACCGAATTCCGGG------ACG-----AGAAAAAACTCG--GGAC MMERVK10C AGTGGTGCCGAAAACCCGGGACAAGAAACATCTTCAGGCACGAGCGAAGACCCCCTGCTACAGGGAGGATTCAGAACTGCATCGCGGGGAAGGAGCGGTTAATAAAGGTTCCCGTAAAAC *** * * ** * ***** * ** *** * CAAGGAAGATCCCTCAT---TCCAGAACCAG--AACTGCGGGTCG-CGGTAATAAAGGTTCCCGTAAAGCAGACTGTTAAGAAGGATTCAACTGTATGAATTCAGAACTTTTC MMERVK10C AGACTGCTGAGAAGGATCCGGCGTGGATTCAGAACTCTTCAGCTGGGGAACGGTGGTAATGAAGGTTCCCGCAAAACAGACTGCTGAGAAGGATCCGACTGCGTGGATTCAGAACTCTTC * * ** * ***** * * * ****** * *** ** ** ****** ********** *** ******* * ******** * **** ** ********** *** IAPEz AGCTGGGGAACGAGAGTACCAGTGAG---------------------------------TACAGCTTTACGAGGTAAGTCTGATCTTG------------------------AACTTTCT MMERVK10C AGCTGGGGAACGGTGGTACCGGTAAGTANAAAGAAATTAATGTGAGTCTCTGAGAGGCATGCTTTCCTACGNAGTAGGTCCGGTAATTGTCGCTGGGGTTGTGTTGCTTGCTTTCTTTCT ************ ***** ** ** * * **** *** *** * * * ****** IAPEz AAGGAAATT-----------------------------CAAGACAG---TCTATCAGAAG---------TAAAGTGGAGAAA-------------------MMERVK10C TGCCTTGATNTTATGCCATTGCCTTAATTGTCANCGCCCTGGACACGAGTGTATCTGCTGGGAATAATTTCACGTAGATAACGCTTTAGTCCAGGACGATC * * **** * **** * * * * ** ** ** Alignment between IAPEz 5'UTR and MMERVK10C 5'UTR 8 RRACH motifs in IAPEz 11 RRACHmotifs in MMERVK10C 5 RRACH motifs at common positions See next page for caption.

. 8 |

 8 See next page for caption.

10 Extended Data Fig. 9 |

 109 See next page for caption.

allele 1 -. 10 |

 110 insertion of 'T' in exon 3 at position: chr4:132,210,772-132,210,773; premature stop codon mutDF1-2-3 allele 1: chr4:132,210,635-132,210,788 (154bp deletion); premature stop codon mutDF2-3 no mutation/deletion mutDF2-3 allele 2: chr4:132,210,772-132,212,108 (1337bp deletion) mutDF1-2-3 allele 1: chr3:16,203,656-16,204,764 (1109bp deletion) mutDF2-3 allele 2: chr3:16,204,739-16,204,770 (32bp deletion) allele 1: chr3:16,203,651-16,204,762 (1112bp deletion) allele 2: 5bp deletion in exon 5 at position: chr3:16,204,766-16,204,770; premature stop codon allele 2: chr4: chr4:132,210,773-132,212,108 (1336bp deletion) YTHDF2 binds to ERV mRNAs. a, Schematic representation showing sgRNA targeting of the Ythdf2 gene. b, Immunoblot showing YTHDF2 protein levels in wild-type and YTHDF2-depleted cells. PCNA served as loading control. Immunoblotting was repeated twice with similar results c, RIP-qPCR showing YTHDF2 enrichment on indicated ERVKs and Nanog in wild-type and Ythdf2-knockout ES cells (two independent clones). Rabbit IgG served as control. Data are mean ± s.d. from three independent experiments. d, Generation of mutant mutYthdf2-3 and mutYthdf1-2-3 ES cells using CRISPR-Cas9. Schematic representation showing sgRNA sequences; mutation/deletion information based on Sanger sequencing is provided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a=P =W +5$ ( )4"8 $ #"4$ "( 6&7 =8 8 4( $ E =#% 88 #2&"#b6&7 '"( # cX dQL bL dYJ TQR L YR L Q\YMP L dYJ TQR L eSQFQdQW L R TX P YVJ Q_ <f&/ $ &"&"&7 / $ =$ "8 2&( $ ""( )4)$ 48! $ &"#C& 964)&$ "C"( & 7 ( ( $ "% <)$ & 4)$ 4&7&"#42!7 B# $ %" =$ #"( $ 8 $ 4&( $ "8( )&!! ! $ &( 7 678 ( ( &"#8 '7 7 ! ( $ "%8'( 42 @ ( $ 2&( 88 8 4( $ E * : %:)"g U=A& "g W + =$ "#$ 4&( $ "%)5()/5 4&7 4'7 &( # ITW L eQVL MNJ J QMP X NFL NFL R P YP X R P X MR L h NW L VX NJ NiX R P R L MNFP YX FR L YW P X MJ QR L NFL OYFKL Nh L P SQL [NX FP R L YVNdQ_

W

  X Qh J KL UQR MW X VQL P SQL R P TUK_ L aNW L ]TYFP X P YP X dQL UYP YL X FMJ TUQL P W QYP OQFP L h YMP NW R L YFUL X FP QW YMP X NFR L UQR X iFL R P W TMP TW QL Q_ i_ L h YMP NW X YJ L FQR P QU L SX QW YW MSX MYJ L FYP TW QL YFUL FTOVQW L Nh L Q\[QW X OQFP YJ L TFX P R L YFUL W Q[J X MYP QR _ QR MW X VQL P SQL W QR QYW MSL R YO[J QL Q_ i_ L YL iW NT[L Nh L P YiiQUL bYR R QW L UNOQR P X MTR L YJ J L P QFNMQW QTR L P STW VQW X L eX P SX FL IW iYFL bX [QL YMP TR L YP X NFYJ L ^NFTOQFP L YFUL [W NdX UQL YL W YP X NFYJ QL h NW L P SQL R YO[J QL MSNX MQ_ L SQFL W QJ QdYFP L UQR MW X VQL P SQL NW iYFX R OLP Y\Y L R NTW MQ L R Q\ L YiQL W YFiQL YFUL YFKL OYFX [TJ YP X NFR _ L P YP QL eSYP L [N[TJ YP X NFL P SQL R YO[J QL X R L OQYFP L P NL W Q[W QR QFP L eSQFL Y[[J X MYVJ Q_ L aNW L R P TUX QR L X FdNJ dX FiL Q\X R P X FiL UYP YR QP R L UQR MW X VQL P SQL UYP YL YFUL X P R L R NTW MQ_ NP QL P SQL R YO[J X FiL [W NMQUTW Q_ L QR MW X VQL P SQL R P YP X R P X MYJ L OQP SNUR L P SYP L eQW QL TR QUL P NL [

  FiL eYR L FNP L W QJ QdYFP L P NL KNTW L R P TUK_ QR MW X VQL P SQL R P TUKL MNFUX P X NFR L h NW L h X QJ UL eNW L [W NdX UX FiL W QJ QdYFP L [YW YOQP QW R L Q_ i_ L P QO[QW YP TW Q L W YX Fh YJ J _ P YP QL P SQL J NMYP X NFL Nh L P SQL R YO[J X FiL NW L Q\[QW X OQFP L [W NdX UX FiL W QJ QdYFP L [YW YOQP QW R L Q_ i_ L J YP X P TUQL YFUL J NFiX P TUQ L QJ QdYP X NF L eYP QW L UQ[P S _ QR MW X VQL P SQL Qh h NW P R L KNTL SYdQL OYUQL P NL YMMQR R L SYVX P YP R L YFUL P NL MNJ J QMP L YFUL X O[NW P Q\[NW P L KNTW L R YO[J QR L X FL YL W QR [NFR X VJ QL OYFFQW L YFUL

  [W NdQFYFMQL X Fh NW OYP X NFL h NW L R [QMX OQFR L YFUL UQR MW X VQL [QW OXP R L P SYP L eQW QL NVP YX FQUL h NW L P SQL eNW L X FMJ TUX FiL P SQL FYOQL Nh L P SQLX R R TX FiL YTP SNW X P K L P SQL UYP QL Nh L X R R TQ L YFUL YFKL X UQFP X h KX FiL X Fh NW OYP X NF _ FUX MYP QL eSQW QL P SQL R [QMX OQFR L SYdQL VQQFL UQ[NR X P QUL P NL [QW OXP L h W QQL YMMQR R L VKL NP SQW L W QR QYW MSQW R _ h L FQeL UYP QR L YW QL [W NdX UQU L UQR MW X VQL SNeL P SQKL eQW QL NVP YX FQUL Q_ i_ L MNJ J QMP X NF L R P NW YiQ L R YO[J QL [W QP W QYP OQFP L YFUL OQYR TW QOQFP L eSQW QL P SQKL eQW QL NVP YX FQUL X _ Q_ L J YVL FYOQ L P SQL MYJ X VW YP X NFL [W NiW YOLYFUL P SQL [W NP NMNJ L h NW L ]TYJ X P KL YR R TW YFMQL IL R P YP QL P SYP L FNL FQeL UYP QR L YW QL [W NdX UQU_ aNW L J YVNW YP NW KL YFX OYJ R L W Q[NW P L R [QMX QR L R P W YX F L R Q\L YFUL YiQL IL R P YP QL P SYP L P SQL R P TUKL UX UL FNP L X FdNJ dQL J YVNW YP NW KL YFX OYJ R _ bW NdX UQL UQP YX J R L NFL YFX OYJ R L NVR QW dQUL X FL NW L MY[P TW QUL X FL P SQL h X QJ UZ L W Q[NW P L R [QMX QR L R Q\L YFUL YiQL eSQW QL [NR R X VJ Q_ L QR MW X VQL SNeL YFX OYJ R L eQW QL MYTiSP L YFUL P W YFR [NW P QUL YFUL eSYP L SY[[QFQUL P NL MY[P X dQL YFX OYJ R L Yh P QW L P SQL R P TUKL X h L X J J QU L Q\[J YX FL eSKL YFUL UQR MW X VQL OQP SNUZ L X h L W QJ QYR QU L RYKL eSQW QL YFUL eSQF L IL R P YP QL P SYP L P SQL R P TUKL UX UL FNP L X FdNJ dQL eX J UL YFX OYJ R _ aNW L J YVNW YP NW KL eNW L eX P SL h X QJ U MNJ J QMP QUL R YO[J QR L UQR MW X VQL YJ J L W QJ QdYFP L [YW YOQP QW R L R TMSL YR L SNTR X Fi L OYX FP QFYFMQ L P QO[QW YP TW Q L [SNP N[QW X NUL YFUL QFU Nh Q\[QW X OQFP L [W NP NMNJ L IL R P YP QL P SYP L P SQL R P TUKL UX UL FNP L X FdNJ dQL R YO[J QR L MNJ J QMP QUL h W NOLP SQL h X QJ U_ UQFP X h KL P SQL NW iYFX YP X NF R L P SYP L Y[[W NdQUL NW L [W NdX UQUL iTX UYFMQL NFL P SQL R P TUKL [W NP NMNJ L IL R P YP QL P SYP L FNL QP SX MYJ L Y[[W NdYJ L NW L iTX UYFMQL eYR L W Q]TX W QUL YFUL Q\[J YX FL eSKL FNP _ bW NdX UQL P SQL P

8 8 36 /

 36 ( pooo087 54/( 2( * >@>f$ 4$ "4 + : 36@B! 6z: p: z* >@>f$ 4$ "4 + C$ "$ 2'28uo= ooo47 7 5 4'"( #8 &4)&"&7 / $ ;)! 4"( &%8k<AH ! $ ( $ 647 7 5& #( 2$"#'!"#8 $ "$ ( $ "8( ) %&( ,$ +<1H l6 11H l($ 7 &( 47 7 8 2 #. $ = $ $ +11H l6 11H >($ 7 &( $ "%7 477 &"#$ $ $ +11H l6 <t ;H l8#( 4( $ "8k<AH ! $ ( $ 6!!'7 &( $ ":q FUX MYP QL P YR L NW L W QR P X FiL R P YP QZ L QdQFP W QJ YP QUL NW L VJ NML UQR X iF_ [QMX h KL P SQL FTOVQW L Nh L VJ NMR L P W X YJ R L NW L Q\[QW X OQFP YJ L TFX P R L [QW L R QR R X NFL YFU NW L R TV¤ QMP L YFUL R [QMX h KL P SQL J QFiP SL Nh L QYMSL P W X YJ L NW L VJ NML X h L P W X YJ R L YW QL VJ NMQU L YFUL X FP QW dYJ L VQP eQQFL P W X YJ R _ P YP QL FTOVQW L YFU NW L P K[QL Nh L dYW X YVJ QR L W QMNW UQUL Q_ i_ L MNW W QMP L VTP P NFL [W QR R L W QR [NFR QL P X OQ L YFUL eSYP L R P YP X R P X MR L eQW QL TR QUL P NL QR P YVJ X R SL P SYP L P SQL R TV¤ QMP R L eQW QL [QW h NW OXFiL P SQL P YR L YR L Q\[QMP QUL Q_ i_ L OQYF L W YFiQ L YFU NW L R P YFUYW UL UQdX YP X NFL YMW NR R

  QMP L X FL P QW ORL Nh L P SQL P YR L NW L R P X OTJ TR L MNFUX P X NFR L X FR P QYUL Nh L [R KMSNJ NiX MYJ L MNFMQ[P R L YFUL X FUX MYP QL eSQP SQW L I®L NW L h YMP NW X YJ L UQR X iFR L eQW QL TR QU_ [QMX h KL dN\QJ eX R QL NW L MJ TR P QW eX R QL YFUL W Q[NW P L YJ J L W QJ QdYFP L [YW YOQP QW R L h NW L MJ TR P QW eX R QL OQP SNUR _ QR MW X VQL P SQL P K[QL Nh L MNW W QMP X NFL YFUL SNeL X P L X R L NVP YX FQUL h NW L OTJ P X [J QL MNO[YW X R NFR L Q_ i_ L a § L a L [QW OTP YP X NFL NW L ^NFP QL YW J N _ Q[NW P L P SQL OQYR TW QR L Nh L UQ[QFUQFMQL TR QUL YFUL P SQL ONUQJ L UQP YX J R L Q_ i_ L bQYW R NFL MNW W QJ YP X NF L [YW P X YJ L MNW W QJ YP X NF L OTP TYJ L X Fh NW OYP X NF _ Q[NW P L P SQL UQ[QFUQFP L dYW X YVJ QL YFUL MNFFQMP X dX P KL OQYR TW Q

Fig. 1 acdefgj

 1 Fig. 1 Impaired Xist upregulation in Spen -/-ESCs upon monolayer differentiation.

Fig. 2 c

 2 Fig. 2 Spen cDNA expression in Spen -/-ESCs rescues Xist expression.

(

  which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Fig. 3 befFig. 4 ac

 34 Fig. 3 SPEN co-localizes with the Xi chromosome very early during monolayer differentiation. Two different states are detectable: early and late SPEN accumulation on the Xi. a SPEN-GFP IF staining (αGFP, green) at different time points of monolayer differentiation (day 0, 1, 2, 3, 5, 7, 10 and 12). White arrowheads mark early SPEN accumulation to the Xi and asterisks late SPEN accumulation. DNA is stained with DAPI (blue). Scale bar: 10 µm.

( 18 d

 18 which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint this version posted December 30, 2020. ; https://doi.org/10.1101/2020.12.30.424676 doi: bioRxiv preprint Percentage of Xist RNA remaining at different time points of actinomycin D treatment (t = 0, 2, 4, 6 and 10 hours) in undifferentiated Wt and Spen -/-ESCs treated with doxycycline. Normalized relative expression to t=0 ± SD, n=2 biological replicates.

Fig. 5 acde

 5 Fig. 5 Tsix regulatory region shows SPEN, HDAC3 and H3K27ac enrichment. SPEN is required to silence Tsix, to allow Xist upregulation upon XCI initiation.

(

  which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint this version posted December 30, 2020. ; https://doi.org/10.1101/2020.12.30.424676 doi: bioRxiv preprintProtein extraction and western blotTo prepare nuclear extracts all procedures were done at 4 o C and buffers supplemented with 1x protease inhibitors (Roche, 4693132001), 15 µM MG-132 (Sigma-Aldrich, C2211) and 0.5 mM DTT. Cells were harvested by scraping in cold PBS, collected and centrifuged (1500 rpm, 5 min, 4 o C). The pellet was incubated in 5x times the pellet volume of Buffer A (10 mM Hepes pH 7.6, 1.5 mM MgCl2, 10 mM KCl) for 10 min, vortexed (30 sec) and centrifuged (3000 rpm, 5 min, 4 o C). Then, the pellet was resuspended in 1,5x times Buffer C (20 mM Hepes pH 7.6, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA) and rotated (30 min, 4 o C). The solution was centrifuged (14000 rpm, 10 min, 4 o C) and the supernatant collected as nuclear extract. The protein concentration was measured using Nanodrop and all samples diluted to the same concentration using Buffer C. NuPAGE™ LDS Sample Buffer (4X) (Thermo Scientific, NP0007) containing 5% b-mercaptoethanol were added to nuclear extracts, and boiled (95 o C, 5 min). For western blot analysis, the NuPAGE™ 3 to 8% Tris-Acetate gels (Invitrogen, EA03755) were used with Tris-Acetate SDS Running Buffer (pH 8.24) and the HiMark Pre-Stained Protein Standard (Invitrogen, LC5699).Wet transfer on a PVDF membrane was done overnight at 4 mA, with the NuPAGE Transfer Buffer (Invitrogen #NP00061), containing 10% methanol and 0.01% SDS. After blocking, the membrane was incubated with the appropriate antibodies: SPEN antibody (Abcam, ab290, 1:2000) and VCP antibody (Abcam, ab11433, 1:20000).

Fluorescent

  labelling was done with dUTP SpectrumRed using the nick translation kit (Abbott, 07J00-001) overnight at 16 o C. The probe was purified with ProbeQuant G-50 Micro Columns (GE, GE28-9034-08), combined with 100 µg mouse tRNA, 20 µg mouse Cot-1 DNA and 100 µg salmon sperm DNA and precipitated with 2 M NaAc (pH 5.6) and 100% EtOH. The pellet was resuspended in 50 µL hybridization mix (50% formamide, 10% dextran sulphate in 2xSSC) and stored at -20 o C. Before use, 20 uL of probe was pre-hybridized (10 min, 75 o C) with 0.5 µg mouse Cot-1 DNA supplemented with 10 mM vanadyl ribonucleoside complex (VRC) (NEB, S1402S) and 0.2 U µL -1 RNAseOUT (Invitrogen, 10777019).

  37 o C. 2.5 mL Glycine 2.5 M were added to the cells (final concentration 0.125 M) to quench the PFA, 5 min RT on a rotator. All buffers from now on contain protease inhibitors (Roche, 4693132001). Cells were washed twice with cold PBS. Then 1x in 10 mL Buffer 1 (10 mM Hepes pH 7.5, 10 mM EDTA, 0.5 mM EGTA, 0.75% Triton X-100) and 1x in Buffer 2 (10 mM Hepes pH 7.5, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA), 10 min rotating at 4 o C. Nuclei were then resuspended in Lysis/Sonication Buffer (150 mM NaCl, 25 mM Tris-HCl pH 7.5, 5 mM EDTA, 1% Triton, 0.1% SDS, 0.5% Sodium deoxycholate) and incubated 30 min on ice. Nuclei were then sonicated 2x 15 min (30'' ON/OFF, max input, ice cold water) in a Bioruptor. A small fraction of the lysate was run on a gel to confirm size population of 100-500 bp. 1 µg of antibody (Cell Signaling, 9733S) was conjugated with 25 µL magnetic beads (Life Technologies, 10004D) for 3h rotating at 4 o C. 25 ug of chromatin was IP'd with the Ab-beads overnight rotating at 4 o C. Beads were washed 2x in standard RIPA buffer (140 mM NaCl, 10 mM Tris-HCl pH 7.5150 mM NaCl, 1 mM EDTA pH8.0, 0.5 mM EGTA pH 8.0, 1% Triton, 0.1% SDS, 0.5% Sodium deoxycholate), 1x in High Salt RIPA (same as standard RIPA but with 500 mM NaCl), 1x LiCl RIPA (same as standard RIPA but with 250 mM LiCl instead of NaCl)and rinsed once with TE, 10 min 4 o C each wash. Chromatin was eluted with 450 µL of Elution Buffer (1% SDS; 0.1M NaHCO3 in H2O) with 22 µl protease K (10 mg mL -1 ) and 5ul RNAse A (10 mg mL -1 ) and shaken

(

  which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint this version posted December 30, 2020. ; https://doi.org/10.1101/2020.12.30.424676 doi: bioRxiv preprint 28 at 1000 rpm for 2 hours at 37 o C first and then 65 o C overnight. DNA was then Phenol-Chloroform extracted and resuspended in 20 µL H20. 0.25 µL was used per PCR to confirm the ChIP's success. The concentration was then measured and a ChIP-seq library was prepared following the manufacturer's instructions (ThruPLEX DNA, Takara Bio) and sequenced on an Illumina HiSeq 2500 sequencer (50 bp paired-end reads).

( 1 Spen -/- 2 Fig. 1 (Fig. 3 (

 1213 Fig.1

(

  which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint this version posted December 30, 2020. ; https://doi.org/10.1101/2020.12.30.

  t❤❛t ✐ts ❢✉♥❝t✐♦♥ ❤❛s ❜❡❡♥ ❝♦♥s❡r✈❡❞ t❤r♦✉❣❤♦✉t ❡✈♦•✉t✐♦♥ ❬❙á♥❝❤❡③✲P✉•✐❞♦ ❡t ❛•✳✱ ✷✵✵✹(✳

	RRM1	RRM2-4	RID RBPID	SPOC
	SPEN/SHARP 3664aa -402kDa					H.sapiens
	SPEN/MINT 3644aa -398kDa					M.musculus
		100%	98%		71%	100%	99%
	SPEN 3583aa -398kDa					G.gallus
		100%	98%	52%	96%	95%
	Spen 3476aa -385kDa				D.rerio
		97%	93%		87%	87%
	Spen 4101aa -455kDa					B.floridae
	87%	61%				65%
	Split-ends 5560aa -600kDa					D.melanogaster
	58%					58%
			DIN-1L 2407aa -385kDa		C.elegans
				20%	25%	30%

TABLE d

 d CONTACT FOR REAGENT AND RESOURCE SHARING d EXPERIMENTAL MODEL AND SUBJECT DETAILSSupplemental Information includes seven figures and four tables and can be found with this article online at https://doi.org/10.1016/j.cell.2018.11.041.

	STAR+METHODS
	KEY RESOURCES TABLE
	REAGENT or RESOURCE	SOURCE	IDENTIFIER
	Antibodies
	anti-LaminB1	Abcam	Cat#ab16048
	anti-HDAC3	Santa Cruz	Cat#sc-376957 X
	Anti-H3K4me3	Merck-Millipore	Cat#05-1339
	anti-H3K4me1	Cell Signaling	Cat#5326S
	anti-H3K27ac	Active Motif	Cat#39685
	anti-H3K27me3	Cell Signaling	Cat#9733S
	anti-H3K9ac	MBL Lifescience	Cat#MABI0305
	anti-pentaH4ac	Merck-Millipore	Cat#06-946
	anti-H2AK119Ub1	Cell Signaling	Cat#8240S
	anti-NCOR1	Abcam	Cat#ab3482
	anti-NCOR2	Abcam	Cat#ab5802
	anti-FLAG	Sigma-Aldrich	Cat#F1804-50UG
	Chemicals, Peptides, and Recombinant Proteins
	RGFP966	Abcam	Cat#ab144819
	BRD6688	Bertin Pharma	Cat#19836.1 mg
	EZ-Link HPDP-Biotin	ThermoFisher Scientific	Cat#21341
	4-Thiouridine	Carbosynth	Cat#NT06186
	Tasquinimod Tubastatin A	Bertin Pharma Bertin Pharma	Cat#17692 H3K9ac, H3K4me3, H3K4me1) Cat#10559	H3K27ac, H4ac,
	PCI-34051	Bertin Pharma	B Analysis Cat#T6325 of	repressive	marks	(H3K27me3,
	LMK235	Bertin Pharma	H2AK119Ub) Cat#14969
	Trypsin/LysC Mix, Mass Spec Grad	Promega	B TT-seq Cat#v5071
	Critical Commercial Assays PyroMark Gold Q24 Ovation Ultralow System V2 1-16	QIAGEN Nugen Technologies	B Analysis of WT and Hdac3 À/À B Analysis of FLAG ChIP-seq on Hdac3 Flag/Flag ESC Cat#970802 cell line Cat#0344-32
	Ovation Universal RNA-Seq System	Nugen Technologies	Cat#7102-08
	Ovation Mouse RNA-Seq System 1-16	Nugen Technologies	Cat#348
	Nick Translation Kit	Roche	Cat#10976776001
	MODified Histone Peptide Array	Active Motif	Cat#13005
	TruSeq Stranded Total RNA Library	Illumina	Cat# RS-122-2201
	Prep Kit with Ribo-Zero Human/Mouse/Rat
	mMACS Streptavidin Kit	Miltenyi Biotec	Cat# 130-074-101
	miRNeasy Micro kit	QIAGEN	Cat# 217084
	MEGAscript T7 Transcription Kit	ThermoFisher Scientific	Cat# AM1333
	Deposited Data
	TT-seq; RNA-seq; ChIP-seq	This study	GSE116480
	IP-MS	This study	PXD011344
	Experimental Models: Cell Lines
	mouse: ESC TX1072	Heard Lab	(Schulz et al., 2014)
	mouse: ESC TX1072-Hdac3À/À#1	Heard Lab	this study
	mouse: ESC TX1072-Hdac3À/À#2	Heard Lab	this study
	mouse: ESC TX1072-Hdac3Flag/Flag	Heard Lab	this study

B Cell Lines d METHOD DETAILS B Doxycycline and inhibitor treatment B Native ChIP-seq B EpiLC induction B RNA FISH B IF with RNA FISH B RNA extraction, reverse transcription, pyrosequencing, qPCR B RNA-seq B TT-seq B CRISPR/Cas9 knockout of Hdac3 B CRISPR/Cas9 FLAG tagging of Hdac3 B HDAC3-FLAG IP-Mass Spectrometry B Anti-FLAG ChIP-seq d QUANTIFICATION AND STATISTICAL ANALYSIS B Time course analysis on TX1072 cell line B Analysis of active histone marks (B Analysis of TXY:Xist and TXY:XistDA B Proteomics data analysis d DATA AND SOFTWARE AVAILABILITY SUPPLEMENTAL INFORMATION (Continued on next page) e1 Cell 176, 182-197.e1-e9, January 10, 2019

2,3,8 , Julia Roensch 2 , Samuel Collombet 1 , Agnès Le Saux 2 , Tomasz Chelmicki 2 , Mikaël Attia 2 , Varun Kapoor 2 , Ye Zhan 4 , Florent Dingli 5 , Damarys Loew 5 , Thomas Mercher 6 , Job Dekker 4,7 & Edith Heard 1,2 *

  Average of input-normalized m 6 A signal intensities along the IAPEz consensus sequence in wild-type (red) and Mettl3 knockout (blue) cells. Vertical black lines denote RRACH motif positions. Discontinued vertical black lines delineate regions of m 6 A enrichment present in wild-type cells and lost in Mettl3-knockout cells. c, Average of m 6 A signal intensities for indicated IAPEz segments in wild-type (red) and Mettl3-knockout (blue) ES cells. Error bars indicate mean ± s.d. of three independent MeRIP-seq. d, Box plot showing log 2 FC in expression of uniquely mapped IAPEz copies grouped according to numbers of 5′ UTR-associated RRACH motifs in Mettl3-knockout versus wild-type cells. Box plots as in Fig. 2d.Only copies with a minimum of ten reads in at least one sample were conserved.

	a Number of m 6 A peaks	15,000 10,000 5,000 0	c Retrotransposon Others Intron 5′UTR Exon 3′UTR log 2 (m 6 -1 2 1 0 A IP/input)	IAPEz	WT	Mettl3 KO	d	3 -1 0 2 2 FC(Mettl3 KO/WT) log 1	31 21 19
	b		4	WT Mettl3 KO		5′ LTR 5′ UTR gag/pro pol 3′ UTR 3′ LTR		RRACH motifs 7 -8 9 -1 0 1 1 -1 8
	A IP/input)		2							WT Mettl3 KO
	log 2 (m 6	0
	-2
										RRACH
			LTR UTR	gag/pro		pol			LTR
			0		2,000	4,000			6,000	7,096
						IAPEz consensus sequence (bp)
	Fig. 3 | IAP mRNAs undergo m 6 A methylation. a, Bar plot showing m 6 A peaks
	within indicated genomic features. b,

  Wtap-and Zc3h13-knockout ES cells grown in serum-free 2i + LIF medium, or after 24 h conversion to serum + LIF medium. Scale bar, 0.5 mm. When replacing 2i + LIF with serum + LIF medium, marked morphological changes were observed in all knockout lines. Experiments were repeated twice with similar results. g, Growth curves of wild-type, Mettl3-, Mettl14-, Wtap-and Zc3h13-knockout ES cells cultured in serum-free 2i + LIF medium, or after conversion to serum + LIF. Data are mean ± s.d. from three independent experiments. When replacing 2i + LIF with serum + LIF medium, the self-renewal ability of Wtap-knockout cells was severely impaired and

	Mettl3-, Mettl14-,
	2, 3, Extended Data
	Figs. 3e-g, 4-7. Western blots for clones indicated in red were repeated at least
	twice with similar results. e, RNA-seq heat maps showing expression of
	selected pluripotency and post-implantation markers in wild-type, Mettl3-
	knockout, Mettl14-knockout, Wtap-knockout and Zc3h13-knockout ES cells.
	Asterisks indicate genes with log 2 FC > 0.75 and FDR < 0.05. Data represent
	averages of two independent RNA-seq. f, Representative images of wild-type,

6 A methyltransferase complex results in deregulation of different retrotransposon families. a, ELISA

  showing normalized m 6 A levels in total RNA in WT and KO ES cells. Data are mean ± s.d.

	(non-DE) retrotransposons. P values were computed using limma and adjusted
	with the Benjamini-Hochberg correction (Methods). c-f, Volcano plots
	showing log 2 FC of retrotransposon expression in Mettl3-, Mettl14-, Wtap-and

of three independent RNA samples. P values were determined by two-sided Student's t-test. b, Volcano plot showing log 2 FC in retrotransposon expression in Mettl14, Wtap and Zc3h13-knockout versus wild-type cells using a random assignment of multi-mapped reads. In red, blue, orange and green are significantly deregulated RepeatMasker annotations belonging to ERVK, ERV1, ERVL and L1 families, respectively. In grey, non-differentially expressed Zc3h13-knockout versus wild-type ES cells using a random assignment of multi-mapped reads. In red, blue, orange and green are significantly deregulated RepeatMasker annotations belonging to ERVK (c), ERV1 (d), ERVL (e) and L1 (f) families, respectively (log FC > 0.75 and FDR < 0.05). In grey, non-differentially expressed retrotransposons. The P values were computed using limma and adjusted with the Benjamini-Hochberg correction.

Analysis of gene expression in mutant ES cells of the m 6 A methyltransferase complex. a, Venn

  Genes with log 2 FC > 0.75 and FDR < 0.05 are marked by an asterisk.

	a
			upregulated genes (n = 941)
			random genes (1000 × randomizations)
		800			P = 0.001	P = 0.017
	c	chr3:34,649,890-34,652,975	1kb P = 1	chr4:55,524,481-55,535,128	1kb	chr6:122,707,144-122,714,854	1kb
	381 2000 MMERVK10C consensus sequence 99 236 1e+05 130 input IP 1e+05 P = 0.6703 P = 0.003 P = 0.1998 Sox2 0 (bp) 1e+06 1e+06 0 -2 2 4 log2 (m 6 A IP/input) Extended Data Fig. 6 | Klf4 5'UTR	R S IN E o th e r D N A 169 318 680 687 P = 0.0919 P = 0.1019 4000	6000	8000	1e+05 1e+05 Nanog RRACH

diagram showing overlap of the upregulated genes (FDR < 0.05, log 2 FC > 0.75) in Mettl3-, Mettl14-, Wtap-and Zc3h13-knockout ES cells as identified by RNA-seq. In blue, genes (n = 941) commonly upregulated in all four knockout ES cells. b, Correlation between gene upregulation and proximity to retrotransposon annotations (-5 kb to +1 kb from the TSS). In blue, genes (n = 941) commonly upregulated in all four knockout ES cell lines. In grey, random genes plotted as mean + s.d. of n = 1,000 randomizations. P values were determined by permutation test. c, Dot plot showing splicing events (normalized counts per million) occurring between exons and RepeatMasker-annotated IAPs in wild-type, Mettl3-, Mettl14-, Wtapand Zc3h13-knockout ES cells. Horizontal lines denote mean (n = 2 independent RNA-seq). d, Heat map showing average expression of selected retrotransposon regulators in wild-type, Mettl3-, Mettl14-, Wtap-and Zc3h13-knockout cells.

Article Extended Data Fig. 7 | IAP mRNAs undergo METTL3-dependent m 6 A methylation. a,

  Left, Venn diagram showing overlap between m6 A peaks identified in wild-type and Mettl3-knockout MeRIP-seq. Right, inputnormalized cumulative distribution of signal intensity for m6 A peaks in wildtype (top), overlapping (middle) and Mettl3-knockout specific (bottom) cells. A set of novel m 6 A peaks was detected in Mettl3-knockout cells; however, they are probably false positives (weak signal intensity compared to canonical wildtype peaks). b, Normalized MeRIP-seq read density in wild-type (red) and Mettl3-knockout (blue) cells across the 5′ UTR, coding sequence and 3′ UTR of mRNA for the genes with at least one m6 A peak. Discontinued lines represent respective inputs. c, UCSC genome browser tracks showing m 6 A distribution (red, normalized read density, RPM) at indicated genes in wild-type and Mettl3knockout ES cells. In black, input RNA. Results represent average signal of three independent MeRIP-seq experiments. d, Inputs (top) and backgroundnormalized m 6 A signal distributions (bottom) across the IAPEz consensus sequence in wild-type (red) and Mettl3-knockout (blue) cells. Vertical black lines denote positions of RRACH motifs. Discontinued vertical black lines denote regions of m 6 A enrichment present in wild-type and lost in Mettl3-knockout cells. e, Average of input-normalized m 6 A signal intensities along the MMERVK10C consensus sequence in wild-type (red) and Mettl3-knockout (blue) cells. Vertical black lines denote RRACH motif positions. Discontinued vertical black lines denote region of m 6 A enrichment present in wild-type and lost in Mettl3-knockout cells. f, Average of m 6 A signal intensities for indicated MMERVK10C sequence segments in wild-type (red) and Mettl3-knockout (red) ES cells. Data are mean ± s.d. of three independent MeRIP-seq experiments. Box plot showing log 2 FC in expression of uniquely mapped IAPEz copies grouped according to number of 5′ UTR-associated RRACH motifs in Mettl14-, Wtap-and Zc3h13-knockout versus wild-type cells. Box plots are as in Fig. 2d. Only copies with a minimum of 10 reads in at least one sample were conserved.

	g, h, Background-normalized m 6 A signal distribution (g) and average of input-
	normalized m 6 A signal intensities across the L1MdA_I consensus sequence in
	wild-type (red) and Mettl3-knockout (blue) cells (h). Vertical black lines denote
	RRACH motif positions. i, Alignment between 5′ UTRs of IAPEz and
	MMERVK10C consensus sequences. IAP-specific, MMERVK10C-specific and
	common RRACH motifs are indicated in blue, yellow and green, respectively.
	j,

8 | Auxin-inducible degron of endogenous METTL3, METTL14 and ZC3H13. a,

  Schematic of TIR1 and SCF1 complex-dependent degradation of endogenously AID-tagged proteins in presence of auxin. b, Immunoblot showing protein levels of endogenously 3×Flag-AID-tagged METTL3 and METTL14 in single and double degron ES cell lines. TIR1-only ES cells were used as control for protein levels. PCNA served as loading control. .d. of three independent auxin inductions). f, Growth curves of TIR1only and AID-METTL3;METTL14 double degron ES cells treated with either DMSO (grey) or auxin (black) for 0-96 h. Data are mean ± s.d. of three independent auxin inductions. Note that contrary to constitutive m 6 Aknockout ES cells, proliferation rate is not altered by acute m6 A loss (similar rate between AID-METTL3;-METTL14 double degron and TIR1-only ES cells).

	c, Immunoblot showing efficiency and reversibility of METTL3 and METTL14	mean ± sHowever, prolonged auxin treatment may have negative effect on the
	depletion after 0-96 h auxin treatment followed by 48 h auxin wash-off. PCNA	proliferation rate. g-i, ZC3H13 auxin-dependent degron. Schematic of ZC3H13
	served as loading control (related to Fig. 4b). d, ELISA showing normalized m 6 A	degron engineering in mouse ES cells (g), immunoblot showing protein levels
	levels in total RNA after 0, 6 and 96 h of auxin-induced degradation of METTL3,	of endogenously 3×Flag-AID-tagged ZC3H13 (h) and degron efficiency in
	METTL14 and METTL3;METTL14 double degron. Data are mean ± s.d. of three	presence of auxin (i). TIR1-only ES cells were used as control for protein levels.
	technical replicates. TIR1-only ES cells served as control. Experiment was	KAP1 served as loading control. j, RT-qPCR showing normalized IAP mRNA
	repeated twice with similar results. e, RT-qPCR showing normalized IAP mRNA	levels relative to 0 h using LTR-specific primers after auxin-induced
	levels relative to 0 h using Δ1-or gag-specific primers after auxin-induced	degradation of ZC3H13. Data are mean ± s.d. from three independent auxin
	degradation of METTL14 (light blue), METTL3 (blue) and METTL3;METTL14	inductions. Immunoblots presented in b, c, h, i were repeated at least twice
	double degron (dark blue). TIR1-only (black) ES cells served as control. Data are	with similar results.

Article Extended Data Fig. 9 | Retrotransposon chromatin profiling in AID- METTL3;METTL14 double degron ES cells using random assignment of multi-mapped reads on full-length elements. a,

  Composite profiles (top) and density plots (bottom) showing enrichment of H3K4me3, H3K27ac and H3K9me3 as determined by CUT&RUN on m 6 A-bound and non-m 6 A bound genes (±2 kb) (gene lists were obtained based on MeRIP-seq results) in AID-METTL3;METTL14 double degron ES cells treated with DMSO or auxin for 96 h. Violin plots and box plots (inside) showing H3K4me3, H3K27ac and H3K9me3 levels at L1MdA_I-IV copies (n = 436 copies) in AID-METTL3;METTL14 double degron ES cells treated with DMSO (grey) or auxin (red) for 96 h. Only copies with a minimum of 10 reads in at least one sample were conserved. Box plots are as in Fig. 2d. P values were determined by two-sided Student's t-test.

		d, Composite profiles showing H3K4me3 (top), H3K27ac (middle) and
		H3K9me3 (bottom) coverage along full-length (>5 kb) L1MdA_I copies
	b, Heat map showing average H3K27ac, H3K4me3 and H3K9me3 levels	(n = 1,400) in AID-METTL3;METTL14 double degron ES cells treated with DMSO
	(log 2 (immunoprecipitate/IgG)) for 56 retrotransposon families in AID-	(control, grey) or auxin (red) for 96 h. Results in a-d represent averages of two
	METTL3;METTL14 double degron ES cells treated with DMSO or auxin for 96 h.	independent CUT&RUN.

c,

  YW P X MX [YFP R L eQW QL FNP L YJ J NMYP QUL X FP NL Q\[QW X OQFP YJ L iW NT[R L R P YP QL R NL IL UQR MW X VQL SNeL [YW P X MX [YFP R L eQW QL YJ J NMYP QUL P NL iW NT[R L YFUL X h L YJ J NMYP X NFL eYR L FNP L W YFUNOL UQR MW X VQL SNeL MNdYW X YP QR L eQW QL MNFP W NJ J QU_

	h L [
	'"4$ "%#&( &5& 47 7 4( #' $ "%t 7 7 '2$"&l$ 1mpuoo&"#t 7 7 '2$"&36&1mvoooA7 &( 8 2 :;)mA05& ! 8 2#"&w$ $ > x0&7 H ;$ 2yA01/ ( 2* ;) 2<$ )14$ "( $ 8 $ 4+ :<j ( ".7 ( =22. &" 5 4&""#' $ "%&)2$G4CA* 6: +* f$ 0&#+ :;) <>1&"&7 / $ 5& ! 8 2#' $ "%36/( pooo087 54/( 2( &"#36@B! 18 ( 5& * 6z: p: z+* >@>f$ 4$ "4 + :
	C>k{* 6o: u: |+ =>( ! * 6z: z: zv+ =1;>0*6p: x: o&+ =}"4#( 8 $ "#( )2&7 7* 6z: o+ =8 &( ' '"( * 6z: u: z+ =0*6n: u: o+ =#%0*6n: pp: n+ = 7 $ 22&* 6n: n|: n+ = %$ "0*6z: z~: o+ =; $ 2k&7 * 6o: ~: ~+ =f5( $ * 6z: p+ =#!;7 * 6p: u: n+ =C>1p* 6p: z: z+ =f5( $ p* 6p: p: + =1;>0 * 6p: v: o4+ =01>;* 6+ =A$ 4& #* 6: p: v: o+ =; '2!(* 6o: n: v+ =;&!1( &( $ ""( 7 7 18 ( 5& * 6n: p+ =36@B! * 6z: p: z+ =A $ 2* 6|: n: o+
	>7 7#&( &%" &( #$ "( )$ ( '#/& &6&$ 7 &.7 "k@}#&( &.& '"#( )"'2.k1@z~uvzv C( )# "D & t "67 6#$ "( ) ( '#/ )t AH m <7 54/( 2( / C0t H .& #"' $ 2&%$ "%

  TUX FiL eSNL W QMNW UQUL P SQL UYP YL YFUL SNe_ FUX MYP QL P SQL R P YW P L YFUL R P N[L UYP QR L Nh L UYP YL MNJ J QMP X NF L FNP X FiL P SQL h W Q]TQFMKL YFUL [QW X NUX MX P KL Nh L R YO[J X FiL YFUL [W NdX UX FiL YL W YP X NFYJ QL h NW L P SQR QL MSNX MQR _ L h L P SQW QL X R L YL iY[L VQP eQQFL MNJ J QMP X NFL [QW X NUR L R P YP QL P SQL UYP QR L h NW L QYMSL R YO[J QL MNSNW P _ L [QMX h KL P SQL R [YP X YJ L R MYJ QL h WNOLeSX MSL P SQL UYP YL YW QL P YQF h L FNL UYP YL eQW QL Q\MJ TUQUL h W NOLP SQL YFYJ KR QR L R P YP QL R NL IL X h L UYP YL eQW QL Q\MJ TUQU L UQR MW X VQL P SQL Q\MJ TR X NFR L YFUL P SQL W YP X NFYJ QL VQSX FUL P SQOL X FUX MYP X FiL eSQP SQW L Q\MJ TR X NFL MW X P QW X YL eQW QL [W Q QR P YVJ X R SQU_ QR MW X VQL P SQL OQYR TW QR L P YQFL P NL dQW X h KL P SQL W Q[W NUTMX VX J X P KL Nh L Q\[QW X OQFP YJ L h X FUX FiR _ L aNW L QYMSL Q\[QW X OQFP L FNP QL eSQP SQW L YFKL YP P QO[P R L P NL W Q[QYP L P SQL Q\[QW X OQFP L h YX J QUL IL R P YP QL P SYP L YJ J L YP P QO[

  (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint this version posted December 30, 2020. ; https://doi.org/10.1101/2020.12.30.424676 doi: bioRxiv preprint

	Fig. 6
										X-linked
	Nap1l2	Linx	Tsx Xite	Tsix	Xist	Jpx Ftx	Slc16a2	Rnf12	genes
	bioRxiv preprint Ch 4:	doi:					.424676
	Exon: 1	2 3				4 5 6	7 8 9 10 11	12	13 14 15 16
		Tsix				Xist
	XCI initiation	Tsix Tsix Tsix	SPEN SPEN	Xist Xist Xist		X-linked gene X-linked gene silencing	SPEN SPEN	Xist stability	establishment of Xi	Spreading and

Cell 176, 182-197, January 10, 2019 ª 2018 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Cell 176, 182-197, January 10, 2019 183

Cell 176, 182-197, January 10, 2019

Cell 176, 182-197, January 10, 2019

independent experiments and two Hdac3 À/À clones for 3 genes sensitive to HDAC3 loss (top) and 3 genes less dependent on its activity (bottom). *p < 0.05, Student's t test. (F and G) Validation of Hdac3 À/À phenotype in day 3 EpiLCs differentiated without DOX. (F) FISH was performed with probes for Xist (p510, red) and an X-linked gene, Huwe1 (green). Scale bar, 5 mm. (G) Also shown is quantification of Huwe1 silencing status in more than 100 nuclei with Xist clouds. c 2 test, *p < 0.05. (H) Screening for HDAC activity compensating for HDAC3 loss using pyrosequencing for AtrX, G6pdX, and Rnf12. The experiment was performed like in (A) but in Hdac3 À/À cells. Shown are averages (± SD) from two independent clones. *p < 0.05, Student's t test. See also FigureS3and TableS1.188 Cell 176, 182-197, January 10, 2019

Cell 176, 182-197, January 10, 2019

Cell 176, 182-197, January 10, 2019

e3 Cell 176, 182-197.e1-e9, January 10, 2019

Cell 176, 182-197.e1-e9, January 10, 2019 e4

Cell 176, 182-197.e1-e9, January 10, 2019 e6

Cell 176, 182-197.e1-e9, January 10, 2019 e8

© The Author(s), under exclusive licence to Springer Nature Limited 2021

ACKNOWLEDGMENTS

We are grateful to Magdalena Zernicka-Goetz and Elizabeth Blackburn for their continuing support of this project. We are thankful to Samuel Collombet, Tomasz Chelmicki, Pierre Gestraud, Aurelie Teissandier, Nicolas Servant, and Emmanuel Barillot for help with the project and members of the Heard lab for helpful discussions. We are grateful to Anton Wutz for sharing his TXY ESC line. We also thank the tissue-imaging platform-PICT-IBiSA (UMR3215/ U934) and the next-generation sequencing platform of Institut Curie. This work was funded by ERC advanced investigator awards (ERC-ADG-2014 671027 to E.H. and ERC-ADG-2015 693023 to P.C.), Labellisation La Ligue, ANR (DoseX 2017: ANR-17-CE12-0029, Labex DEEP: ANR-11-LBX-0044, ABS4NGS: ANR-11-BINF-0001, and part of the IDEX PSL: ANR-10-IDEX-0001-02 PSL to E.H.), a Sir Henry Wellcome Postdoctoral Fellowship (201369/Z/16/Z; to J.J.Z.), Volkswagen Foundation funding (to P.C.), and Cell 176, 182-197, January 10, 2019 195 Acknowledgements We thank K. Ancelin for support throughout the project and help with in vivo experiments; J. Barau, D. Holoch and R. Margueron for support and help with the project; M. Carrara for critical reading of the manuscript; and members of the Heard laboratory for discussions. We thank E. Nora for sharing the OsTIR1 and AID-targeting plasmids; T. Honjo for sharing the Spen flox mouse line; O. Masui for sharing the Xist-Bgl stem-loop targeting constructs and L. Lavis for sharing Halo-JF646 and JF549 with us. We also thank the imaging platform (A. Dauphin and PICT-IBiSA (UMR3215/U934)) and the protein purification and sequencing platforms of Institut Curie as well as L. Villacorta, J. Provaznik and V. Benes of GeneCore at EMBL. This work was funded by a Boehringer Ingelheim doctoral fellowship (20017-2019 to F. Dossin), an ERC Advanced Investigator award (ERC-ADG-2014 671027 to E.H.), Labellisation La Ligue (to E.H.), ANR (DoseX 2017: ANR-17-CE12-0029, Labex DEEP: ANR-11-LBX-0044, ABS4NGS: ANR-11-BINF-0001, and part of the IDEX PSL: ANR-10-IDEX-0001-02 PSL to E.H.), a Sir Henry Wellcome Postdoctoral Fellowship (201369/Z/16/Z to J.J.Z.), 'Région Ile-de-France' and Fondation pour la Recherche Médicale grants (to D.L.), EMBO long-term fellowships (ALTF 549-2014 to I.P., ALTF 301-2015 to T.C.), and Fondation pour la Recherche Medicale (SPF 20140129387 to I.P.) and NIH (HG003143 to J.D.) grants. J.D. is an investigator of the Howard Hughes Medical Institute. Acknowledgements We thank the members of the Bourc'his laboratory for their support; M. Greenberg and A. Shkumatava for critical reading of the manuscript; G. Cristofari and J. J. Zylicz for suggestions; I. Pinheiro for help with FACS analysis; E. Nora for targeting plasmids; and M. Schultz and M. Borensztein for assistance with cell culture and immunofluorescence experiments. We acknowledge the ICGex NGS platform of the Institut Curie -supported by grants ANR-10-EQPX-03 (Equipex) and ANR-10-INBS-09-08 (France Génomique) from the Agence Nationale de la Recherche-and the Cell and Tissue Imaging Platform (PICT-IBiSA) of Institut Curie -member of the French National Research Infrastructure France-BioImaging (ANR-10-INBS-04). The laboratory of D.B. is part of the LABEX DEEP (ANR-11-LABX-0044, ANR-10-IDEX-0001-02). This work was supported by the Fondation Bettencourt Schueller, the Association Robert Debré pour la Recherche Médicale (ARDRM), the Fondation pour la Recherche Médicale (FRM) and the Association de Recherche contre le Cancer (ARC-PJA-20191209637). T.C. was a recipient of an EMBO postdoctoral fellowship; E.R. and M.D were supported by PhD fellowships from la Ligue contre le Cancer and from Région Ile-de-France, respectively. Acknowledgements We thank Anniek Meesters and Esther Sleddens-Linkels for technical help; Kristian Helin for providing the EZH2 antibody; Martine M. Jaegle for sharing with us a floxed-puromycin resistance cassette and all the members of the Erasmus MC Developmental Biology department for useful discussions. T.R.F. is supported by an Erasmus MC grant (Mrace). B.F.T., H.M.B., E.T. and J.G. are supported by the Oncode Institute. T.B. is supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) -TRR81-A12 and BO 1639/9-1, the Behring-Röntgen foundation and Excellence Cluster for Cardio Pulmonary System (ECCPS) in Giessen. B.D.G. is supported by a research grant of the University Medical Center Giessen and Marburg (UKGM) and by a Prize of the Justus Liebig University Giessen".

Software and Algorithms Bowtie2 (2.2.5) (Langmead and Salzberg, 2012) http://bowtie-bio.sourceforge.net/bowtie2/index.shtml TrimGalore (v0.4.0) Felix Krueger lab https://github.com/FelixKrueger/TrimGalore Cutadapt (1.8.2) (Martin, 2011) https://github.com/marcelm/cutadapt SNPSplit (0.3.2) (Krueger and Andrews, 2016) http://www.bioinformatics.babraham.ac.uk/projects/ SNPsplit/ Bedtools (2.25.0) (Quinlan and Hall, 2010) http://bedtools.readthedocs.io/en/latest MACS2 (2.0.10) (Zhang et al., 2008) https://github.com/taoliu/MACS/ Subread -featureCounts (1.5.1) (Liao et al., 2014) http://subread.sourceforge.net/ DeepTools (3.0.2) (Ramı ´rez et al., 2014) http://deeptools.ie-freiburg.mpg.de STAR (2.3.0) (Dobin and Gingeras, 2015) https://github.com/alexdobin/STAR Samtools (1.3) (Li et al., 2009) https://github.com/samtools/samtools Tophat (2.1.0) (Trapnell et al., 2009) https://github.com/infphilo/tophat R (3.4.0) R Core Team http://www.R-project.org/ edgeR (R package) (Robinson et al., 2010) https://bioconductor.org/packages/release/bioc/html/ edgeR.html Limma (R package) (Ritchie et al., 2015) http://bioconductor.org/packages/release/bioc/html/ limma.html drc (R package) (Ritz et al., 2015) https://cran.r-project.org/web/packages/drc/index.html Proteome Discoverer (v 2.2) ThermoFisher Scientific N/A myProMS (Poullet et al., 2007) N/A Bowtie2 (2.2.5) (Langmead and Salzberg, 2012) http://bowtie-bio.sourceforge.net/bowtie2/index.shtml TrimGalore (v0.4.0) Felix Krueger lab https://github.com/FelixKrueger/TrimGalore Data availability RNA-seq, Hi-C and CUT&RUN data used in this study have been deposited in the Gene Expression Omnibus under accession number GSE131784. Source Data for Figs. 1-3 and Extended Data Figs. 1, 3, 4 are provided with the paper, either in the form of supplementary tables or source data files.

Data availability

The raw imaging data that support the findings of this study are available from the corresponding authors on request owing to size considerations. All sequencing data derived from CRISPR-Cas9 screens I and II, RNA-seq, MeRIP-seq and CUT&RUN have been deposited in the Gene Expression Omnibus (GEO) under accession number GSE145616. Previously published data were downloaded from GEO: Mettl3 control and Mettl3 knockout nuclear RNA-seq: (GSE133585) 19 . Source data are provided with this paper.

C( )#7 %/ 0!7 $ 4&( 1'! 1 $ ,k1@z~uvzv2v>2( )/7 &( $ " %'7 &( ( )8 &( 8"#%"' ( 6$ ' ( &" 4 $ !( : 1'.1 $ , k1@z~uno 03>H m , k1C~nz~u|oj;@10!z k1C~nz~u|zj;@10!p k1C~nz~u|pC( ( 7 n{}@10!z k1C~nz~u|nC( ( 7 n{}@10!p k1C~nz~u|~C( ( 7 z~{}@10!z k1C~nz~u|uC( ( 7 z~{}@10!p k1C~nz~u|vj;>A{}@10!z k1C~nz~u|xj;>A{}@10!p k1C~nz~u||s4n)zn{}@10!z k1C~nz~u|s4n)zn{}@10!p k1@z~unzu 0t AH m , k1C~nz~vuvt "!'( j; k1C~nz~vuxt "!'( {} k1C~nz~vu|2v>j; !z k1C~nz~vu2v>j; !p k1C~nz~vvo2v>j; !n k1C~nz~vvz2v>Cn{} !z k1C~nz~vvp2v>Cn{} !p k1C~nz~vvn2v>Cn{} !n k1@z~uvzu 0t 1A0D & , k1C~nppp~|14 "t mz!' k1C~nppp~14 "t mpH .7 & ( k1C~npppuo14 "t mn.7 & ( uG k1C~npppuz14 "t m~.7 & ( xG k1C~npppup14 "t mu.7 & ( G k1C~npppun14 "t t & 7 /H .7 & ( !z k1C~npppu~14 "t t & 7 /H .7 & ( !p k1C~npppuu14 "t t & 7 /.7 & ( !z k1C~npppuv14 "t t & 7 /.7 & ( !p k1C~npppux14 "t t 7 &( H .7 & ( !z k1C~npppu|14 "t t 7 &( H .7 & ( !p k1C~npppu14 "t t 7 &( .7 & ( !z k1C~npppvo14 "t t 7 &( .7 & ( !p k1@zuvoox r;?0r3

k1C~xz|||t "!'(>t GH C@;;-n, H C@;;-z~##* "t >>+ k1C~xz||ot "!'(>t GH C@;;-n, H C@;;-z~##* t >>+ k1C~xz||zt A, ln{~2n>t GH C@;;-n, H C@;;-z~##* "t >>+0!z

Author contributions F. Dossin and E.H. conceived the experiments and F. Dossin performed them unless stated otherwise. T.M. provided the Spen-knockout mice and J.J.Z. performed the embryo experiments. E.H. and I.P. supervised the work. J.R. established the Xist stem-loop cell line and acquired live-cell images. F. Dossin, J.R. and V.K. analysed live-cell imaging experiments. V.K. wrote the image analysis software. F. Dossin

Competing interests

The authors declare no competing interests.

Additional information

Supplementary information is available for this paper at https://doi.org/10.1038/s41586-020-

Reporting Summary

Nature Research wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Research policies, see Authors & Referees and the Editorial Policy Checklist.

Statistics

For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.

n/a Confirmed

The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly

The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.

A description of all covariates tested

A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code

Policy information about availability of computer code

Data collection

All microscopy images were acquired either with an Inverted Confocal Spinning Disk Roper/Nikon for fixed cells or a Super-resolution microscope OMX (Applied Precision Incorporation, DeltaVision) for live cells. Sequencing data was collected using the Illumina platform. Pyrosequencing data was collected using the PyroMark Q24 System from Qiagen. QPCR was performed on a ViiA 7 Real-Time PCR System from Thermo Fisher Scientific. Mass Spectrometry was performed on Thermo Scientific Orbitrap Fusion Tribrid MS from Thermo Fisher Scientific. Western blot (chemiluminescent) images were collected using a ChemiDoc MP from BioRad.

Data analysis

Trimgalore (v 0.4.4), STAR (2.5.3a), SNPsplit (v 0.3.2), picard (v2.18.2), DESeq2 (v1.18.1), R (3.4.1), featureCount (1.6.3), Fiji (2.0.0), dplyr (0.7.4), readr (1.1.1), tidyr (0.8.0), ggplot2 (2.2.1), macs2 (v 2-2.1.2.1), Subread (v1.28.1), HiC-Pro (v2.11.0) For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data Policy information about availability of data

All manuscripts must include a data availability statement. This statement should provide the following information, where applicable:

-Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability

All data generated in this study are available on GEO database under the number GSE131784.

nature research | reporting summary

October 2018

Field-specific reporting

Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences

For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design

All studies must disclose on these points even when the disclosure is negative.

Sample size

Sample size was not predetermined. For mouse experiments, we used a sample size commonly used and accepted for basic statistical inference while using an justifiable number of mice. For tissue culture based experiments, at least two independent clones were systematically assessed for each genotype. Typically, for the SPOC tethering experiment, 4 independent clones were characterized with very little variation being observed between them.

Data exclusions No data were excluded from the analysis, with the exception of live imaging analysis, from which dying cells, as well as cells which moved outside from the field of view during movie acquisition were removed.

Replication

All attempts at replication were successful and noted in the relevant figure legend.

Randomization Samples were not randomized, given that samples were grouped according to their respective genotypes.

Blinding

For most experiments, no blinding was performed as most measurements were derived from third party machines/softwares, and hence not affacted by subjective interpretation. For RNA FISH image analysis (counting of Xist RNA clouds and X-linked gene pinpoints), each experimental condition was blinded from the first author F. Dossin during counting.

Validation

The HaloTag antibody is validated for western blot in Fig. 1b, with specific signal disappearing upon auxin treatment, and reappearing upon removal of auxin from the culture medium. The V5 antibody is validated for Western blot in Extended Data Fig. 1a, with specific signal being observed only in cells expressing Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Article

Transposon element-based analysis was performed using only uniquely mapped reads by retrieving reads with NH tag equal to 1. To avoid confounding effects between expression from retrotransposon copies and from genes, elements overlapping genes in the same direction were removed from RepeatMasker annotations. FeatureCounts v.1.5.1 was used for the quantification and only copies with at least 10 reads in at least one sample were conserved. RRACH motifs were searched into the 5′ UTR sequences of individual IAP copies using RSAT dna pattern. Division into three categories of RRACH motif number (7-8, 9-10 and 11-18) was performed to homogenize the number of IAP copies per category.

To estimate intron retention between genes and single IAP copies, reads alignment was performed using specific parameters to report only uniquely mapped reads with STAR v.2.7.0a 36 (-outFilterMultimap-Nmax 1-outSAMmultNmax 1). Unannotated splice junctions detected by STAR was annotated with GENCODE v.18 and IAP LTR elements from RepeatMasker annotation to retrieve splicing events between a gene and an IAP element. The number of uniquely mapped reads crossing the splicing events was calculated for each sample and normalized by the library size.

MeRIP-seq analysis

Due to the addition of 3 nucleotides on 5′-end of the second sequencing read (R2) from the Pico v.2 SMART Adaptor, paired-end reads were trimmed using Trim Galore v.0.4.4 with the options: -three_prime_clip_ R1 3-clip_R2 3 (http://www.bioinformatics.babraham.ac.uk/projects/ trim_galore/). Reads were aligned onto the mouse ribosomal sequence (GenBank: BK000964.3) using Bowtie v.1.2 allowing at most three mismatches 42 . Previously unmapped reads were aligned onto the mouse reference genome (mm10) using STAR v.2.6.0c reporting randomly one position, allowing 4% of mismatches (-outFilterMultimapNmax 5000-outSAMmultNmax 1-outFilterMismatchNmax 999-outFilter-MismatchNoverLmax 0.04). PCR duplicates were removed using STAR with the option -bamRemoveDuplicatesType UniqueIdenticalNot-Multi. Bigwig files were produced with deepTools v.2.5.3 43 using the option -normalizeUsingRPKM. Peaks enriched in the MeRIP sample over the input control were defined using MACS2 peak-caller 44 with a genome size of 994,080,837 bp 20 and the FDR threshold of 5%. Reads were extended to 200-bp-long fragments. Only peaks called in at least two replicates were used for downstream analysis. Peaks intensity was calculated using FeatureCounts v.1.5.1 38 and normalized to background (reads not falling into peaks) and to peak length. GENCODE v.18 was used to define 5′UTR, 3′UTR, intronic and exonic regions. Retrotransposon annotations (RepeatMasker) were downloaded from UCSC table browser. Genes overlapping with at least one peak were used to calculate coverage along the genic region (5′UTR, coding sequence and 3′UTR) with trumpet R package 45 . Mapped reads onto the Mouse reference genome overlapping with IAP and L1MdA_I elements were extracted as single-end reads and mapped to the full-length IAP (GenBank: M17551.1) and L1MdA_I consensus sequences with Bowtie2 v.2.2.9 [START_REF] Donohoe | The pluripotency factor Oct4 interacts with Ctcf and also controls X-chromosome pairing and counting[END_REF] with these parameters: -local -N 1. Coverage along the consensus sequence was normalized to background (reads not falling into peaks) as was done previously for peak intensities. Rolling mean was calculated for a window of 50 bp to smooth the signal. RRACH motif was searched into the IAP consensus sequence using RSAT dna pattern. Intragenic peaks in the wild-type condition were used to define m 6 A-bound genes. In addition, a threshold of 1 RPKM in the input wild-type samples was put to be sure that the gene is expressed. Genes with RPKM higher than 1 and not overlapping with a m 6 A peak were defined as non-m 6 A bound genes.

CUT&RUN analysis

Paired-end reads were trimmed using Trim Galore v0.4.4. The alignment was performed onto a concatenated genome using the mouse reference genome (mm10) and the Escherichia coli genome (str. K-12 substr. MG1655, GenBank: NC_000913) with STAR v.2.7.0a 36 reporting randomly one position, allowing 6% of mismatches (-outFilterMulti-mapNmax 5000-outSAMmultNmax 1-outFilterMismatchNmax 999-outFilterMismatchNoverLmax 0.06). PCR duplicates were removed using Picard v.2.6.0 (http://broadinstitute.github.io/picard/). Reconstructed transposon annotation was used as input for quantification with FeatureCounts v.1.5.1 38 using reads mapped onto the mouse genome. Normalization of counts per million was performed using as library size the number of mapped reads onto the mouse genome.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.

Author contributions

Competing interests

The authors declare no competing interests.

Additional information Supplementary information

The online version contains supplementary material available at https://doi.org/10.1038/s41586-020-03135-1.

Correspondence and requests for materials should be addressed to T.C. or D.B. Peer review information Nature thanks Miguel Branco and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer review reports are available. Reprints and permissions information is available at http://www.nature.com/reprints. helped review and edit the manuscript. J.W.M.M. and J.G. were responsible for the founding acquisition.

RNA-seq and ChIP-seq datasets were generated in the Erasmus MC Center for Biomics led by W.v.IJ.

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

g Quantification of (f), showing the percentage of nuclei with Xist clouds. Average percentage ± SD, n=2 biological replicates, 150-500 nuclei quantified per replicate. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Supplementary Fig. 3 Spen cDNA rescue ESC lines characterization. |Related to Fig. 2. a Schematic overview of the generation of Spen -/-ESC lines able to stably express the Spen cDNA from the ROSA26 locus. These lines were made using a sgRNA targeting the ROSA26 locus and a vector coding for the Spen cDNA and a hygromycin resistance cassette (HygroR) 25 .

b Strategy to identify correct Spen cDNA rescue clones by PCR on gDNA. Specific 3' Spen cDNA integration in the ROSA26 locus. Primer on the Flag-tag present in the 5' Spen cDNA end to identify those clones containing the Spen cDNA vector. Specific 5' and 3' integration of the PuroR cassette in the Spen locus to identify the Spen -/-line. RFLP analysis on Spen exon 13 to determine the presence of Spen in the ROSA26 locus and its absence in the Spen -/-cells; the Spen rescue cDNA sequence is C57BL/6 (B6). Verification of the presence of two X chromosomes per ESC line, making use of a length polymorphism on the X chromosome. M = DNA ladder. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Methods

Cell culture

Mouse ESCs were grown on male feeder cells and medium containing DMEM (Gibco), 15% Foetal Calf Serum (FCS), 0.1 mM non-essential amino acids (NEAA), 100 U mL -1 penicillin, 100 ug mL -1 streptomycin, 0.1 mM 2-mercaptoethanol (Gibco) and 1000 U mL -1 LIF. Previous to monolayer differentiation cells were plated in non-gelatinized plates to eliminate feeder cells. Then, plated at specific densities per time point in differentiation medium composed of IMDM-glutamax (Gibco), 15% FCS, 0.1 mM NEAA, 100 U mL -1 penicillin, 100 µg mL -1 streptomycin, 37.8 µL L -1 monothioglicerol and 50 mg mL -1 ascorbic acid. When appropriate, medium was supplemented with 2 μg mL -1 doxycycline.

Gene editing using the CRISPR/Cas9 technology Different female F1 2-1 hybrid (129/Sv-Cast/Ei) ESC lines with different genetic modifications to interrogate various aspects of XCI were targeted in this study (Supplementary Table 2). To generate Spen heterozygous and homozygous knockout clones two single-guide RNAs (sgRNA) targeting the 5' (5'-AGTGCGCTTCGTCACTGCAC-3') and 3' (5'-TCCTCCCGCCCCGACGCGGA-3') region of the Spen ORF were cloned in the Cas9-GFP pX458 vector (Addgene plasmid #48138). Compatible 5' and 3' Homology arms (HA) of approximately 500 bp were amplified by PCR from mouse gDNA and cloned in the pCR-Blunt-II-TOPO vector with a NdeI restriction site in-between the 5' and 3' HA. This site was used to insert a Puromycin resistance (PuroR) cassette flanked by loxP sites. The ROSA26 locus was targeted using the pX458 vector coding for a sgRNA (5'-CGCCCATCTTCTAGAAAGAC-3') compatible with the pFD46 expression vector 25 , coding for the Spen cDNA and a hygromycin resistance cassette. To create an endogenous Spen C-terminal enhanced GFP (eGFP) knock-in, a sgRNA targeting the 3' end of Spen ORF (5'-GATTGTCATTGCCTCGGTG-3') was cloned in the Cas9-PuroR pX459 vector (Addgene plasmid #62988). The donor template was made using a gblock from Integrated DNA Technologies coding for compatible 5' and 3' HA of 600 bp with a NheI and AscI restrictions sites in-between the 5' and 3' HA, which were used to insert an eGFP in frame with the Spen coding sequence. The appropriate plasmid combinations were transfected into ESCs using lipofectamine 2000 and plated at low density to obtain single colonies, when appropriate medium was supplemented with 1 µg mL -1 puromycin (Sigma-Aldrich, P8833) for 48-72 hours or 250 μg mL -1 hygromycin B (Invitrogen, 10687010) for 7 days. Colonies were screened by PCR for correct integration of the desired construct (Supplementary Table 3). Positive clones were further characterized by western blot, RT-qPCR and/or FACS, and the presence of 2 X chromosomes and correct karyotype was also assessed.

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

day 0 day 5 day 0 day 5

Tsix-Stop Tsix-Cherry

% allelic expression

M

Supplementary Fig. 6 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted December 30, 2020. ; https://doi.org/10.1101/2020.12.30.424676 doi: bioRxiv preprint

ABSTRACT

In female placental mammals, dosage compensation of X-linked gene expression is achieved early during development through transcriptional inactivation of one of the two X chromosomes (XCI). This process is dependent on Xist, a long non-coding RNA which coats and silences the X chromosome from which it is transcribed. The mechanisms through which Xist initiates transcriptional silencing during XCI remain however completely unknown. In 2015, several studies identified that the SPEN protein binds Xist RNA directly, and its implication in mediating gene silencing was reported. However, its precise function and mechanism(s) of action during XCI are unclear.

During my PhD, I made use of a conditional loss of function approach, the auxin inducible degron, to acutely deplete SPEN in mouse embryonic stem cells (mESCs) undergoing XCI.

Using this approach, I demonstrate that SPEN is absolutely necessary for chromosome-wide Xist-mediated gene silencing during initiation of XCI. Furthermore, using conditional Spen KO mouse embryos, I show that SPEN is also required for the transcriptional inactivation of the paternal X chromosome during imprinted X inactivation. Depleting SPEN in differentiated cells, in which XCI has been established, reveals that SPEN is neither required to maintain gene silencing nor to preserve the spatial organization of the inactive X chromosome.

By combining fixed and live cell imaging of Xist and SPEN, I show that SPEN colocalizes with Xist RNA, and accumulates on the X chromosome, immediately upon Xist upregulation, suggesting that SPEN can initiate gene silencing very early on during XCI. Profiling SPEN chromatin binding sites reveals that SPEN is recruited to promoters and enhancers of active genes specifically. The magnitude of SPEN recruitment to X-linked promoters dictates the efficiency with which these genes will be silenced. Remarkably, SPEN disengages from chromatin after gene silencing, indicating that active transcription required for SPEN's association with chromatin.

Using a functional complementation approach, I identify the SPOC domain as the effector of SPEN's gene silencing activity during XCI. Artificial tethering of SPOC to Xist RNA results in transcriptional repression along the entire X chromosome, demonstrating that SPOC contains all the sufficient potential to instruct gene silencing during XCI. I further characterize the protein interactors of SPOC using mass spectrometry and reveal that SPOC interacts with several protein complexes involved in repressing transcription, including the NCoR/SMRT (histone deacetylation), the NuRD (nucleosome remodeling/histone deacetylation) and the m6A writing (governing mRNA fate) complexes. Finally, several transcription initiation and elongation factors are found to interact with SPOC, as well as the RNA polymerase II (RNAPII) transcription machinery. I identify that SPOC interacts directly and specifically with the C-terminal domain (CTD) of RNAPII only when the latter is phosphorylated on Ser5, and determine the 3D structure of the SPOC/RNAPII-CTD Ser5-P complex at 1.8Å resolution. These results suggest that SPEN could directly repress transcription during XCI by interfering with RNAPII-CTD Ser5-P templated processes.

Altogether, my PhD work reveals that SPEN is essential for chromosome-wide transcriptional silencing during XCI, both in mESCs and in vivo. Following Xist upregulation, SPEN is immediately recruited to active gene promoters and enhancers, silences transcription, and subsequently disengages from chromatin. Through its RRMs and SPOC domains, SPEN acts as a molecular integrator, bridging Xist with histone deacetylases, nucleosome remodelers, RNA methyltransferases and most importantly, the transcription machinery. KEYWORDS epigenetics -X chromosome inactivation, SPEN/SPOC, functional genomics, structural biology, mouse embryonic stem cells,