Antoine Hocher

Myriam Ruault

Petra Kaferle

Marc Descrimes

Mickaël Garnier

Antonin Morillon

Angela Taddei

Mickael Garnier

Expanding heterochromatin reveals discrete subtelomeric domains delimited by chromatin landscape transitions

Introduction

Heterochromatin classically designates chromosomal domains that remain condensed throughout the cell cycle (Emil Heitz 1928). In contrast to gene specific repressors, heterochromatin silences genes independently of their DNA sequence [START_REF] Talbert | Spreading of silent chromatin: inaction at a distance[END_REF]. Heterochromatin is prevalent in eukaryotic genomes and is key to processes including gene dosage compensation, cell differentiation, speciation and genome stability [START_REF] Grewal | Heterochromatin revisited[END_REF].

Telomeres and a portion of subtelomeres are associated with heterochromatin in many species [START_REF] Louis | Subtelomeres[END_REF]. Subtelomeres are genomic domains that are particularly difficult to define. While they often exhibit structural and functional properties, such as the presence of specific gene families, chromatin marks or a relatively fast gene turnover, there is no strict definition that segregates all these properties between subtelomeres and the core genome [START_REF] Louis | Subtelomeres[END_REF].

Transcriptional silencing generally initiates at defined loci and propagates by selfrecruitment mechanisms [START_REF] Grunstein | Molecular model for telomeric heterochromatin in yeast[END_REF][START_REF] Hoppe | Steps in assembly of silent chromatin in yeast: Sir3-independent binding of a Sir2/Sir4 complex to silencers and role for Sir2-dependent deacetylation[END_REF][START_REF] Grewal | Heterochromatin revisited[END_REF][START_REF] Gartenberg | The nuts and bolts of transcriptionally silent chromatin in Saccharomyces cerevisiae[END_REF]. The coupling of histone modifying enzymes to nucleosomes allows the specific binding of silencing effectors and drives the formation of heterochromatin domains [START_REF] Richards | Epigenetic codes for heterochromatin formation and silencing: Rounding up the usual suspects[END_REF][START_REF] Wang | New Insights into the Regulation of Heterochromatin[END_REF]. However, the spread of heterochromatin must be limited to prevent encroaching on euchromatin [START_REF] Donze | Braking the silence: How heterochromatic gene repression is stopped in its tracks[END_REF].

In budding yeast, the silent information regulator (SIR) proteins, Sir2 Sir3 and Sir4, implement stable repression at the two cryptic mating type loci (HML and HMR) and semistable repression of genes near telomeres [START_REF] Gartenberg | The nuts and bolts of transcriptionally silent chromatin in Saccharomyces cerevisiae[END_REF][START_REF] Grunstein | Epigenetics in Saccharomyces cerevisiae[END_REF][START_REF] Aparicio | Modifiers of position effect are shared between telomeric and silent mating-type loci in S. cerevisiae[END_REF][START_REF] Moazed | Silent information regulator protein complexes in Saccharomyces cerevisiae: a SIR2/SIR4 complex and evidence for a regulatory domain in SIR4 that inhibits its interaction with SIR3[END_REF][START_REF] Rine | Four genes responsible for a position effect on expression from HML and HMR in Saccharomyces cerevisiae[END_REF][START_REF] Rudner | A nonhistone protein-protein interaction required for assembly of the SIR complex and silent chromatin[END_REF][START_REF] Rusche | The establishment, inheritance, and function of silenced chromatin in Saccharomyces cerevisiae[END_REF]. The SIR complex is recruited at these loci by a combination of specific DNA binding proteins that have functions outside silencing. At telomeres, the Repressor activator Rap1 binds the degenerated telomeric sequence TG 1-3 [START_REF] Shampay | DNA sequences of telomeres maintained in yeast[END_REF], and recruits the SIR complex through direct interaction with Sir3 and Sir4. This recruitment is reinforced by additional interactions between Sir4 and the Ku heterodimers [START_REF] Tsukamoto | Silencing factors participate in DNA repair and recombination in Saccharomyces cerevisiae[END_REF][START_REF] Roy | Separation-of-function mutants of yeast Ku80 reveal a Yku80p-Sir4p interaction involved in telomeric silencing[END_REF]) Once nucleated, the activity of Sir2, a conserved histone deacetylase, creates favorable binding sites for Sir3, which preferentially binds deacetylated H4K16. Iterative cycles of Sir2 mediated histone deacetylation and Sir3 binding allow the self-propagation of the SIR complex on chromatin until a barrier is eventually reached [START_REF] Grunstein | Epigenetics in Saccharomyces cerevisiae[END_REF][START_REF] Gartenberg | The nuts and bolts of transcriptionally silent chromatin in Saccharomyces cerevisiae[END_REF].

Boundaries restrict silent domains at the cryptic mating type loci [START_REF] Donze | The boundaries of the silenced HMR domain in Saccharomyces cerevisiae[END_REF][START_REF] Donze | RNA polymerase III and RNA polymerase II promoter complexes are heterochromatin barriers in Saccharomyces cerevisiae[END_REF]. A tRNA gene confines the Sir complex to HMR [START_REF] Donze | The boundaries of the silenced HMR domain in Saccharomyces cerevisiae[END_REF] while directional nucleation restricts silencing at HML [START_REF] Bi | The yeast HML I silencer defines a heterochromatin domain boundary by directional establishment of silencing[END_REF]. About half of subtelomeres have a Y' middle repeat isolated from SIR spreading by the transcription factor Reb1. At these subtelomeres, adjacent internal TG repeats associated with the middle repetitive sequence core X can act as relays of silent chromatin propagation [START_REF] Pryde | Limitations of silencing at native yeast telomeres[END_REF]Fourel et al, 2002: Thurtle andRine 2014;[START_REF] Ellahi | The chromatin and transcriptional landscape of native saccharomyces cerevisiae telomeres and subtelomeric domains[END_REF]. Beyond these last nucleation sites, the spread of silencing is rather limited ranging from hundreds of base pairs to few kb and no boundary has been identified so far. However, Sir3 was recently shown to expend inward chromosomes in G1 arrested cells [START_REF] Mitsumori | Analysis of novel Sir3 binding regions in Saccharomyces cerevisiae[END_REF]. Although numerous factors such as nuclear pore complex components and transcription factors display barrier properties in boundary assays, their physiological role in vivo remains to be explored [START_REF] Oki | Barrier proteins remodel and modify chromatin to restrict silenced domains[END_REF]). The collective action of chromatin modifying enzymes implements chromatin states that potentially decrease Sir3 affinity for nucleosomes. In addition to the acetylation of H4K16 by the SAS-I complex, acetylation of histone H3 tails by Gcn5 and Elp3, methylation of H3K4 and H3K79, and H4K16ac-dependent incorporation of the H2A.Z histone variant were all proposed to contribute to limit Sir3 spreading at subtelomeres [START_REF] Gartenberg | The nuts and bolts of transcriptionally silent chromatin in Saccharomyces cerevisiae[END_REF]. In mutants lacking those enzymes or marks the SIR complex propagates further away from the telomeres [START_REF] Suka | Sir2p and Sas2p opposingly regulate acetylation of yeast histone H4 lysine16 and spreading of heterochromatin[END_REF][START_REF] Sperling | Histone H3 N-terminus regulates higher order structure of yeast heterochromatin[END_REF][START_REF] Kristjuhan | Spreading of Sir3 protein in cells with severe histone H3 hypoacetylation[END_REF][START_REF] Meneghini | Conserved histone variant H2A.Z protects euchromatin from the ectopic spread of silent heterochromatin[END_REF]. However, the respective contribution of each mechanism to heterochromatin restriction and what further limits the spread of silencing in those mutants remains unknown.

A key parameter regulating heterochromatin dynamics, function and spatial distribution is the concentration of silencing factors. For instance, increasing Sir3 dosage in budding yeast expands subtelomeric silent domains toward the chromosome core (Renauld et al. 1993a) and increases telomere clustering (Ruault et al. 2011). However, extension of silencing domains was monitored at few subtelomeres and the dose-dependency of heterochromatin propagation remains qualitative (Renauld et al. 1993a;[START_REF] Hecht | Spreading of transcriptional repressor SIR3 from telomeric heterochromatin[END_REF][START_REF] Strahl-Bolsinger | SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin in yeast[END_REF][START_REF] Katan-Khaykovich | Heterochromatin formation involves changes in histone modifications over multiple cell generations[END_REF]. Here we examine the impact of expressing the silencing factor Sir3 at varying levels genome-wide.

Results

Saturation of extended silent domains upon SIR3 overexpression

To systematically examine the impact of elevated Sir3 on the genome, we generated yeast strains that overexpress SIR3 at stable, different and non-overlapping levels. We replaced the endogenous SIR3 promoter with three different promoters, generating strains that produced 9ൈ-Sir3 (pscADH1-SIR3, hereafter denominated "pADH-SIR3"), 16ൈ-Sir3 (pscTEF1-SIR3, "pTEF-SIR3"), and 29ൈ-Sir3 (pscTDH3-SIR3, "pGPD-SIR3") as determined by Western blot (Fig S1A), and fluorescence quantification of live cells expressing Sir3-GFP (Fig 1A,Fig S1B). FACS profiles of wild-type and pGPD-SIR3 strains were largely similar, suggesting that the cell cycle was unaffected by overexpression of SIR3 (Fig S1C).

We probed genome-wide Sir3 binding by ChIP-chip using our antibody raised against the fulllength native (untagged) protein (Ruault et al. 2011).We obtained a high signal to noise ratio (above 300) in the vicinity of known nucleation sites, namely TG repeats, ARS consensus sequence of the X-core elements (ACS) and the two cryptic mating type loci, in agreement with previous studies [START_REF] Ellahi | The chromatin and transcriptional landscape of native saccharomyces cerevisiae telomeres and subtelomeric domains[END_REF][START_REF] Teytelman | Highly expressed loci are vulnerable to misleading ChIP localization of multiple unrelated proteins[END_REF][START_REF] Radman-Livaja | Dynamics of Sir3 spreading in budding yeast: secondary recruitment sites and euchromatic localization[END_REF][START_REF] Sperling | Histone H3 N-terminus regulates higher order structure of yeast heterochromatin[END_REF]. To rule out potentially confounding effects due to well documented ChIP artifacts, we compared the binding of Sir3 within subtelomeres to its binding at highly transcribed genes, a known source of artefactual signal [START_REF] Teytelman | Highly expressed loci are vulnerable to misleading ChIP localization of multiple unrelated proteins[END_REF][START_REF] Park | Widespread misinterpretable ChIP-seq bias in yeast[END_REF][START_REF] Kasinathan | High-resolution mapping of transcription factor binding sites on native chromatin[END_REF]. Finally, we compared Sir3 binding to that of the chromatin binding deficient mutant Sir3-A2Q as an additional control. In both cases, the signature of hyper-chipable loci was negligible compared to Sir3 binding signal at subtelomeres (Fig S1D ,S1F), providing confidence in the integrity of our data. On average in wild-type cells, we detected Sir3 binding up to 2,6 kb away from the last telomeric element (ACS), with some weaker signal at sites previously reported as secondary nucleation sites, consistent with previous studies [START_REF] Lieb | Promoter-specific binding of Rap1 revealed by genome-wide maps of protein-DNA association[END_REF][START_REF] Sperling | Histone H3 N-terminus regulates higher order structure of yeast heterochromatin[END_REF][START_REF] Ellahi | The chromatin and transcriptional landscape of native saccharomyces cerevisiae telomeres and subtelomeric domains[END_REF][START_REF] Radman-Livaja | Dynamics of Sir3 spreading in budding yeast: secondary recruitment sites and euchromatic localization[END_REF]. Elevation of Sir3 levels expanded the distance of Sir3 binding to ~12 kb in 9ൈSir3 cells and up to ~19 kb in 16ൈ or 29ൈSir3 cells (Fig 1B). Sir3 spreading was similar in 16ൈ and 29ൈSir3 cells. Sir3-GFP nuclear background levels almost doubled in 29ൈSir3 compared to 16ൈSir3 cells (Fig S1E), arguing that Sir3 binding to chromatin reached saturation. The constitutive overexpression of Sir3 submerged most of the secondary recruitment sites that were present in wild-type cells leading to the formation of extended continuous Sir3 bound domains at most subtelomeres. We observed that Sir3 binding increased at few euchromatic (non subtelomeric) sites upon Sir3 overexpression such as YAT1 or YFR017W (Fig S1F and table S1) but chose not to pursue this further as in most cases changes in Sir3 binding were not associated with changes in gene expression in agreement with previous reports [START_REF] Marchfelder | SIR-dependent repression of non-telomeric genes in Saccharomyces cerevisiae?[END_REF]. In parallel, we monitored telomere foci in function of Sir3 concentration by live microscopy imaging of Rap1-GFP (Fig 1C -E). In wild type cells, telomeres cluster together in 3 to 5 foci located at the nuclear periphery [START_REF] Gotta | The clustering of telomeres and colocalization with Rap1, Sir3, and Sir4 proteins in wild-type Saccharomyces cerevisiae[END_REF]. However, upon Sir3 overexpression using the Gal1p promoter, most of the telomeres group together in the center of the nucleus (Ruault et al. 2011a). In the range of Sir3 concentration probed, we observed that telomere clustering increased non-linearly as a function of Sir3 levels and this reached saturation at levels between 9-16ൈSir3. Most cells (78%) had at least 3 Rap1-GFP foci in the WT strain while 64-76% of cell had one or two foci in strains overexpressing Sir3 9-fold or more (Fig 1D). Increased foci intensity paralleled the decrease in foci number (Fig 1E,Left), consistent with increased telomere grouping in cells overexpressing Sir3. Furthermore, the proportion of nuclear Rap1-GFP within foci increases from 13.6% in WT cells to a maximum of 21.6-22.2 % for Sir3 dosage above Right). Together, this suggests that not all telomeres are clustered within Rap1 foci in WT cells at a given time, and that elevated Sir3 levels increase the total number of telomeres within clusters, a process eventually reaching saturation. Thus, increased Sir3 dosage expands Sir3 genome binding and telomere clustering until they reach saturation between 9-16ൈSir3. However, individual telomeres showed different stereotypical behaviours in response to increased Sir3 dosage. We classified telomeres based on their response to Sir3 dosage elevation (see Methods). One example of each class is displayed on (Fig 1F) and full data is shown on (Fig S1G). "Fragile" subtelomeres (6/26) displayed increased Sir3 spreading and plateaued at 9ൈSir3. "Progressive" subtelomeres (13/26) displayed gradually increased Sir3 spreading between 9-16ൈSir3 and then plateaued at 16ൈSir3. "Resistant" subtelomeres (4/26) displayed increased Sir3 spreading and plateaued at 16ൈSir3. Finally, "insensitive" subtelomeres (3/26) did not expand in response to elevated Sir3 levels. The expanded Sir3 domains showed diverse lengths in all categories, ranging from 7-25 kb (HM excluded), independently of chromosomal arm length or middle repeat content (Fig S1H -I).

Sir3 spreading extends Silent Domains

Overexpression of SIR3 repressed subtelomeric transcription, as expected. Given that overexpression of the point mutant sir3-A2Q, which leads to telomere clustering but only Most genes within ESDs are not highly transcribed in WT cells, suggesting that Sir3 spreading might be limited by transcription. However, highly expressed genes like IRC7 (Fig 2C) and DLD3 (Fig 3B) were not excluded from ESDs and were repressed upon SIR3 overexpression.

Both genes belong to the top 10% of most expressed genes and to the top 20% of most frequently transcribed genes in wild-type cells [START_REF] Pelechano | A complete set of nascent transcription rates for yeast genes[END_REF]. Similarly, at 7 subtelomeres at least one gene within the ESD had higher read density than the gene adjacent to the ESD (Fig 2E). Furthermore, genes found immediately before and after the end of ESDs showed comparable transcript levels (Fig 2F). Therefore, transcriptional activity per se did not appear sufficient to limit Sir3 spreading when Sir3 is over-abundant.

The limitation of Sir3 spreading could be the consequence of the counter selection of cells silencing essential genes as ESDs did not contain any and 3 ESDs ended right before three essential genes. However, we do not favour this hypothesis for two main reasons. First, we did not detect significant decreases in mRNA levels for these genes upon Sir3 overexpression. Second, none of these genes show any haplo-insufficiency phenotype [START_REF] Deutschbauer | Mechanisms of haploinsufficiency revealed by genome-wide profiling in yeast[END_REF] arguing that a weak repression would likely not be counterselected. We thus conclude that extended Sir3 spreading led to efficient gene silencing of the underlying genes and that gene activity did not account for the end of ESDs.

ESDs are not limited by distance from the telomere or by barrier TF elements To test whether the distance from the telomere limits Sir3 spreading, we compared Sir3 spreading at a WT telomere VIIL against a 15 kb truncated version (Fig 3A). In both cases, Sir3 binding ended within the HXK2 promoter, with a somewhat sharper decline rate in the truncated strain. This suggests that the Sir3 spreading boundary is either defined relative to the chromosome core or is a local feature. Focusing on silent domain ends, we quantified the slope of Sir3 binding at each subtelomere in the strains overexpressing SIR3, when sufficient data were available (24/32 subtelomeres). We found that the slope at the end of a silent domain did not correlate with the distance from the telomere (i.e nucleation point) and there was no correlation with the groups defined based on sensitivity to Sir3 dosage (Fig S3A). Thus, when Sir3 is in excess, the delineation of the ESD did not depend on the distance from the nucleation site. To investigate whether DNA sequence-specific barrier elements confine Sir3 ESDs within subtelomeres, we evaluated the available binding data of 10 transcription factors (TF) with proposed barrier activity (Adr1, Gcn4, Rgt1, Hsf1, Sfp1, Reb1, Abf1, Leu3, Swi5: [START_REF] Harbison | Transcriptional regulatory code of a eukaryotic genome[END_REF]), Rap1: (Rhee and Pugh 2012),Tbf1: [START_REF] Preti | The Telomere-Binding Protein Tbf1 Demarcates snoRNA Gene Promoters in Saccharomyces cerevisiae[END_REF]). At 12 subtelomeres, we identified bound TF sites at genes corresponding to the ESD limit (Fig S3C). However, each of these TFs was also bound elsewhere within the ESD (Fig 3B) indicating that they were not sufficient to limit the spreading of Sir3. Only the three subtelomeres categorized as insensitive to Sir3 levels (group 4), contained known barrier elements flanking Sir3 bound domains: a tRNA gene at subtelomere IIL, a previously identified barrier sequence homologous to the left barrier of HML [START_REF] Bi | Domains of gene silencing near the left end of chromosome III in Saccharomyces cerevisiae[END_REF] at the subtelomere XIR and the I silencer at subtelomere IIIL (Fig S3B). Thus, in most cases, none of the previously identified barrier elements that we could probe was sufficient to block Sir3 spreading.

Sir2 does not limit the majority of extended silent domains

We considered that Sir3 spreading might be limited by the capacity of Sir2 to deacetylate H4K16. We first monitored the genome-wide occupancy of Sir3 in strains overexpressing Sir2. We found that Sir2 overexpression had a weaker impact than 9ൈSir3 overexpression (Fig S4A). Sir3 spreading in cells co-overexpressing Sir2 and Sir3 or overexpressing Sir3 alone were identical at most subtelomeres (19/26), as illustrated by their mean ChIP profile (Fig 4A). In the remaining 7 cases Sir3 spreading was increased by co-overexpression of Sir2, slightly extending the average profile of Sir3 binding (Fig S4B). It is noteworthy that the further extended silent domains remained devoid of essential or tRNA genes. Thus, Sir2 activity did not generally limit the spread of heterochromatin, even when Sir3 is in excess.

ESDs encompass known domains of Sir3 extension

We compared how Sir3 bound domains extend upon overexpression to known situations of Sir3 binding extension: Sir3 spreading in H3 tail mutants [START_REF] Sperling | Histone H3 N-terminus regulates higher order structure of yeast heterochromatin[END_REF] and in cells blocked in G1 by alpha-factor treatment [START_REF] Mitsumori | Analysis of novel Sir3 binding regions in Saccharomyces cerevisiae[END_REF]. As shown on Fig 4, ESDs encompass the domains bound by Sir3 in H3 tail mutants or in G1 blocked cells. Although in the H3 tail mutant Sir3 bound domains increased only at half subtelomeres, in these cases Sir3 binding profiles were very similar to those observed upon SIR3 overexpression (Fig 4B and Fig S4B). In contrast, Sir3 binding in G1 blocked cells appeared to cover domains identical to ESDs but with a binding signature qualitatively different, as only a low magnitude binding signal is observed in the extended Sir3 binding domains.

Together, this shows that Sir3 bound domains in G1 blocked cells or in H3∆4-30 are contained within ESDs, although Sir3 is not overexpressed in these conditions. This suggests that the same determinants of Sir3 restriction are at play in all these contexts. Finally, the similarities of Sir3-bound domains in those conditions may indicate that ESDs correspond to chromosomal features that exist independently of Sir3 dosage.

ESDs coincide with a pre-existing chromatin landscape

To identify potential chromatin determinants of ESDs, we analyzed the genome-wide distribution of 27 histone marks or variants [START_REF] Weiner | High-Resolution Chromatin Dynamics during a Yeast Stress Response[END_REF][START_REF] Schulze | Linking Cell Cycle to Histone Modifications: SBF and H2B Monoubiquitination Machinery and Cell-Cycle Regulation of H3K79 Dimethylation[END_REF]). We first computed the correlation between Sir3 binding signals and histone modifications across the first centromere proximal 50 kb flanking X-core elements (Fig 5A). Consistent with previous results, we observed the expected anti-correlation between Sir3 binding and H4K16 acetylation in WT cells with a Pearson correlation coefficient of -0.45 (Fig 5A,and exemplified in Fig 5B). Correlation between Sir3-A2Q binding and histone marks ranged from -0.25 to 0.2, providing a negative control. Generally, in wild-type cells Sir3-bound nucleosomes were depleted of most histone marks, with the exception of H4R3 methylation and H2A phosphorylation, which were enriched within silent domains (Fig S5A), as reported earlier [START_REF] Yu | The role of protein arginine methylation in the formation of silent chromatin[END_REF][START_REF] Szilard | Systematic identification of fragile sites via genomewide location analysis of gamma-H2AX[END_REF]. We observed that Sir3 binding signal was better correlated with several histone marks in all conditions corresponding to extended binding of Sir3 (H3 tail mutants, G1 blocked cells, SIR3 overexpression and SIR2 and SIR3 cooverexpression) than in asynchronous wild-type cells. Namely, Sir3 binding signal correlated better with histone H3 methylation and histone H2A phosphorylation (Fig 5A ,B), the highest correlation values being with Sir3 binding signal in cells co-overexpressing SIR2 and SIR3 (Pearson correlation coefficient of -0.49 with H3K79me3 and 0.72 with H2AP). While Sir3 binding in G1 blocked cells remained negatively correlated with H4K16 acetylation status, this anti-correlation was much weaker in H3 tail mutants and even lower in strains overexpressing SIR3. This suggests that H4K16 acetylation might limit Sir3 binding in G1 blocked cells but not in H3 tail mutants or upon SIR3 overexpression. To investigate a potential link between chromatin and the consequences of SIR3 overexpression, we compared histone marks enrichment at the flanks of wild type silent domains among the fragile, progressive and robust subtelomere groups defined in Fig 1 . We observed that some histones marks are on average differentially enriched in the three groups (Fig S5A). The most pronounced differences (Kruskal-Wallis, p-val < 10 -4 ) concerned H3K79me2 and the acetylation of the H4 tail lysines (K5,8,12 but not K16: ANOVA, p-val = 0.55) that showed higher levels within ESDs of progressive telomeres than at fragile telomeres and even higher levels at robust telomeres. As acetylation of H4K5,8,12 reduces Sir3 affinity for H4 in a cumulative manner [START_REF] Carmen | Acetylation of the yeast histone H4 N terminus regulates its binding to heterochromatin protein SIR3[END_REF], those differences could contribute to the differential spreading observed between those groups of subtelomeres upon overexpression of SIR3. Similarly, Sir3 H3K79me2 could limit Sir3 spreading at progressive and robust telomeres when Sir3 is overexpressed 9-fold but not above. H3K79me, and the histone variant H2A.Z, previously reported as antagonistic to SIR spreading, showed low levels in wild type silent domains similar to the bulk of the genome but were enriched within the ESDs and returned to background levels past ESDs (Fig 5A ,C). This suggested that these chromatin features did not efficiently block Sir3 spreading when Sir3 is over-abundant or in G1 phase.

In contrast, H3K4me3, H3K36me3 and H3K79me3 were only present after the ESD terminus (Fig 5C ,S5B). We reasoned that the longer intergenes present within subtelomeres might bias our analysis, artificially leading to the depletion of marks associated to gene bodies. To control for this potential artifact, we separated promoter nucleosomes (-3, -2, -1) from gene body nucleosomes and obtained essentially the same results (Fig S5C).

Thus, the subtelomeric chromatin landscape exhibits more similarities with Sir3 binding when it is extended than with Sir3 bound region in wild-type asynchronous population.

In a complementary approach, we focused on Sir3 binding signal at the ends of ESDs. We classified each subtelomere according to Sir3 binding signal's area under the curve (AUC), computed on the logistic-like fit of the Sir3 binding signal in ESDs (see Methods). At the ten telomeres showing the highest AUC, some histone marks displayed sharp transitions, particularly H3K79me3 and H2AS129P (Fig 6A). In contrast, the ten subtelomeres with the lowest Sir3 binding AUC at ESD ends showed rather smooth changes both for Sir3 binding and for these marks (Fig 6A and S6A). Thus, ESDs correlated with a pre-existing chromatin landscape defined by specific histone modifications, low levels of H3K79me3 and H3K36me3 and high levels of H2AP.

The methyl transferase Dot1 is essential for viability when SIR3 is overexpressed.

As H3K79 methylation has been shown to impair the binding of Sir3 to histone peptides and to nucleosome in vitro [START_REF] Altaf | Interplay of chromatin modifiers on a short basic patch of histone H4 tail defines the boundary of telomeric heterochromatin[END_REF][START_REF] Wang | Heterochromatin protein Sir3 induces contacts between the amino terminus of histone H4 and nucleosomal DNA[END_REF], it appeared as a good candidate to limit ESDs. To test this hypothesis, we overexpressed SIR3 in this absence of Dot1, the only methyltransferase responsible for H3K79 mono-, di-and tri-methylation. We found that the dot1∆ GPD-SIR3 strains were sick and generated suppressors upon streaking. To avoid artifacts due to these potential escapers, we selected dot1∆ GPD-SIR3 clones in the presence of 5 mM nicotinamide (NAM), which inhibits Sir2 activity and thus silencing [START_REF] Bitterman | Inhibition of silencing and accelerated aging by nicotinamide, a putative negative regulator of yeast Sir2 and human SIRT1[END_REF]. After the selection of positive clones, we assessed the growth of these mutants on medium without NAM, allowing silencing to initiate [START_REF] Osborne | The establishment of gene silencing at single-cell resolution[END_REF]. Above 9-fold overexpression of SIR3, dot1∆ mutants exhibited growth defects that were proportional to Sir3 amounts and rescued by Sir2 inhibition (Fig 6B). In contrast, the H3K4 and H3K36 methyltransferases Set1 and Set2, and the histone deacetylase Rpd3 were all dispensable to maintain cell growth in presence of high Sir3 dosage (Fig S6B). A dot1∆ strain overexpressing the spreading-defective Sir3-A2Q point mutant was viable, further supporting that the requirement for Dot1 is to restrict the spread of Sir3 and not the clustering of telomeres (Fig S6B). Furthermore, co-overexpression of DOT1 and SIR3 led to loss of silencing, showing that H3K79 methylation prevails on Sir3 binding even when SIR3 is overexpressed (Fig S6C).

H3K79 methylation protects euchromatin from the spread of silencing

To identify H3 and H4 histone residues involved in the limitation of Sir3 spreading, we set a genetic screen based on the Synthetic Gene Array [START_REF] Dai | Strain construction and screening methods for a yeast histone H3/H4 mutant library[END_REF]. H3K79R was the sole histone point mutant having growth defects that could be rescued by 5 mM NAM treatment (Fig 6C ,S6D) indicating that H3K79 methylation plays a key role in limiting Sir3 spreading. In contrast, the H4K16R mutant had non-significant growth defects (Fig 6C ,S6E), consistent with a rather subtle influence of H4K16 on the maximal extent of silencing upon SIR3 overexpression.

Next, we observed Sir3 binding events that led to cell lethality in DOT1 deleted mutants overexpressing SIR3. To do so, cells were first grown in the presence of NAM, and then released into fresh medium for 8 hours. Sir3 binding in cells overexpressing SIR3 released from NAM was very similar to what we observed in cells grown in the absence of NAM (Pearson correlation coefficient, 0.9). In contrast, in dot1∆ mutants overexpressing SIR3, Sir3 spread beyond ESDs at several subtelomeres and bound numerous euchromatic sites (Fig 6D ,E). The subtelomeric extension of Sir3 spreading encompassed 4 essential genes as shown for subtelomeres 3L, 3R and 2L (Fig 6D andS6E) likely accounting for the lethality of this strain. In addition, Sir3 overcame the three previously identified barriers including the tRNA gene present at the border of subtelomere 2L that was insensitive to Sir2 and Sir3 overexpression in otherwise wild-type cells (Fig S6E). To define the inhibitory effect of each H3K79 methylation state on Sir3 binding, we compared Sir3 binding in dot1 mutants overexpressing SIR3 to the levels of mono, di and trimethylation of H3K79 deposited by Dot1 in WT cells. We observed that in the absence of Dot1, Sir3 was binding loci that were enriched for H3K79 tri-methylation but depleted for H3K79 mono-and di-methylation in wild type cells (Fig 6F). This suggests that it was the trimethyl state of H3K79 that inhibited Sir3 binding and so prevented heterochromatin formation within euchromatin.

Accordingly, SIR3 overexpression was also lethal in rad6∆ cells (Fig S6F), these lack H3K4me3 and H3K79me3 but not H3K79me1 and 2 [START_REF] Ng | Lysine methylation within the globular domain of histone H3 by Dot1 is important for telomeric silencing and Sir protein association[END_REF][START_REF] Schulze | Linking Cell Cycle to Histone Modifications: SBF and H2B Monoubiquitination Machinery and Cell-Cycle Regulation of H3K79 Dimethylation[END_REF]). We thus conclude that H3K79me1 and me2 could contribute to but are not sufficient for blocking Sir3 binding within euchromatin.

ESDs coincide with discrete domains that segregate subtelomeric features.

We identified discrete subtelomeric domains corresponding to the maximal extension of Sir3 bound domains. We next sought to identify regulators of genes found in these domains by screening a compendium of over 700 mutants [START_REF] Kemmeren | Large-scale genetic perturbations reveal regulatory networks and an abundance of gene-specific repressors[END_REF]. We classified subtelomeric genes into four different groups (1) genes or pseudo-genes associated with middle repeat elements (telomeric), (2) genes bound by Sir3 in WT cells, (3) genes bound by Sir3 only upon SIR3 overexpression and (4) genes bound Sir3 only upon co-overexpression of SIR2 and SIR3. Groups 3 and 4 correspond to ESDs. We also considered the group of genes located within 10 kb from the end of ESDs and located between 10-20 kb from ESD ends as a control.

For each mutant, we tested if the proportion of differentially expressed genes (|log 2 (FC)|>2) within a subtelomeric domain was higher than expected by chance, considering the effect of each mutation on the rest of the genome. We identified genes whose mutation affects specific subtelomeric subdomains (Fig 7A). As expected, deletion of any of the SIR had localized effects within the telomeric and WT Sir3 bound domains.

Our analysis confirmed mutants previously known to affect subtelomeric transcription.

Telomerase components and the nucleoporin NUP170 [START_REF] Van De Vosse | A role for the nucleoporin Nup170p in chromatin structure and gene silencing[END_REF]) up regulates specifically the most telomeric group of genes in our analysis, while the mediator complex tails proteins Med2, Med15 and Gal11 [START_REF] Peng | The tail-module of yeast Mediator complex is required for telomere heterochromatin maintenance[END_REF][START_REF] Lenstra | The Specificity and Topology of Chromatin Interaction Pathways in Yeast[END_REF]), the Hda1/2/3 complex, and the general repressors Tup1/Cyc8 specifically affect genes located within ESDs (Fig 7A). Importantly, the localized enrichment for downregulated genes in rpd3 or sas2/4/5 mutants did not extend beyond the ESDs. This enrichment for downregulated genes was likely the consequence of increased spreading of the SIRs in those mutants. Therefore, SIR dependent silencing in those mutants seemed not to extend beyond ESDs, further reinforcing the notion that ESDs represent the maximal extension of SIR dependent silencing. Lastly, enrichment for downregulated genes in the tup1 and Ssn6 mutants decreased abruptly at the end of ESDs (Fig S7A -B) and no mutant had enriched impact on the genes in the 10 kb immediately adjacent to ESDs. Thus, the domains defined by the saturated expansion of silent chromatin encompassed the subtelomeric domains in which known mutants affecting subtelomeres have an effect.

Lastly we probed how the ESD point of view segregates subtelomeric properties as compared to other definitions of subtelomeres: distance from telomeres, from the first essential gene, the end of HAST domains (defined as Hda1 affected subtelomeric regions, [START_REF] Robyr | Microarray deacetylation maps determine genome-wide functions for yeast histone deacetylases[END_REF] or subtelomeres based on synteny conservation across close relative species [START_REF] Yue | Contrasting evolutionary genome dynamics between domesticated and wild yeasts[END_REF]. ESDs often extended within the core chromosome as defined by synteny, more than 10 kb at five subtelomeres (

Discussion

The Sir complex has been a model for chromatin complex propagation and gene silencing for decades. Pioneer studies demonstrated that increasing the dose of Sir3 extends silenced domains at subtelomeres (Renauld et al. 1993b;[START_REF] Pryde | Limitations of silencing at native yeast telomeres[END_REF], a property common to several heterochromatin complexes. However there has been controversy on the generality of this finding at natural telomeres [START_REF] Pryde | Limitations of silencing at native yeast telomeres[END_REF][START_REF] Ellahi | The chromatin and transcriptional landscape of native saccharomyces cerevisiae telomeres and subtelomeric domains[END_REF], and the details of this process remain unclear. Here, we systematically studied the impact of increasing Sir2 and Sir3 dosage on the propagation of the SIR complex and on genome-wide transcription. Gradual overexpression of Sir3 revealed that the spreading of Sir3 over subtelomeres reached saturation at Sir3 levels between 9 and 16ൈ suggesting the existence of fixed borders. Similarly, telomere clustering increased at 9ൈ and 16ൈ Sir3 levels but not above. Yet, the change in chromosome organization imposed by telomere hyperclustering in the center of the nucleus (Ruault et al. 2011) had a very minor impact on gene expression per se as shown by overexpression of the silencing deficient sir3-A2Q point mutant. Conversely, spreading of wild-type Sir3 was systematically associated with decreased transcript levels of the underlying genes. This is in contrast with the situation observed in wild-type cells where Sir3 is found at rather discrete loci close to nucleation sites and with a limited effect on gene expression as previously reported (Thurtle and Rine, 2014;Takahashi, 2011). Thus, although Sir3 spreading and impact on gene expression appears limited in laboratory strains and under standardized growth conditions it has the potential to spread over several kb creating domains of silent chromatin that we named ESDs for extended silent domains. Unexpectedly, the response to increase in Sir3 levels differed among subtelomeres, and the concentration of Sir3 required for maximal spreading varied. Additional spreading extended up to 30 kb in-between subtelomeres, independently of middle repeat elements or chromosomal arm length and expression levels of the underlying genes. However, the domains covered by Sir3 upon overexpression shared similar chromatin marks, suggesting that the chromatin landscape dictated the extent of Sir3 spreading. Indeed, we identified H3K79me3 as the most efficient barrier to prevent heterochromatin propagation. Finally, by revealing the maximal subtelomeric domains accessible to Sir complex in viable cells, our work uncovered previously unknown, discrete subtelomeric domains that isolated the structural and functional features associated with subtelomeres.

Different categories of Sir chromatin antagonism

Although our data suggest the existence of fixed borders, our search for punctual barrier elements did not retrieve convincing candidates. Furthermore, native binding sites for transcription factors that block silencing when tethered to chromatin [START_REF] Oki | Barrier proteins remodel and modify chromatin to restrict silenced domains[END_REF] were not efficient barriers to Sir3 spreading. We also observed extension of Sir3 binding within regions that were enriched with chromatin marks previously reported as antagonistic to its spreading, such as histone variant H2A.Z [START_REF] Guillemette | Variant histone H2A.z is globally localized to the promoters of inactive yeast genes and regulates nucleosome positioning[END_REF][START_REF] Martins-Taylor | H2A.Z (Htz1) controls the cell-cycledependent establishment of transcriptional silencing at Saccharomyces cerevisiae telomeres[END_REF][START_REF] Meneghini | Conserved histone variant H2A.Z protects euchromatin from the ectopic spread of silent heterochromatin[END_REF]) and mono-methylated H3K79 [START_REF] Altaf | Interplay of chromatin modifiers on a short basic patch of histone H4 tail defines the boundary of telomeric heterochromatin[END_REF]. Histone tail acetylation appears to limit spreading of Sir3 when Sir3 is present in a limiting amount. Indeed, we observed that acetylation levels of H4K5,8,12 are higher within ESDs that resist

End of extended silent domains: the specific role of Dot1

We observed that the end of extended silent domains coincides with a major histone mark transition zone, characterized by an increased enrichment of H3K4me3, H3K36me3 and H3K79me3. Deletion of SET1 or SET2, the genes encoding the enzymes responsible for the H3K4me3 and H3K36me3, respectively, had no impact on cell growth upon Sir3 overexpression. In contrast, deletion of DOT1, which encodes the only H3K79 methyltransferase in budding yeast, was lethal in this condition. Yet viability in this strain could be rescued by inhibiting Sir2 indicating that loss of H3K79 methylation was responsible for the deadly spreading of elevated Sir3 in the absence of Dot1. Accordingly, when dot1∆ strain overexpressing SIR3 were released from Sir2 inhibitor, Sir3 spread beyond ESDs invading 4 essential genes. In these conditions, Sir3 was also binding numerous euchromatic sites that were enriched for H3K79me3 (but not H3K79me1 or 2) in wild-type cells. H3K79me3 is anti-correlated with H4K5,8 and 12 acetylation [START_REF] Weiner | High-Resolution Chromatin Dynamics during a Yeast Stress Response[END_REF], which also have the potential to limit Sir3 spreading (Carmen et al, 2002 and above). This suggests that H3K79me3 was protecting nucleosomes hypoacetylated on H4K5,8 and 12 from Sir3 binding at euchromatic sites. As Dot1 is responsible for mono-, di-and tri-methylation of H3K79 [START_REF] Stulemeijer | Dot1 histone methyltransferases share a distributive mechanism but have highly diverged catalytic properties[END_REF] this raises the question of the relative contribution of these marks for blocking Sir3 spreading. In vitro, all three H3K79 methyl marks reduce Sir3 affinity for reconstituted nucleosomes [START_REF] Behrouzi | Heterochromatin assembly by interrupted Sir3 bridges across neighboring nucleosomes[END_REF][START_REF] Martino | Reconstitution of yeast silent chromatin: multiple contact sites and O-AADPR binding load SIR complexes onto nucleosomes in vitro[END_REF]).

Here we show that upon overexpression, Sir3 spread over domains enriched for H3K79me1 and me2, implying that these marks did not block Sir3 spreading in vivo although they slow down silencing establishment (Katan-Khaykovich and Struhl 2005; [START_REF] Osborne | The establishment of gene silencing at single-cell resolution[END_REF]. In contrast, H3K79me3 signal increases abruptly where Sir3 spreading stopped. In the absence of Dot1 and upon overexpression of SIR3, we observed that Sir3 predominantly binds loci that were initially enriched for H3K79 tri-methylation state. Accordingly, mutants in which H3K79me3 is abolished but not H3H79me and H3K79me2 [START_REF] Ng | Lysine methylation within the globular domain of histone H3 by Dot1 is important for telomeric silencing and Sir protein association[END_REF][START_REF] Schulze | Linking Cell Cycle to Histone Modifications: SBF and H2B Monoubiquitination Machinery and Cell-Cycle Regulation of H3K79 Dimethylation[END_REF]) were sensitive to SIR3 overexpression. This is consistent with the observation that Sir3 associate with H3K79 mono and di methylation at an active subtelomeric reporter gene in vivo [START_REF] Kitada | Mechanism for epigenetic variegation of gene expression at yeast telomeric heterochromatin[END_REF]. This is also in good agreement with crystal structure data predicting that the potential of H3K79 to form hydrogen bonds with the BAH domain of Sir3 would progressively decrease with H3K79 methylation to be abolished by H3K79me3, thereby decreasing Sir3 affinity to nucleosomes [START_REF] Armache | Structural Basis of Silencing: Sir3 BAH Domain in Complex with a Nucleosome at 3.0 A Resolution[END_REF]Arnaudo et al. 2013). All together our work demonstrates that H3K79 methylation, predominantly the tri-methyl state, restricts silencing within subtelomeric regions thus protecting euchromatin. As the occupancy of this mark is independent of transcription rate [START_REF] Schulze | Linking Cell Cycle to Histone Modifications: SBF and H2B Monoubiquitination Machinery and Cell-Cycle Regulation of H3K79 Dimethylation[END_REF], this offers the attractive possibility of preventing heterochromatin spreading independently of transcription.

Subtelomeric specificities

In most organisms, specific features of chromosome ends extend beyond telomeres, within domains generally referred to as subtelomeres [START_REF] Louis | Subtelomeres[END_REF]. In budding yeast, several viewpoints enable the identification of diverse subtelomeric features including a lower gene density, and a faster evolution than the core genome [START_REF] Yue | Contrasting evolutionary genome dynamics between domesticated and wild yeasts[END_REF]. The chromatin landscape also exhibits specific features within domains located proximal to chromosome ends [START_REF] Matsuda | Highly condensed chromatins are formed adjacent to subtelomeric and decondensed silent chromatin in fission yeast[END_REF][START_REF] Millar | Genome-wide patterns of histone modifications in yeast[END_REF][START_REF] Robyr | Microarray deacetylation maps determine genome-wide functions for yeast histone deacetylases[END_REF]. The first is undoubtedly the presence of heterochromatin, which has a unique signature in terms of histone marks. However, specific properties associated with chromosome ends often extend beyond heterochromatic domains [START_REF] Matsuda | Highly condensed chromatins are formed adjacent to subtelomeric and decondensed silent chromatin in fission yeast[END_REF][START_REF] Millar | Genome-wide patterns of histone modifications in yeast[END_REF].

At most S.cerevisae subtelomeres, Hda1-affected subtelomeric (HAST) domains [START_REF] Robyr | Microarray deacetylation maps determine genome-wide functions for yeast histone deacetylases[END_REF] and Htz1 activated (HZAD) domains [START_REF] Guillemette | Variant histone H2A.z is globally localized to the promoters of inactive yeast genes and regulates nucleosome positioning[END_REF]) lie contiguous to SIR silenced chromatin. In addition, phosphorylation of H2AS129 and monomethylation of H3K79 also extend further away than SIR silenced domains. Here we show that ESDs possess a consistent chromatin signature. Namely these domains are enriched for H2AP, Htz1 and depleted of tri-methylated histone H3 which levels show sharp transition at the end of ESDs. Consistently, Htz1 sensitive genes are enriched in these domains. Furthermore, considering the end of ESDs as a boundary between subtelomeres and the core genome segregates genes sensitive to the depletion of chromatin modifiers such as Hda1, Tup1/Ssn6 or Sas2 better than other definitions of subtelomeres (Fig S7D ,S7E). Similarly, ESDs segregated transition of histone marks such as H3K73me3 better than other definitions of subtelomeric domains. Thus, ESDs coincide with discrete subtelomeric domains isolating structural and functional features and could provide an alternative definition of subtelomeres. Furthermore, domains defined by ESD contain genes than can collectively be repressed in non-stressful conditions, a notion consistent with the idea that subtelomeres contain genes required for response to stressful environments [START_REF] Louis | Subtelomeres[END_REF].

At most subtelomeres that we could analyse ESDs are broader or coincide with subtelomeres defined based on synteny conservation across related species (Fig S7A). We and others recently showed that chromatin states impact on efficiency and outcome of both homologous recombination and nucleotide excision repair [START_REF] Guintini | Repair of UV-induced DNA lesions in natural Saccharomyces cerevisiae telomeres is moderated by Sir2 and Sir3, and inhibited by yKu-Sir4 interaction[END_REF][START_REF] Batté | Recombination at subtelomeres is regulated by physical distance, double-strand break resection and chromatin status[END_REF]. This raises the question of whether the specific chromatin state associated with subtelomeric domains uncovered in this study contributes to the particular evolution of those regions.

Contribution of telomere proximity to subtelomeric properties

A central question in the biology of subtelomeres is to what extent the properties of subtelomeres are due to their proximity to telomeres or a mere consequence of their gene content? Several studies demonstrated that the SIR complex contributes to the localization of enzymes to subtelomeres. For example, subtelomeric localization of the Okazaki fragment processing protein Dna2 is severely reduced in sir mutants [START_REF] Choe | Dynamic localization of an Okazaki fragment processing protein suggests a novel role in telomere replication[END_REF]. In addition, the kinase Tel1 responsible for H2A phosphorylation in subtelomeric regions is present at telomeres but H2AP levels depend mainly on the integrity of the SIR complex [START_REF] Kitada | ??H2A is a component of yeast heterochromatin required for telomere elongation[END_REF]. Regions enriched for H2AP coincide with ESDs suggesting that either Sir3 acts remotely, or binds these regions at least transiently in wild-type. Accordingly, profiling of Sir3 binding in G1 arrested cells showed low levels of Sir3 binding within ESDs [START_REF] Mitsumori | Analysis of novel Sir3 binding regions in Saccharomyces cerevisiae[END_REF]). Thus, Sir3 might influence the chromatin landscape in subtelomeric regions. How the transient presence of Sir3 during the G1 phase of the cell cycle could stabilize H2A phosphorylation is unclear. One attracting possibility is that Sir3 acts by recruiting a so far unidentified factor that would remain associated to chromatin through the whole cell cycle.

Conclusion

By taking the opposite approach to depletion studies, our work describes the dose dependency of budding yeast heterochromatin. In the presence of a large excess of silencing factors, ectopic nucleation of heterochromatin remains limited and does not impact euchromatic transcription. In contrast, we observed the extension of subtelomeric silent domains and characterized their maximal extension along with the antagonistic factors that have been overcome, such as H2A.Z or H3K79me. By scanning chromatin properties associated with Sir3 maximal binding, we uncovered major subtelomeric histone mark transition zones that functionally protect euchromatin from the spread of silencing. The long-term contribution of heterochromatin to the peculiar properties of subtelomeres will require further study.

Methods

Media and Growth conditions

The strains used in this study are listed in Table S2. Yeast cells were grown on YP with 2% glucose, raffinose or galactose. Unless notified, all the strains used in this study were grown at 30 °C with shaking at 250rpm.

Yeast transformation protocol

Cells were seeded on liquid medium and grown to 0,8<OD 600 <1,2. 3 ODs (~3x10 7 yeast cells) of cells were taken and washed with 1ൈ TEL (10 mM EDTA pH 8, 100 mM Tris pH8, 1M Lithium Acetate), then 3μl of SSDNA (Sigma ref: D9156-5ML), DNA template (0,5μl if plasmid DNA, 5μl of digested plasmid or PCR product), 300μl of 1ൈ TEL and 45% PEG-4000 solution were added. The mix was put 30 min at 30 °C and heat shocked at 42°c for 15 minutes. Lastly, cells were plated on appropriate selective medium.

Rap1 foci analysis

The image analysis is performed with a slightly modified version of the dedicated tool from [START_REF] Guidi | Spatial reorganization of telomeres in long-lived quiescent cells[END_REF]. These modifications regard the quantification of foci and aim at providing a more accurate estimation of the quantity of fluorescence held inside each focus. The Gaussian fitting approach has been replaced by a template matching framework with a bank of 100 symmetric 2D Gaussian kernels with standard deviations ranging from 0.5 to 7 pixels.

The position of each template is determined as the maximum of normalized cross correlation whereas the most suitable template for a single focus is selected by minimizing the sum of square differences between the Gaussian template and the data within a circular mask of radius twice the standard deviation. The foci are then defined as spherical objects with radii of two times the standard deviations of the matched templates. All foci that could not be fitted were considered as a cube of dimension 5ൈ5ൈ5. Variation of the box size did not affect overall results. The foci intensity can thus be measured as the sum of the fluorescence signal inside its sphere. Furthermore, the proportion of intensity from a nucleus held inside each of its foci is also computed.

Sir3-GFP quantification

Quantification of Sir3-GFP signal was carried using microscopy. Briefly cells were segmented on the basis of trans signal using a modified version of CellX, and the intensity of 30 Z-stacks deconvolved imaged was summed. Deconvolution was carried using MetaMorph. For each cell the intensity/pixel was measured and normalized by the WT average.

Western blots

Protein extracts were prepared from 2 ODs of exponentially growing cultures (O.D ~1) using the post-alkaline extraction method (Kushnirov et al. 2000). For immunoblotting, we used custom-made rabbit polyclonal antibodies raised against full length Sir3 [1:10000 dilution] (Ruault et al. 2011).

FACS

Cell cycle profiles were obtained on a Accury FACS machine using CYTOX as DNA staining agent and analyzed using FlowJoX.

SGA screen

Query strain was obtained by transforming strain yAT-1949 with pGAL-SIR3-HPH, integrated within TRP1. The query strain was crossed with the collection of histone point mutants (Dai and Boeke 2012) following the selection steps described in [START_REF] Tong | Systematic genetic analysis with ordered arrays of yeast deletion mutants[END_REF], with selection media adapted to respective genotypes. Each cross was done in quadruplicate on 1536format plates. Once double mutants were acquired, they were transferred to one of the following medium: double mutant selection medium (glucose), double mutant selection medium (glucose) + 5 mM NAM, double mutant selection medium (galactose) or double mutant selection medium (galactose) + 5 mM NAM. All strains were grown at 30 o C and imaged after 2 days. Image analysis and scoring were done with SGAtools (Wagih et al.

2013)

, where mutants growing on glucose media served as controls. Only significant changes were considered (p-val< 0.001) and a last significance threshold was chosen to only keep mutants which score's absolute value was > 0.2.

Dilution Assays

Cells were grown overnight in YPD 5 mM NAM before dilution. 5-fold serial dilutions are shown. Plates were grown for 2-3 days at 30°C .

Pellet preparation for ChIP

A total of 20 O.D equivalent of exponentially growing cells were fixed in 20 mL with 0.9 % formaldehyde for 15 min at 30°C, quenched with 0.125 M glycine and washed twice in cold TBS 1ൈ pH 7.6. Pellets were suspended in 1mL TBS 1ൈ, centrifuged and frozen in liquid nitrogen for -80°C storage. For NAM release experiments, Cells were grown overnight in YPD 5 mM NAM before dilution at 0.2 OD in YPD and allowed to grow for 8 hours (O.D ~1.5).

Chromatin immunoprecipitation

All following steps were done at 4°C unless indicated. Pellets were re-suspended in 500 µL of lysis buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA pH 8, 16.7 mM Tris pH8, 167 mM NaCl, 0.5 % BSA, 0.02 g.L -1 tRNA and 2.5 µL of protease inhibitor from SIGMA P1860) and mechanically lysed by three cycles of 30s, intensity 6ms-1 with 500 µm zirconium/silica beads (Biospec Products) using a Fastprep instrument (MP Biomedicals). Each bead beating cycle was followed by 5 min incubation on ice. The chromatin was fragmented to a mean size of 500 bp by sonication in the Bioruptor XL (Diagenode) for 14 min at high power with 30 s on / 30 s off and centrifuged 5 min at 13 000 rpm. 10 µL were kept to be used as Input DNA. Cleared lysate was incubated overnight with 1 µL of polyclonal antibody anti-Sir3 (Ruault et al. 2011). 50 µL of magnetic beads protein A (NEB) were added to the mixture and incubated for 4h at 4°C. Magnetic beads were washed sequentially with lysis buffer, twice with RIPA buffer (0.1% SDS, 10 mM Tris pH7.6, 1 mM EDTA pH8, 0,1% sodium deoxycholate and 1% Triton X-100), twice with RIPA buffer supplemented with 300 mM NaCl, twice in LiCl buffer (250 mM LiCl, 0.5% NP40, 0.5 % sodium deoxycholate), with TE 0.2% Triton X-100 and with TE. Input were diluted 10ൈ with elution buffer (50 mM Tris, 10 mM EDTA pH8, 1%SDS) and beads were re-suspended in 100 µL elution buffer. A reversal cross-linking was performed by heating samples overnight at 65°C. Proteins were digested with proteinase K in presence of glycogen and the remaining DNA was purified on QIAquick PCR purification columns. Finally, samples were treated with RNase A 30 min at 37°C.

ChIP-chip preparation and hybridation

Samples used for ChIP-chip have all been analysed by qPCR prior to microarray hybridization.

For microarray hybridization 4/5 of the immunoprecipitated DNA and of the DNA from the input were ethanol precipitated and re-suspended in 10µL of water (Gibco). Purified material was amplified, incorporating amino-allyl-dUTP using as described in [START_REF] Guidi | Spatial reorganization of telomeres in long-lived quiescent cells[END_REF]. The size of amplified fragments (~500 bp) was assessed by gel electrophoresis. For each sample 1.5 µg of amplified DNA was coupled either with Cy5 (immunoprecipitated sample) or Cy3 (input sample) and hybridized on 44k yeast whole genome tiling array (Agilent) as described in [START_REF] Guidi | Spatial reorganization of telomeres in long-lived quiescent cells[END_REF].

Microarray data acquisition, analysis and visualization

Microarray was imaged using a Agilent DNA microarray scanner and quantified using GenePix Pro6.1 as described in [START_REF] Guidi | Spatial reorganization of telomeres in long-lived quiescent cells[END_REF].

Genome wide data analysis

Unless mentioned otherwise, data analysis was carried using R (R Core Team 2016). All datasets were lifted over to SacCer3 when required. Histone marks data were obtained from [START_REF] Weiner | High-Resolution Chromatin Dynamics during a Yeast Stress Response[END_REF]. Sir3 binding in H3 tail mutants from [START_REF] Sperling | Histone H3 N-terminus regulates higher order structure of yeast heterochromatin[END_REF], nucleosome turnover from [START_REF] Dion | Dynamics of replication-independent histone turnover in budding yeast[END_REF]. Transcriptome data were downloaded from the website supporting the publication [START_REF] Kemmeren | Large-scale genetic perturbations reveal regulatory networks and an abundance of gene-specific repressors[END_REF]. Subtelomere definition was obtained from [START_REF] Yue | Contrasting evolutionary genome dynamics between domesticated and wild yeasts[END_REF]. Z-scores were computed using the R scale function.

Criterion for the clustering of subtelomeres shown in Fig 1 were the following: First, the spreading end point was computed as the most subtelomeric probes with Sir3 binding Zscore >1 flanked by more than 5 probes with Z-score < 1. We then applied the following criterions: "Fragile" subtelomeres: d(TEF-ADH)<2 kb<d(ADH-WT) ; "Unextendable": d(TEF-ADH), d(ADH-WT) <2 kb ; "Robust": d(ADH-WT) <2 kb<d(TEF-ADH) ; "Progressive": d(TEF-ADH), d(ADH-WT) >2 kb. Of note, 2 subtelomeres (VIIR, XVIR) which classification was too sensitive to a given threshold were manually curated. Euchromatic binding sites shown as supplemental table 1 were computed as sites away from ESDs (or present at more than 50 kb from a telomere when ESD are not defined) at which at least two neighbouring probes are bound (z-score >0.5) by Sir3 upon overexpression of SIR3 (computed the signal obtained from the W303 strain yAT1254). Downsampling of [START_REF] Sperling | Histone H3 N-terminus regulates higher order structure of yeast heterochromatin[END_REF], [START_REF] Mitsumori | Analysis of novel Sir3 binding regions in Saccharomyces cerevisiae[END_REF]) and [START_REF] Weiner | High-Resolution Chromatin Dynamics during a Yeast Stress Response[END_REF] Area under the curve was exactly computed on the fitted signal of Sir3 binding in strains overexpressing SIR2 and SIR3, 10 kb before the end of silent domains and 5 kb after.

Mutants showing localized effects were identified with using the hypergeometric distribution, function phyper with Bonferroni correction for multiple testing (n=703 being the number of mutants studied showing significant transcription changes).

Linear model fitting of histone mark transition zones: For this plot subtelomeres differing between W303 and BY background were excluded (n=3, TELIR, TELVIIL, TELXIVR). For each viewpoints all subtelomeres in which the viewpoint is defined were considered. First, the length on which fitting was applied was optimized as the length giving the highest R-squared around ESDs. Window size ranging from 1 to 5 kb (100 bp step) were tried for each histone mark probed. Fitting of data on windows of defined size 20 kb around viewpoints with 50 bp steps was carried with the R lm() function.

RNA-seq

Total RNA from a 25mL culture of exponentially growing yeasts were extracted using phenolchloroform. Banks were constructed using the kit SOLiD Total RNA-seq, with minor modifications: RNA are Zinc fragmented and fragments with size ranging from 100 to 200 nt selected by gel purification. After reverse transcription, only fragment of size > 150nt are kept. Paired end (50 + 35) sequencing was done by the Institut Curie plateform. Differential expression was called using edgeR [START_REF] Robinson | edgeR: differential expression analysis of digital gene expression data[END_REF], with a false discovery rate inferior to 0.1.

Data access

ChIP-chip and RNA-seq data from this study have been submitted to the Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/) under the accession numbers GSE106499, and GSE104391, respectively. (A) Quantification of Sir3 levels by integration of Sir3-GFP signal in strains expressing SIR3-GFP under different promoters as indicated. (B) ChIP-chip against Sir3 was carried in strains expressing SIR3 under different promoters as indicated. Moving average of Sir3 binding (block = 1000 bp, window = 10 bp) at telomeres (with the exception of TELIIIL and TELIIIR that contain HM loci) as a function of distance from the ARS consensus sequence (ACS) within the last telomeric element. Enrichment is shown as the standardized IP over Input (See mat and meth). (C) Rap1-GFP foci grouping in strain differing for Sir3 levels. Cells were grown in YPD overnight, diluted to OD600 nm= 0. (A) Pearson correlation matrix between Sir3 binding and histone marks, SIR3 oe corresponds to yAT1254 and SIR2 & SIR3 oe to yAT1668. Histone modification data from [START_REF] Weiner | High-Resolution Chromatin Dynamics during a Yeast Stress Response[END_REF] for all marks except H3K79me2 [START_REF] Schulze | Linking Cell Cycle to Histone Modifications: SBF and H2B Monoubiquitination Machinery and Cell-Cycle Regulation of H3K79 Dimethylation[END_REF]). (B) Genome browser visualization of Sir3 binding in WT, pGPD-SIR2 pGPD-SIR3 strains, in G1 blocked cells, in H3∆4-30 mutants and selected histone modifications (from [START_REF] Weiner | High-Resolution Chromatin Dynamics during a Yeast Stress Response[END_REF] in WT strains at TELVIR. (C) Distribution of selected histone marks relative to H3 (data from [START_REF] Weiner | High-Resolution Chromatin Dynamics during a Yeast Stress Response[END_REF] along wild type silenced domains and within the contiguous subtelomeric domains accessible to Sir3 upon overexpression. For comparison, the distributions of those marks within the 5 kb contiguous to the end of extended silent domains as well as within euchromatin (i.e. ESD excluded, n=49313) are shown. Sir3 enrichement

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  binds to telomeric repeats (Fig 2A-B), did not affect global transcription of subtelomeres (Fig S2A), repression was attributed to Sir3 binding to chromatin and not clustering of telomeres. However, 22 genes showed transcriptional changes common to the overexpression of SIR3-A2Q and SIR3, i.e potentially caused by telomere clustering, including 20 euchromatic genes (Fig S2D). Those transcriptional changes could be the consequences of changes in spatial localization or alternatively due to the sequestering of specific factors within the telomere hyper-clusters. The extension of Sir3-bound domains upon SIR3 overexpression systematically repressed underlying transcripts genome-wide (Fig 2C for the Tel6R and 2D genome wide) providing a parallel assessment of the validity of binding events measured by ChIP-chip. Repression was largely independent of initial transcript level (Fig 2D) and of coding status (e.g. the right subtelomere of Chromosome VI, Fig 2C, Fig S2B). These extended silent domains (ESDs) included 100 genes that were not bound by Sir3 in WT cells. The logarithm of transcriptional repression was linearly proportional (R 2 =0.71) to the Sir3 binding signal, reflecting the absence of silencing escapers (Fig 2D). Analysis of reads mapping to multiple loci indicated that entire gene families, characteristic of subtelomeres and Y' elements, were on average repressed upon SIR3 overexpression (Fig S2C, S2E), suggesting that the subtelomeric regions devoid of chip probes are collectively silenced within ESDs.

  Fig S7C), showing that ESD constituted a different definition of subtelomeres. ESD and HAST domains ended at similar location at 8 subtelomeres (<3 kb) but HAST domains, which are only defined at 22 subtelomeres, generally extended beyond ESD (Fig S7D). Furthermore, the transitions of the H2AS129ph and H3K79me3 histone marks (Fig 7B) are evidently sharper from the ESD end viewpoint than from the distance from X-core sequence or syntenically defined subtelomere end. Quantification of H3K79me3 transition by systematic fitting of a linear model around the transition zones confirmed that ESDs better captures H3K79me3 subtelomere to euchromatic transitions than syntenically derived subtelomere or HAST domain ends (Fig 7C). Similar quantification for H2S129P transition showed than ESDs and HASTs comparably segregated H2AS129P although ESDs included four more subtelomeres, both viewpoints being more efficient than syntenically defined subtelomere ends (Fig S7E). Thus, probing the maximal extent of silencing domains revealed discrete subtelomeric domains delimited by histone mark transition zones and provides a new definition of subtelomeres (Fig 7D).

  to intermediate levels of SIR3 overexpression than at other ESDs. Furthermore, H3 tail mutants show increase Sir3 spreading at some subtelomeres within ESDs (Fig 4). Consequently, our work indicates that histone tail acetylation, H2A.Z, and specific transcription factors likely buffer the spread of the SIR rather than block it (Fig 7D).

  data to the 44k microarray probes for Fig 4B and 5A was done using R, visual inspection of the data confirmed that downsampling was carried without errors. In details, all signal located in between the mi-distance to the previous and the next probe were average and allocated to the central probe. Average telomeric profiles were done by computing the mean of the signal over 10 kb windows separated by 10 bp. The limits of Extended silent domains were computed as the first probes possessing 5 neighboring probes 18 that have Z-score inferior to 1, starting from the telomere. Fitting of Sir3 binding data was done using MATLAB fitting toolbox using Bisquare robustess option. The function used is f(x)=K/(1+exp(-r(-x+t0)))+1, with the following fitting parameters for K,r, and t0: lower bounds: [10 0.0001 1000], Starting point: [10 0.0001 1000], upper bounds: [200 0.01 40000].

Figure 1 :

 1 Figure 1: Increasing Sir3 dosage leads to telomere clustering and SIR spreading saturation.

  2 and imaged at OD600 nm= 1. (D) Quantification of Rap1-GFP foci distribution in images from C. (E) Left: Distribution of Rap1-GFP signal attributed to the brightest foci in each nucleus; Right: Distribution of the relative amount of Rap1 measured within foci relative to total nuclear Rap1 signal. (F) Stereotypical examples of Sir3 binding in function of Sir3 dosage. Enrichment corresponds to standardized Sir3 binding (z-score). The number of subtelomeres within each group is shown into bracket.

Figure 2 :

 2 Figure 2: Sir3 extended domains are silenced and restricted to subtelomeres. (A) Representative Rap1-GFP images of exponentially growing strains with different Sir3 amount or expressing the SIR3-A2Q point mutant allele. (B) Chromosome wide binding of Sir3 in the same strains as in A and blow-up on subtelomere VIR. Enrichment is shown as the standardized IP over Input and scale is thresholded at 15 for visualization purposes. (C) Total RNAseq read density and corresponding transcriptional fold change along subtelomere VIR in indicated exponentially growing (OD600 nm~1) strains. (D) Sir3 binding and corresponding transcription changes of subtelomeric genes (Distance from chromosome end <50 kb) upon overexpression of SIR3. Genes showing infinite fold change values were excluded from this plot. Color code indicates if a gene is annotated as within ESDs (see math and meth) and shade indicates significance (FDR<0.1) of the detected changes. Read density in WT cells is proportional to the disk area. Black line corresponds to linear fitting of the data, and corresponding R-squared value is shown. (E) Exemplification of the 7 subtelomeres at which a gene within the ESD shows larger transcript amount than the genes located at the end of the domain. (F) Read density of genes located before and after the end of extended silent domains compared to genome wide distribution, statistical test: Wilcoxon test, paired values.

Figure 3 :

 3 Figure 3: End of extended silent domains is defined locally and independently of transcriptional activity. (A) Sir3 binding at native and truncated TELVIIL, x coordinates correspond the native telomere TELVIIL. (B) Sir3 binding at TELVL in WT and Sir3 overexpressing (pGPD-SIR3) strains. Transcription factor (TF) binding and DNase I hypersensitive sites along TELVL are shown.Figure 4: ESDs encompass known domains of Sir3 extension. (A) Moving average of Sir3 binding (block = 1000 bp, window = 10) at the end of ESDs in the indicated conditions or mutants. (B) Genome browser visualization of Sir3 binding at subtelomere IIR, all data are shown as Z-score with a lower bound of -1 and an upper bound of 12.

Figure 4 :

 4 ESDs encompass known domains of Sir3 extension. (A) Moving average of Sir3 binding (block = 1000 bp, window = 10) at the end of ESDs in the indicated conditions or mutants. (B) Genome browser visualization of Sir3 binding at subtelomere IIR, all data are shown as Z-score with a lower bound of -1 and an upper bound of 12.

Figure 5 :

 5 Figure 5: Extension of silent domains predicts major subtelomeric chromatin transitions.

Figure 6 :

 6 Figure 6: H3K79 methylation is key to sustain viability upon Sir3 overexpression. (A) Moving average of Sir3 binding at telomeres (10 kb windows, 10 bp step). The top and bottom 10 telomeres with regards to Sir3 signal in strains overexpressing SIR2 and SIR3 were plotted separately. H3K79me3 data were obtained from Weiner et al. 2015. Blue lines indicate genome wide lower and higher quartiles for each mark. Red line corresponds to the local smoothing of histone modification data. (B) Dilution assay to probe viability of dot1 mutants upon overexpression of SIR3. Cells were constantly grown in presence of 5 mM NAM prior to this assay. Cells were grown overnight, and 0.5 OD of cells were plated in 5ൈ serial dilutions on YPD or YPD 5 mM NAM. (C) Growth score of selected histone point mutants on galactose plates (Sir3 inducing conditions) with or without NAM compared to glucose plates (Sir3 dosage is WT). (D) Genome browser visualization of Sir3 binding in pGPD-SIR3 and dot1∆ pGPD-SIR3 strains 8 hours after being released from 5 mM NAM. KRR1 and CDC39, labelled in red, are essential genes. H3K79 methylation enrichment were obtained from Weiner et al. 2015 for H3K79me and H3K79me3 and from Schulze et al. 2009 for H3K79me2 (Mat score is shown). (E) Moving average of Sir3 binding (block = 1000 bp, window = 10 bp) at the end of ESDs in pGPD-SIR3 and dot1∆ pGPD-SIR3 strains 8 hours after being released from 5 mM NAM. (F) Dot plot showing Sir3 enrichment in the indicated strains and condition against the enrichment for each methylation level of H3K79. H3K79 methylation data were obtained from the same source as in E and averaged at each ChIPchip probe.

Figure 7 :

 7 Figure 7: Extension of silent domains reveals new aspects of subtelomeric structuration. (A) Localized effects of mutations affecting subtelomeric transcription. The different subtelomeric subdomains are defined according to Sir3 binding. The number of genes within each domain is into brackets. Mutant names are positioned according to the domain(s) within which the proportion of genes up or down-regulated (log(FC)>1 or < -1) is significantly elevated (Hyper-geometric law, with Bonferonni correction n=703). (B) Distribution of H3K79me3 and H2AS129ph (obtained from Weiner et al. 2015) relative to different subtelomeric viewpoints. Blue lines indicate genome wide lower and higher quartiles for each mark. Red line corresponds to the LOESS smoothing of histone modification data. (C) Quantification of H3K79me3 transition in function of different genomic viewpoints. Shown are the results of a linear model fitting of the histone mark enrichment data (residuals standard deviation, slopes, R 2 and p-values) over 2.35 kb windows every 50 bp. (D) Model depicting how extending silent domains enables to uncover consistent subtelomeric domains delimited by chromatin mark transitions.

  A) Western Blot anti-Sir3 in the strains used in Fig 1 for ChIP-chip. (B) Representative examples of Sir3 fluorescence in strains overexpressing Sir3-GFP. (C) FACS profile of Sir3 binding (Zscore) growing WT and pGPD-SIR3 strains. (D) ChIP signal at highly expressed genes in the indicated strains. PolII enrichment data were obtained from Szilard et al. 2010. For comparison, subtelomeric binding signal is generally much higher (light blue probes at TELVL and TELVIR panel F. (E) Quantification of Sir3-GFP nuclear background in strains overexpressing SIR3-GFP. (F) Representative images of loci bound by Sir3 within euchromatin, light blue color indicates the probes included in Extended silent domains. Scale: 0-300. (G) Sir3 binding in function of Sir3 dosage at individual subtelomeres classified as in main Fig 1F. 6 subtelomeres are not shown due to insufficient data. (H) Chromosomal arm length versus spreading of Sir3. (I) Subtelomere groups as defined in Fig 1F in function of chromosomal arm length and telomeric middle repeat content. Supplemental_Fig_S2: (A) Transcriptional changes in sir3-A2Q mutants versus sir3∆ mutants. (B) Transcription of ncRNAs within subtelomeres, color code is identical to the main Fig 2D. (C) Average Read density at Y' elements. (D) All transcriptional changes coined significant by EdgeR within code indicates log2(FC). (E) Transcriptional changes of genes from subtelomeric families. Supplemental_Fig_S3: (A) Example of fitting of the ChIP-chip data, function used is shown on the graph. Right: Inferred slope versus position of inflexion point. (B) Examples of identified barrier at three subtelomeres at which Sir3 spreading did not extend when Sir3 dosage was increased. (C)

  Moving average of Sir3 binding at telomeres (with the exception of TELIIIL and TELIIIR, which contain HM loci) as in Fig 1F, in the indicated genotypes. Representative examples of Sir3 binding. (B) Sir3 binding at individual subtelomeres. Enrichment corresponds to standardized Sir3 binding (z-score). Origin of external dataset is indicated. 6 subtelomeres are not shown due to insufficient data.

  Distribution of selected histone marks relative to H3 (data from[START_REF] Weiner | High-Resolution Chromatin Dynamics during a Yeast Stress Response[END_REF], except H3K79me2 from Schulze et al. 2009) at the flanks of WT silent domain ends (5kb) at the three groups of subtelomeres that are eventually sensitive to SIR3 overexpression. (B) Distribution of selected histone marks relative to H3 (data from[START_REF] Weiner | High-Resolution Chromatin Dynamics during a Yeast Stress Response[END_REF] except H3K79me2, from Schulze et al. 2009) along wild type silenced domains and within the contiguous subtelomeric domains accessible to Sir3 upon overexpression. As a control, the distribution of those marks within the 5 kb contiguous to the end of extended silent domains as well as the genome wide distribution of those. (C) Identical as B but focusing on nucleosomes localized over gene bodies. Supplemental_Fig_S6: (A) Drop assays probing viability in the presence or absence of 5 mM NAM. Protocol is identical to the one of Fig 6B. Genotypes are as indicated. (B) dot1 mutants overexpressing are viable. (C) DOT1 overexpression counteracts SIR3 overexpression. WT strains have an ADE2 reporter gene located at telomere VL. (D) SGA score of all histone point mutants probed, colored points pass our significance criterion and are colored according to their respective behavior (rescued by NAM treatment, sick in all conditions). (E) Genome browser visualization of Sir3 binding in pGPD-SIR3 and dot1∆ pGPD-SIR3 strains 8 hours after being released from 5 mM NAM. tRNA tL(UAA)B1 is labelled in red. H3K79 methylation enrichment were obtained from Weiner et al. 2015 for H3K79me and H3K79me3 and from Schulze et al. 2009 for H3K79me2 (Mat score is shown). (F) Drop assays probing viability in the presence or absence of 5 mM NAM in rad6 mutants. Cells were grown over night in YPD + 5 mM NAM and release 3h in YPD before plating on YPD or YPD + NAM.

  #Supplemental_Table_S1 #List of detected binding sites of Sir3 within euchromatin. All non-subtelomeric Sir3 binding s -score >0.5) by Sir3 upon overexpression of SIR3) together with the change in transcript l

	#Supplemental table 2: yeast strains used d in this study.
	sites (n=18) (defined as euchromatic site at which at least two neighbouring probes are bou RNA levels and Significance levels (read density) for the flanking genes in WT versus SIR3 overexpressing or SIR3 dele eted strains. ID Mating T. nd (zSir3 binding 191 alpha
	Coordinates log2(Mutant/WT) this study Mitsumori et al. 1254		alpha	False Discovery Rate (EdgeR) Sperling et al.
	Site # SIR3 oe vs WT SIR3-A2Q oe vs WT sir3 vs WT Chromosome Start Stop sir3 vs WT WT pADH-SIR3 pTEF-SIR3 pGPD-SIR3 (W303) Flanking gene(s) SIR3 oe vs WT SIR3-A2Q oe vs WT Type H3∆4-30 G1 Extended upon oe
	1 n.s 0,1	1256	chrI no	0,0	44488 1of2 alpha	45151 -0,1 yes	YAL053W n.s yes	Intergene n.s yes
	1 2 3 4 4 5 6 7 8 8 9 10 11 11 12 0,3 -0,4 0,3 0,1 0,2 -0,4 -0,2 -0,7 -0,2 0,0 -1,3 -1,0 0,0 0,0 n.s n.s n.s CN1 n.s 1,8E-02 n.s -n.s n.s n.s n.s n.s n.s n.s n.s CN11 CN11 CN17 CN4 CN7 CN7	2487 2627 2629 2554 1667 2476 2987 2986 2056 1668 779 3443 3442 3441	chrI chrI chrII chrIII chrIII chrIII chrIV chrIV chrVI chrVI chrVIII chrX chrX chrX chrXI no yes no no no no no yes yes yes 1of2 1probeonly -0,1 0,2 -0,1 0,1 -0,9 -0,9 --0,1 -0,7 0,4 0,3 -0,1 -1,0 0,1 -0,1 yes yes 1of2 no no no no yes yes	44488 189742 428552 30013 30013 50934 436567 1430227 181554 181554 175722 360277 683821 683821 68881 1of2 yes yes a a no a no a yes a 1of2 yes yes yes 1of2 1of2 yes yes yes a a a a a a a a a	45151 192296 428610 30532 30532 52028 436626 1430284 184062 184062 175767 360518 683880 683880 68932 -0,2 0,2 -0,2 -0,4 -1,1 -0,1 --0,4 0,1 0,2 0,2 -0,6 0,3 0,1 -0,2 yes yes yes no no yes yes yes yes yes no no yes yes yes	YAL054C YAR035W YBR092C YCL054W YCL055W YCL040W -YDR488C YFR017C YFR018C YHR033W YJL043W YJR137C YJR138W YKL198C n.s n.s n.s n.s n.s n.s -n.s n.s n.s n.s n.s 4,2E-04 n.s n.s yes yes yes yes yes yes yes yes yes yes yes 1of2 yes yes yes	Intergene Gene body Gene body Intergene Intergene Gene body Flanks 2 tRNAs Gene body Gene body Gene body Gene body Gene_body Intergene Intergene Gene body n.s n.s n.s n.s n.s n.s -n.s n.s n.s n.s n.s 6,7E-04 n.s n.s yes yes no yes yes yes yes yes yes yes yes yes yes yes yes
	13 0,2 n.s	3004	chrXII 1of2 yes	0,1	1012859 yes alpha	1012911 0,7 yes	YLR438W n.s yes	Gene body n.s yes
	14 4,7 7,0E-45		chrXII no	4,8	1020034 no	1022147 -3,6 yes	YLR442C 1,5E-81 yes	Gene body 1,6E-85 no
	15 -0,9 n.s CN25	3123	chrXIII 1of2	0,2	757329 yes alpha	758041 -0,2 yes	YMR244W n.s yes	Gene body n.s yes
	16 0,1 n.s		chrXIV no	0,0	25008 no	25466 -0,1 yes	YNL327W n.s yes	Gene body n.s no
	17 -0,1 n.s	3180	chrXV no	0,4	480828 1of2 alpha	480875 0,1 no	YOR084W n.s yes	Gene body n.s yes
	18 -0,1 n.s		chrXVI 1of2 oneprobe 0,0	520221 yes	520280 0,1 yes	YPL016W n.s yes	Intergene n.s yes
	18 -1,6 n.s	3181	chrXVI 1of2 oneprobe -0,8	520221 yes alpha	520280 0,1 yes	IRC15 4,0E-02 yes	Intergene n.s yes
		3182			alpha
		3183			alpha
		3184			alpha
		2838			alpha
		2841			alpha
		3301			1N
		3333			1N

Acknowledgments 19

We thank T. Rio Frio, S. Baulande and P. Legoix-Né (NGS platform, Institut Curie), A. Lermine (bioinformatics platform, Institut Curie), P.Lebaccon (PICT@Pasteur microscopy platform) for support. We warmly thank Jacob Swadling and Alexander Esin for helping out with the language. We also thank Camille Gautier for help with multiple mapping reads analysis and Marta Kwapisz for RNA-seq library preparation. We thank Valérie Borde for help with microarray experiments, David Sitbon, Valérie Borde and Arnaud De Muyt for fruitful discussions, David Sitbon and Ann Ehrenhofer-Murray for critical reading of the manuscript. This work has benefited from the facilities and expertise of the NGS platform of Institut Curie (supported by the Agence Nationale de la Recherche [ANR-10-EQPX-03, ANR10-INBS-09-08] and the Canceropôle Ile-de-France). A. Morillon's lab is supported by the Agence Nationale de la Recherche (DNA-life) and the European Research Council (EpincRNA starting grant, DARK consolidator grant). A. Taddei's lab is supported by funding from the Labex DEEP (ANR-11-LABX-0044_DEEP and ANR-10-IDEX-0001-02 PSL), from the ANR DNA-Life (ANR-15-CE12-0007) and FRM grant DEP20131128535. AH was supported by fellowships from the ENS PhD program and the FRM (FDT20160435654).

Disclosure Declaration

None of the authors have any competing interests