Myriam Authors

Ruault

Vittore F Scolari

Luciana Lazar-Stefanita

Antoine Hocher

Isabelle Loïodice

Camille Noûs

Romain Koszul
email: romain.koszul@pasteur.fr

Angela Taddei
email: angela.taddei@curie.fr

Sir3 mediates long-range chromosome interactions in budding yeast RUNNING TITLE (50 characters max): Sir3 proteins bridge distant loci together

Keywords: heterochromatin, yeast, nuclear organization, telomeres, nucleolus, Hi-C

Physical contacts between distant loci contribute to regulate genome function. However, the molecular mechanisms responsible for settling and maintaining such interactions remain poorly understood. Here we investigate the well conserved interactions between heterochromatin loci. In budding yeast, the 32 telomeres cluster in 3-5 foci in exponentially growing cells. This clustering is functionally linked to the formation of heterochromatin in subtelomeric regions through the recruitment of the silencing SIR complex composed of Sir2/3/4. Combining microscopy and Hi-C on strains expressing different alleles of SIR3, we show that the binding of Sir3 directly promotes long range contacts between distant regions, including the rDNA, telomeres, and internal Sir3 bound sites.

Furthermore, we unveil a new property of Sir3 in promoting rDNA compaction. Finally, using a synthetic approach we demonstrate that Sir3 can bond loci belonging to different chromosomes together, when targeted to these loci, independently of its interaction with its known partners (Rap1, Sir4), Sir2 activity or chromosome context. Altogether these data suggest that Sir3 acts as a molecular bridge that stabilizes long-range interactions.

Introduction

The 3D organization of a genome in space and time can potentially impact its functions. However, the mechanisms underlying this organization and its dynamics remain poorly understood. Contacts between distant loci can for instance influence and/or regulate biological functions, as exemplified by the enhancer-promoter interactions observed in metazoa. Another example observed from yeast to man is provided by the clustering of repeated sequences sequestering silencing factors and thus forming repressive subcompartments [START_REF] Meister | Building silent compartments at the nuclear periphery: a recurrent theme[END_REF].

The formation of these subcompartments in the nuclear space has several functional consequences.

Firstly, it allows for a more robust and specific repression of the associated sequences.

Subcompartments allow a local concentration of silencing factors, and consecutively, sequester these factors from the outside, limiting their action elsewhere in the genome. Secondly, the spatial proximity of the clustered sequences favor recombination events between them [START_REF] Batté | Recombination at subtelomeres is regulated by physical distance, double-strand break resection and chromatin status[END_REF]. Finally, since these sequences can be located on different chromosomes, or separated by long distances along the same chromosome, subcompartments affect the global genome folding in 3D space. Therefore, understanding the molecular mechanisms driving and regulating these long-range interactions provides insights on several DNA mechanisms, including genetic regulation, the maintenance of chromosomal stability, or also compaction and folding throughout the cell cycle. Chromatin tethering or co-localization involve a variety of direct or indirect molecular mechanisms, such as cohesinmediated loop formation [START_REF] Nasmyth | How might cohesin hold sister chromatids together?[END_REF][START_REF] Rao | A 3D Map of the Human Genome at Kilobase Resolution Reveals Principles of Chromatin Looping[END_REF], or anchoring of heterochromatin to the nuclear lamina [START_REF] Falk | Heterochromatin drives compartmentalization of inverted and conventional nuclei[END_REF]). However, protein-mediated direct bridging of distant loci positioned on the same or different chromosomes remain scantily described experimentally.

In exponentially growing cells, the 32 telomeres of S. cerevisiae cluster within 3 to 5 foci mainly found at the nuclear periphery, where the silencing information regulators Sir2, Sir3 and Sir4, forming the SIR complex, concentrate [START_REF] Gotta | The Clustering of Telomeres and Colocalization with Rapl, Sir3, and Sir4 Proteins in Wild-Type Saccharomyces cerevisiae[END_REF]. These clusters represent a well-documented example of a repressive nuclear subcompartment [START_REF] Gartenberg | The Nuts and Bolts of Transcriptionally Silent Chromatin in Saccharomyces cerevisiae[END_REF][START_REF] Kueng | SIR Proteins and the Assembly of Silent Chromatin in Budding Yeast[END_REF]). This organization is regulated by the physiological state of the cells, as illustrated in long lived quiescent cells, in which telomeres regroup into a unique, large cluster in the center of the nucleus [START_REF] Guidi | Spatial reorganization of telomeres in long-lived quiescent cells[END_REF].

At the molecular level, the SIR complex is recruited at the telomeric and sub-telomeric TG1-3 repeats by the binding of the transcription factor Rap1, whose C-terminal end contains binding domains for the silencing factors Sir3 and Sir4 [START_REF] Gartenberg | The Nuts and Bolts of Transcriptionally Silent Chromatin in Saccharomyces cerevisiae[END_REF]. The SIR complex is also recruited at the silent mating type loci (HM loci) through different DNA binding proteins, such as Orc1 and Abf1, which -like Rap1 -have also other functions in the cell [START_REF] Haber | Mating-Type Genes and MAT Switching in Saccharomyces cerevisiae[END_REF].

Furthermore, genome-wide ChIP experiments revealed that Sir3 can also be found at the promoters of some subtelomeric seripauperin genes [START_REF] Radman-Livaja | Dynamics of Sir3 spreading in budding yeast: secondary recruitment sites and euchromatic localization: Dynamics of Sir3 spreadingin budding yeast[END_REF]), and at a handful of discrete and telomere-distal loci not associated with specific functions (so far) [START_REF] Hocher | Expanding heterochromatin reveals discrete subtelomeric domains delimited by chromatin landscape transitions[END_REF][START_REF] Mitsumori | Analysis of novel Sir3 binding regions in Saccharomyces cerevisiae[END_REF][START_REF] Radman-Livaja | Dynamics of Sir3 spreading in budding yeast: secondary recruitment sites and euchromatic localization: Dynamics of Sir3 spreadingin budding yeast[END_REF][START_REF] Sperling | Histone H3 N-terminus regulates higher order structure of yeast heterochromatin[END_REF][START_REF] Takahashi | Dot1 and Histone H3K79 Methylation in Natural Telomeric and HM Silencing[END_REF][START_REF] Teytelman | Highly expressed loci are vulnerable to misleading ChIP localization of multiple unrelated proteins[END_REF]).

In addition, Sir2 is found at the rDNA locus where it protects rDNA repeats from recombination.

Although Sir3 was initially thought to associate to the nucleolus only in aged cells [START_REF] Kennedy | Redistribution of Silencing Proteins from Telomeres to the Nucleolus Is Associated with Extension of Life Span in S. cerevisiae[END_REF][START_REF] Sinclair | Accelerated Aging and Nucleolar Fragmentation in Yeast sgs1 Mutants[END_REF], it was later found to interact with the rDNA by chromatin immunoprecipitation (ChIP) in exponentially growing cells [START_REF] Radman-Livaja | Dynamics of Sir3 spreading in budding yeast: secondary recruitment sites and euchromatic localization: Dynamics of Sir3 spreadingin budding yeast[END_REF]).

Once recruited the SIR complex has the ability to spread along the chromatin fiber and to repress the transcription of underlying genes by the RNA polymerase II. This spreading results from the association of the histone deacetylase activity of Sir2 with the affinity of Sir3 for unmodified nucleosomes, linked by Sir4 interactions with both Sir2 and Sir3. The N-terminal BAH (bromo-adjacent homology) domain of Sir3 plays a major role in binding nucleosomes through an extensive surface of interaction, which is stabilized by the constitutive N-terminal acetylation of Sir3 [START_REF] Arnaudo | The N-terminal acetylation of Sir3 stabilizes its binding to the nucleosome core particle[END_REF][START_REF] Armache | Structural Basis of Silencing: Sir3 BAH Domain in Complex with a Nucleosome at 3.0 A Resolution[END_REF]).

According to these structural studies, acetylation of H4K16 or methylation of H3K79 would reduce nucleosome binding affinity for Sir3. Reciprocally H4K16 deacetylation by Sir2 increases the affinity of the Sir2/3/4 complex, favoring its spreading on adjacent nucleosomes [START_REF] Oppikofer | A dual role of H4K16 acetylation in the establishment of yeast silent chromatin: Dual role of H4K16 ac in yeast silencing[END_REF]. In vivo, SIR spreading is limited by the titration of SIR proteins, especially Sir3, and by H3K79me3, which prevents Sir3 binding in euchromatic regions. Indeed, overexpression of Sir3 leads to the formation of extended silent domains (ESD) covering subtelomeric regions, and delimited by transition zones where H3K79me3 levels abruptly increase [START_REF] Hocher | Expanding heterochromatin reveals discrete subtelomeric domains delimited by chromatin landscape transitions[END_REF].

A consequence of the limiting amount of SIR proteins is that loci associated with these proteins compete for these limiting pools [START_REF] Buck | Action of a RAP1 carboxy-terminal silencing domain reveals an underlying competition between HMR and telomeres in yeast[END_REF][START_REF] Michel | Spontaneous rDNA copy number variation modulates Sir2 levels and epigenetic gene silencing[END_REF][START_REF] Smith | Distribution of a Limited Sir2 Protein Pool Regulates the Strength of Yeast rDNA Silencing and Is Modulated by Sir4p[END_REF]. Telomere clustering, by increasing the local concentration within these subcompartments, could thus favor SIR spreading within subtelomeric regions. Reciprocally, telomere clustering as well as long-range interactions between HM loci depend on SIR recruitment at these loci [START_REF] Gotta | The Clustering of Telomeres and Colocalization with Rapl, Sir3, and Sir4 Proteins in Wild-Type Saccharomyces cerevisiae[END_REF][START_REF] Miele | Yeast Silent Mating Type Loci Form Heterochromatic Clusters through Silencer Protein-Dependent Long-Range Interactions[END_REF]. Furthermore, Sir3 overexpression leads to increased telomere clustering in addition to increased SIR spreading [START_REF] Ruault | Clustering heterochromatin: Sir3 promotes telomere clustering independently of silencing in yeast[END_REF]. A Sir3 point mutation (A2Q) enables to disentangle telomere clustering and SIR spreading. This mutation prevents the N-terminal acetylation of Sir3 and thus its ability to bind nucleosomes and spread along subtelomeric regions, yet Sir3-A2Q promotes telomere clustering [START_REF] Hocher | Expanding heterochromatin reveals discrete subtelomeric domains delimited by chromatin landscape transitions[END_REF][START_REF] Ruault | Clustering heterochromatin: Sir3 promotes telomere clustering independently of silencing in yeast[END_REF][START_REF] Sampath | Mutational Analysis of the Sir3 BAH Domain Reveals Multiple Points of Interaction with Nucleosomes[END_REF]. This led us to propose that Sir3 is the bridging factor between telomeres [START_REF] Meister | Building silent compartments at the nuclear periphery: a recurrent theme[END_REF][START_REF] Ruault | Clustering heterochromatin: Sir3 promotes telomere clustering independently of silencing in yeast[END_REF]).

Here we directly test this hypothesis by exploring the role of Sir3 on the global organization of the genome by combining genome-wide capture of chromosome conformation (Hi-C) and microscopy.

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RESULTS

Sir3 impacts genome organization

We previously showed that Sir3 is a limiting factor for telomeres clustering. Indeed, while the telomere-bound protein Rap1 fused to GFP in living cells forms 3-5 foci at the nuclear periphery in wild-type cells, only weak residual foci can be detected in sir3∆ cells (see Figure 1A and [START_REF] Ruault | Clustering heterochromatin: Sir3 promotes telomere clustering independently of silencing in yeast[END_REF]). These weak foci possibly result from the random encounter of telomeres at the nuclear periphery [START_REF] Zimmer | W303-1 derived strains : MATa ade2-1 can1-100 leu2-3,112 his3-11,15 trp1-1 ura3-1 (W303-1) yAT69 MATa adh4::URA3-4xUASG-(C1-3A)n yAT340 MATa ade2-1::ADE2 rap1::GFP-RAP1(LEU2) sik1::SIK1-mRFP(KanMX) yAT788 MATa ade2-1::ADE2 his3::GFP-LacI-SIR3(HIS3) sir3∆::KanMX yAT821 MATa adh4::URA3-4xUASG-(C1-3A)n pHIS3-GFP-LacI-SIR3(HIS3) yAT921 MATa adh4::URA3-4xUASG-(C1-3A)n pHIS3-GFP-LacI-SIR3(HIS3) sir3∆::KanMX yAT1004 MATa ade2-1::ADE2 net1::NET1-GFP(HIS3MX) sik1::SIK1-mRFP(KanMX) yAT[END_REF][START_REF] Hozé | Spatial telomere organization and clustering in yeast Saccharomyces cerevisiae nucleus is generated by a random dynamics of aggregation-dissociation[END_REF], due to Sir3 independent telomere anchoring at the nuclear envelope [START_REF] Hediger | Live Imaging of Telomeres: yKu and Sir Proteins Define Redundant Telomere-Anchoring Pathways in Yeast[END_REF][START_REF] Tham | Localization of Yeast Telomeres to the Nuclear Periphery Is Separable from Transcriptional Repression and Telomere Stability Functions[END_REF][START_REF] Schober | Yeast telomerase and the SUN domain protein Mps3 anchor telomeres and repress subtelomeric recombination[END_REF]). On the contrary, Sir3 overexpression leads to the grouping of telomeres in larger foci, or hyperclusters, located in the center of the nucleus [START_REF] Ruault | Clustering heterochromatin: Sir3 promotes telomere clustering independently of silencing in yeast[END_REF]. Moreover, overexpression of the sir3-A2Q mutant, whose Nterminal substitution blocks its acetylation, thus preventing silencing [START_REF] Wang | Importance of the Sir3 N Terminus and Its Acetylation for Yeast Transcriptional Silencing[END_REF]) and spreading in subtelomeric regions [START_REF] Hocher | Expanding heterochromatin reveals discrete subtelomeric domains delimited by chromatin landscape transitions[END_REF], also resulted in the formation of telomere hyperclusters (Figure 1A and (Ruault et al. 2011)). Whereas telomere clusters tend to dissociate in wild-type during mitosis (Figure 1A), hyperclusters mediated by Sir3 overexpression persist throughout the cell cycle (right panel of Figure 1A).

To explore the influence of Sir3 on the 3D organization of the genome we generated genome-wide Hi-C contact maps [START_REF] Lieberman-Aiden | Comprehensive Mapping of Long-Range Interactions Reveals Folding Principles of the Human Genome[END_REF]) of cells either depleted for Sir3, or carrying different sir3 alleles (Methods). To prevent unwanted influence(s) of the loop organization of mitotic chromosomes characterized in cycling cells [START_REF] Dauban | Regulation of Cohesin-Mediated Chromosome Folding by Eco1 and Other Partners[END_REF][START_REF] Garcia-Luis | FACT mediates cohesin function on chromatin[END_REF], Hi-C libraries were obtained from G1 elutriated daughter cells [START_REF] Diamond | Separation and enrichment of cell populations by centrifugal elutriation[END_REF]. The contact maps of wild-type cells revealed an enrichment of contacts between telomeres as well as between centromeres (red dots on the map), as previously reported [START_REF] Cournac | Normalization of a chromosomal contact map[END_REF][START_REF] Duan | A three-dimensional model of the yeast genome[END_REF]. The enrichment in telomere-telomere contacts almost disappears in the absence of Sir3 (sir3∆ background; Figure 1B, black arrowheads), whereas centromere-centromere contacts remain apparent (black * on the map). In the strain overexpressing Sir3, inter-subtelomeres contact frequency was strongly increased (Figure 1B) to the expense of contacts with other part of the genome (white lines in between red dots), while frequency of inter-centromere contacts appeared unchanged compared to a WT strain (black * on the map and Supplemental Fig S1A). These differences can be visualized more quantitatively by computing the log2ratio plot between mutants and WT maps (Figure 1C). In these maps, the color scale reflects the balance of contacts between the two conditions: the bluer, the more contacts in the WT; the redder, the more contacts in the mutant. The comparison of WT and sir3∆ maps results in the visualization of inter-subtelomeres contact ratio as blue pixels dots, as expected from the drop in inter-subtelomeres contact frequency observed in sir3∆ strain compared to WT. Conversely, the red stripes expanding from, and bridging subtelomeric regions indicate that the declustered telomeres are now able to contact more other regions of the genome in the absence of Sir3. This reflects the fact that telomeres are now less constrained in the absence of clustering (see also [START_REF] Muller | Characterizing meiotic chromosomes' structure and pairing using a designer sequence optimized for Hi-C[END_REF]) for a similar effect during meiosis prophase).

In strains overexpressing the silencing deficient Sir3-A2Q protein, the frequency of inter-telomere contacts also increased compared to WT, in agreement with the microscopy, although not to the extent of the strains overexpressing Sir3 (red dots in Figure 1B). The ratio map of the Sir3-A2Q overexpressing cells versus WT cells shows an inverted pattern compared to the sir3∆ vs. WT, with red dots at the level of the telomeres and blue lines in between telomeres (Figure 1C). A similar but stronger pattern was observed when comparing WT and Sir3 overexpressing strains in G1. This pattern was even more contrasted for G2/M cells, as telomere contacts are barely detectable in the wild-type strain while they remained high in number upon Sir3 overexpression (Figure 1B and 1C right panels and Supplemental Fig S1B). Altogether, these data show that contacts between subtelomeres decrease in the absence of Sir3, while they increased upon Sir3 or Sir3-A2Q overexpression, in good agreement with microscopy.

Sir3 binding coincides with inter-subtelomere contacts

To determine whether Sir3 overexpression has an impact on global chromosome compaction, we plotted the contact probability curve, 𝑝(𝑠). As expected, the curves display an enrichment in contacts for distances ~10 kb to 100 kb in G2/M compared to G1 samples (Figure 2A), reflecting the condensation of chromosomes during mitosis [START_REF] Schalbetter | SMC complexes differentially compact mitotic chromosomes according to genomic context[END_REF][START_REF] Lazar-Stefanita | Cohesins and condensins orchestrate the 4D dynamics of yeast chromosomes during the cell cycle[END_REF]).

However, different amounts of Sir3 did not significantly modify the compaction of the genome measured at the global level in G1 phase, as assessed by p(s) curves (Figure 2A).

We then focused on telomere-telomere contacts. We first compared the frequency of inter-telomere contacts as a function of the distance to telomeres, with the frequency of inter-centromeres contact as a function of the distance to centromeres (Supplemental Fig S2A). The frequency of contacts shows a very similar decay when the distances from telomeres or centromeres increase in wild-type cells. This is probably reflecting a similar flexibility of chromosome arms in these regions. In Sir3 or Sir3-A2Q overexpressing cells, we observed a higher frequency of inter-telomere contacts, while intercentromere contacts remain unchanged. However, assessing subtelomeric interactions in the telomere proximal regions was limited by the high variability of the alignment coverage in subtelomeric regions. We thus plotted the ratio of inter-subtelomere contacts between a given strain and a sir3∆ strain as a function of the distance to the telomeres to conceal these sequence biases (Figure 2B). The ratio of the signal in the sir3∆ strain measures the effects of the Sir3 protein on the contact probability and concomitantly conceals the Sir3-independent biases present in all strains. For both the wild-type or the Sir3-A2Q overexpressing strains, ratios were the highest near the telomeres and decreased slowly when moving away from it. In good agreement with our microscopy data, intersubtelomere contacts were higher in the Sir3-A2Q overexpressing strain than in the wild-type. Sir3 overexpression led to an even higher ratio of inter-subtelomere contacts that decay slowly with the distance to telomere over the first 10 kb before decreasing sharply up to 35 kb towards the centromeres. The extent of high level of inter-subtelomere contacts is comparable to the extent of Sir3 spreading as analyzed by ChIP ( [START_REF] Hocher | Expanding heterochromatin reveals discrete subtelomeric domains delimited by chromatin landscape transitions[END_REF] and Figure 2C), suggesting that Sir3 bound regions mediate these contacts.

Indeed, in wild-type cells, Sir3 binds to DNA 2.6 kb away on average from the last telomeric element.

In strains overexpressing Sir3, it can be detected as far as 20 kb away from the telomere, generating large Sir3-bound regions, dubbed extended silent domains or ESDs, [START_REF] Hocher | Expanding heterochromatin reveals discrete subtelomeric domains delimited by chromatin landscape transitions[END_REF] and Figure 2C). In contrast, Sir3-A2Q detection is limited to the TG repeats, even when overexpressed.

To assess the contribution of Sir3 binding to trans-subtelomere contacts, we computed the cumulated amount of trans contacts between subtelomeric loci as a function of their distance from their respective ESD (Methods). We observed a general increase of trans-subtelomere interactions for all loci located between the telomeres and the ESD boundaries, which we identify as the signature of the telomere hypercluster. Past the ESD border, trans-interactions decrease sharply, further suggesting that in Sir3 overexpressed conditions, Sir3 bound regions (ESDs) define a specific compartment isolated from the rest of the chromosomes. We also notice a persistence of trans contacts along the diagonal reflecting a co-alignment of subtelomeric regions adjacent to the ESDs (seen as the increased contact frequency between regions at equivalent genomic distances from the ESD boundary). These regions are possibly maintained in close proximity by the constraint imposed by the telomere hypercluster to the chromatin polymeric backbone. This co-alignment of chromosome arms is also visible in the cumulated interchromosome contact maps of the 100 kb subtelomeric regions and is very similar to the one observed in pericentromeric regions in G2/M phase (Supplemental Fig S1B and (Schalbetter et al. 2017)). In wild type cells or Sir3-A2Q overexpressing cells, we observed a progressive decrease of contacts from the telomere proximal region to the telomere distal region, probably reflecting the bonding of subtelomeres through shorter regions upstream of the ESD boundary (Figure 2D-E and Supplemental Fig S2A). Also, in WT cells, the intensity of the contacts is generally lower, reflecting the presence of more than one telomere cluster, possibly due to heterogeneity at the single cell level as a result of random encounters of different couples of telomeres (Figure 1A).

At the individual chromosome level, to visualize the correlation between Sir3 spreading and contacts between different subtelomeres, we built differential contact maps at an higher level of detail using the Serpentine algorithm [START_REF] Baudry | Serpentine: a flexible 2D binning method for differential Hi-C analysis[END_REF], which adapts the resolution on each position of the contact map to the data coverage (Figure 2E, see Supplemental Fig S2B for maps comprising all chromosomes). The Serpentine map comparing the Sir3 overexpressing cells with the sir3∆ showed a strong increase of inter-subtelomere contacts between ESDs of Chromosome VI and Chromosomes I, III and V (dark red regions at the corners of the maps, Figure 2E). As a consequence, these subtelomeric regions make less contact with non-subtelomeric regions. In the wild-type vs sir3∆ Serpentine maps we observed a similar, though less pronounced, trend. Thus, contacts between subtelomeric regions coincide with Sir3 binding, suggesting that Sir3 binding, which is the epigenetic information setting on the linear chromosomes, also defines the boundary of the telomere clusters in 3D (Figure 2F).

Sir3 but not Sir3-A2Q accumulates at the rDNA upon overexpression

We next asked whether non-subtelomeric Sir3 bound regions interact with subtelomeres. Sir3 binds to the rDNA through a mechanism that requires Sir2 deacetylase activity [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] Radman-Livaja | Dynamics of Sir3 spreading in budding yeast: secondary recruitment sites and euchromatic localization: Dynamics of Sir3 spreadingin budding yeast[END_REF]. In agreement with this work, live microscopy showed that a functional endogenously expressed yeGFP-tagged version of Sir3 colocalize with an endogenously expressed TagRFP-T-tagged rDNA bound protein Net1 (Figure 3A and Supplemental Fig S3A-C).

To identify precisely the region bound by Sir3 in a rDNA repeat unit, we performed ChIP experiments (Methods). Sir3 was found preferentially enriched in the non-transcribed regions (NTS1 and NTS2), and, to a lesser extent, around the RDN25 transcriptional unit and the promoter of the 37s rDNA precursor transcript (Figure 3B). Probing Sir3 binding in a strain overexpressing Sir3 we observed a strong accumulation of Sir3 over the rDNA unit, with a stronger signal at NTS1 and NTS2, showing that Sir3 concentration was limiting for binding not only at telomeres but also at the rDNA locus.

Consistently with previous work [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] we did not observe Sir3 binding in the absence of Sir2, even upon Sir3 overexpression. In contrast to wild-type Sir3, the Sir3-A2Q mutant that has lost its affinity to bind nucleosomes was not detected at the rDNA by ChIP. Together these results suggest that Sir3 accumulates at the rDNA through its affinity for unacetylated nucleosomes resulting from Sir2 activity (Figure 3B).

Sir3 promotes interaction of the telomeres with the rDNA locus

A 4C like analysis using the rDNA locus as a viewpoint (Methods) showed that when Sir3 is expressed at its endogenous level in exponential phase, the rDNA locus makes significant long range contacts with only two loci, located outside Chromosome XII (Figure 3C). When the rDNA locus was less active (overnight cultures), we observed that long-range contacts between the rDNA and other regions of the genome increased to 11, corresponding mostly to rDNA-telomere interactions (10 out of 11). In contrast to the contacts observed in exponential phase, those were lost in the absence of Sir3 (Figure 3C). These observations suggest that the activity of the rDNA prevents these Sir3 dependent contacts during exponential phase. In strains overexpressing Sir3, the rDNA locus now shows during exponential phase significant contacts with all telomeres, indicating that the increased amount of Sir3 at both types of loci favored their trans-contacts. When Sir3-A2Q was overexpressed, the loci in contacts with the rDNA remained similar to WT exponentially growing cells, but significant contacts with telomeres were not detected after an overnight culture. Therefore, rDNA-telomere contacts require Sir3 binding to the rDNA. Note that these analyses are averaged over a cell population, and don't necessarily reflect the contacts of the rDNA in each independent cell of the population.

To observe these contacts at the single cell level in strains expressing different sir3 alleles, we imaged cells expressing Rap1-GFP (telomere foci) and Sik1-mRFP (involved in pre-rRNA maturation and thus staining the nucleolus). Images were taken from overnight cultured cells enriched in rDNA-telomere interactions. In a wild-type strain, telomere foci are located at the nuclear periphery and don't interact with the nucleolus (Figure 3D). In contrast, telomeres in a strain overexpressing Sir3 group together and position in close contact with the nucleolus. More specifically, the telomere hypercluster is set within the nucleolus, excluding the Sik1 protein (magnification shown at the bottom of Figure 3D). The Sir3-A2Q mediated telomere hypercluster, on the other hand, didn't interact with the nucleolus, in agreement with Hi-C maps. We next measured the 3D-distance between the brightest telomere clusters and the nucleolus at a single cell level using NucLoc [START_REF] Berger | High-resolution statistical mapping reveals gene territories in live yeast[END_REF]. The distance between the brightest cluster of telomeres and the center of mass of the nucleolus was shorter in strains overexpressing Sir3 (with a median distance of 0.6 µm) compared to the wild type strain (1.2 µm) (Figure 3E). In strains overexpressing the Sir3-A2Q mutant, the distance was intermediate (median distance: 0.9 µm) reflecting the localization of the hypercluster at the center of the nucleus without interacting with the nucleolus, again in agreement with 4C maps. Also in agreement with 4C plots, the physiological state of the cells influenced the level of association between the telomere hypercluster and the nucleolus. Indeed, these two subnuclear compartments that are tightly associated after an overnight culture in Sir3 overexpressing cells dissociate upon dilution in fresh medium, and re-associate when cells reach saturation (Supplemental Fig S3D-E).

The number of rDNA repeats also influenced telomere hypercluster-rDNA contacts: fewer contacts were measured in cells carrying a short rDNA (25 copies) compared with cell with high copy number (190 repeats) in the Sir3 overexpressing mutant (Supplemental Fig S3F).

These results show that contacts between the rDNA and telomeres are modulated by 1) the amount of available Sir3 and 2) the transcriptional activity at the rDNA, which prevents these interactions.

Sir3 overexpression compacts the rDNA and inverts the spatial organization of the nucleolus

Since Sir3 has a strong impact on the spatial organization of the telomeres, we asked whether the accumulation of Sir3 at the rDNA locus could modify its spatial organization. In wild-type cells the rDNA labelled by Net1-GFP appears as a filament (Figure 4A-B), as previously reported [START_REF] Straight | Net1, a Sir2-Associated Nucleolar Protein Required for rDNA Silencing and Nucleolar Integrity[END_REF].

In Sir3 overexpressing cells, rDNA compaction varies with the physiological state of the nucleolus (Supplemental Fig S4A), with an increasing compaction from early to late exponential phase. As expected from cells grown overnight, the compacted rDNA is overlapping with the telomere hypercluster in most cells (Figure 4A bottom panel). In contrast, cells overexpressing the Sir3-A2Q mutant, that cannot bind the rDNA, show a filamentous rDNA (Figure 4A), suggesting a direct role for Sir3 in compacting the rDNA.

We next investigated the consequence of overexpressing Sir3 on nucleolus organization by imaging strains expressing the rDNA bound protein Net1 labeled with GFP, as well as Sik1-mRFP, staining the nucleolus. The GFP-labelled rDNA filament surrounds the Sik1 signal in wild-type cells or in cells overexpressing Sir3-A2Q (Figure 4B). This organization was inverted in Sir3 overexpressing cells, with the Net1-GFP signal located in the center of the nucleolus and surrounded by the Sik1 signal. The compaction of the rDNA as well as the inversion of the nucleolar organization were both independent of the formation of telomere hyperclusters, as they were observed in sir4∆ cells overexpressing Sir3

(Supplemental Fig S4B-C).
Finally, to get a better idea of the arrangement of the telomere hypercluster and the nucleolus when they interact, i.e. when the activity of the nucleolus is low (overnight cultures), we made triple labelled strains that allowed us to image three-ways combination of the telomere hypercluster (Rap1), Sir3, the rDNA (Net1) and the nucleolus (Sik1), in Sir3 overexpressing strains (Figure 4C). In those conditions, the Sir3 signal from the telomeres was indistinguishable from the rDNA bound one, corresponding to a large focus that overlapped with both the Net1 and Rap1 signals ( binds and coalesces the telomeres into a hypercluster, while accumulating at the rDNA as well and increasing its compaction. When the activity of the nucleolus is low, the regions of the rDNA that are Sir3-bound interact with the telomere hypercluster, bringing them together into a "super" Sir-enriched subcompartment. The part of the nucleolus dedicated to pre-RNA processing is excluded from this subcompartment and is self-organizing between the Sir3 enriched subcompartment and the nuclear periphery.

Ectopic Sir3-bound regions coincide with long range and trans-contacts

In addition to the telomeres and the rDNA, small peaks of Sir3 binding are detected by ChIP at a few discrete sites located internally on chromosomal arms [START_REF] Hocher | Expanding heterochromatin reveals discrete subtelomeric domains delimited by chromatin landscape transitions[END_REF][START_REF] Mitsumori | Analysis of novel Sir3 binding regions in Saccharomyces cerevisiae[END_REF][START_REF] Sperling | Histone H3 N-terminus regulates higher order structure of yeast heterochromatin[END_REF][START_REF] Takahashi | Dot1 and Histone H3K79 Methylation in Natural Telomeric and HM Silencing[END_REF]. Although some of these sites are known ChIP artefacts [START_REF] Teytelman | Highly expressed loci are vulnerable to misleading ChIP localization of multiple unrelated proteins[END_REF], others are not, as no enrichment was seen in the Sir3-A2Q expressing strain and Sir3 enrichment increased upon Sir3 overexpression [START_REF] Hocher | Expanding heterochromatin reveals discrete subtelomeric domains delimited by chromatin landscape transitions[END_REF].

The Serpentine maps revealed that some of those internal sites are also in contact with other Sir3bound loci, especially in cells that have slowed down cell division (i.e. overnight cultures). This was clearly visible on the Serpentine map of Chromosome I and VI when comparing cells overexpressing Sir3 to the ones overexpressing the Sir3-A2Q mutant protein, that is not recruited at these internal sites (Figure 5A). Especially, the YAT1 and IGD1 genes, respectively located on Chromosome I and VI (38 kb and 60 kb from the closest telomere), showed increased contact frequencies with the subtelomeres belonging to the same chromosome. This was also true for the YDL007C-A ORF located 13 kb away from the centromere of Chromosome IV, which interacts with the subtelomere IVL, thus counteracting the global Rabl-like organization of this chromosomal arm (Figure 5A). The Serpentine map indeed also revealed increased cis-interactions along the left arm of Chromosome IV, possibly reflecting a loop bridging YDL007C-A and the telomere in some of the cells. Further, we observed increased contact frequencies between the SIR3 gene, where Sir3 is recruited upon overexpression, and the subtelomeres located 56 kb downstream on the right arm of Chromosome XII (Supplemental Fig S5A), possibly hinting at a negative feedback mechanism regulating SIR3 expression. This mechanism would be different from one previously proposed [START_REF] Renauld | Silent domains are assembled continuously from the telomere and are defined by promoter distance and strength, and by SIR3 dosage[END_REF], where Sir3 spreading extends up to SIR3, as we do not observe such a continuous spreading of Sir3 from the telomeres up to the SIR3 locus (Supplemental Fig S5A).

However, not all Sir3 bound sites showed preferential interactions with telomeres, maybe owing to other constraints associated with cis-associated loci. Some of the internal Sir3 bound sites, that interact with the subtelomeres of their own chromosome in cis-, also interact in transwith other subtelomeres. Figure 5B presents the contacts between Chromosomes IV, V and VI and the rest of the genome, and emphasizes the ability of internal Sir3 bound sites to contact any subtelomere of the genome. No other transcontacts outside Sir3 bound regions were observed in these maps (Supplemental Fig S5B). However, Hi-C maps of the sir3∆ strain show apparent contacts between the FLO1, FLO9 and FLO11 genes located respectively in telomere-proximal regions of Chromosome I's right arm, left arm, and IX's right arm (Supplemental Fig S5C). Further analysis at the read level revealed that the apparent interaction between FLO1 and FLO9 could stem from an alignment artefact. However, this is not the case for FLO1-FLO11 interaction (Supplemental Fig S5D). In strains expressing Sir3 or Sir3A2Q, this interaction probably also occurs but is almost impossible to distinguish from Sir3 mediated interactions between subtelomeres.

In summary, we observed that upon Sir3 overexpression, increased Sir3 binding at internal sites smaller than 4 kb is accompanied by increased contacts between these sites and subtelomeric regions suggesting a direct involvement of Sir3 (Figure 5A-C). We do not observe increased interactions between these sites and subtelomeres when comparing the wild type and sir3Δ strains. However, we detect an increased compaction of these loci resulting in a higher frequency of contact between these sites and their flanking regions (Supplemental Fig S5E). It is possible that Sir3 retains a role of segregation and silencing on these sites without affecting the large-scale chromosome architecture in the tested wild type conditions. Alternatively, increased bridging between these loci and telomeres might be present in the cell population, but not significant enough to be detectable by our assay.

Artificial arrays of Sir3 are sufficient to promote trans-interactions

To directly test whether Sir3 is sufficient to bond 2 ectopic loci together, independently of silencing and clustering of telomeres, we hijacked the GFP-LacI / LacO array system to target Sir3 to specific loci harboring LacO arrays by expressing GFP-LacI fused to Sir3 (Figure 6A). The fusion of GFP-LacI in the Nterminus of Sir3 abolishes its silencing function, but its clustering function remains fully functional (Supplemental Fig S6A-B). LacO arrays were introduced at LYS2 (Chr II) and LEU2 (Chr III) loci in a strain unable to form telomere foci (rap1-17 sir3∆) to prevent the recruitment of GFP-LacI-Sir3 to telomeres.

We measured the pairing of the two arrays in different strains after an overnight culture. In a strain expressing the GFP-LacI, the two foci were paired in 6% of the cells (Figure 6B). When we expressed GFP-LacI-Sir3 the percentage of paired loci increased to 28%. This increased pairing was independent of Sir2 activity as 25% of the cells had a paired array when grown in the presence of the splitomicin Sir2 inhibitor. The Sir3-induced increased pairing was also independent of Sir4, since the percentage of pairing was similar in a WT and sir4∆ strain expressing GFP-LacI-Sir3. Because these experiments were performed in strains expressing a rap1 allele truncated for the Rap1-Sir3 interaction domain, we can infer that the Sir3 mediated pairing is also Rap1 independent. Therefore, this synthetic approach demonstrated that Sir3 can bond loci belonging to different chromosomes together, independently of its interaction with Rap1, and Sir4, and Sir2 activity, or chromosome context (Figure 6C). This strongly suggests that Sir3-Sir3 interactions provide the physical link that holds together Sir3 bound loci.

Discussion:

Here we investigated the impact of the silencing factor Sir3 on the regulation of the yeast genome 3D folding. We show that Sir3 is both necessary and limiting for telomere clustering.

Sir3 mediates interactions between telomeres.

Hi-C contact maps clearly show that interactions between subtelomeric regions decrease in the absence of Sir3, and increase upon Sir3 overexpression. These interactions do not require Sir3 spreading since overexpressing Sir3-A2Q, whose binding is limited to the telomeric TG repeats, is sufficient to increase subtelomeric contacts. However, contacts rapidly decrease away from telomeres in the Sir3-A2Q strain, whereas in the strain overexpressing the WT version of Sir3 they remain strong up to over ~20 kb along the subtelomeres. In addition, when Sir3 is overexpressed the increased contacts coincide with Sir3 enrichment along the subtelomeres. This suggests that the (limited) subtelomeric contacts observed in the Sir3-A2Q strain are an indirect consequence of the Sir3dependent bonding of chromosomes by their telomeres, whereas the spreading of WT Sir3 directly promotes the subtelomeric contacts. Extended Sir3 spreading is also associated with increased interaction in cispossibly reflecting the compaction resulting from heterochromatin formation.

Sir3 impacts nucleolar organization.

Another consequence of overexpressing Sir3 is its impact on the organization of the nucleolus.

Although Sir3 association with the rDNA has been documented [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] Radman-Livaja | Dynamics of Sir3 spreading in budding yeast: secondary recruitment sites and euchromatic localization: Dynamics of Sir3 spreadingin budding yeast[END_REF], its function at this locus remained elusive. Our experiments show that increasing Sir3 association with the rDNA increases its compaction, especially when the rDNA activity is low (i.e. in slow growing cells). This compaction is accompanied by an inversion of the nucleolar organization: while the rDNA is found at the periphery of the nucleolus in wild-type cells, it becomes nested within the nucleolus upon Sir3 overexpression (Figure 4). In addition, in these conditions both Hi-C and microscopy show that the rDNA associates with telomeres in a Sir3 dependent manner. Although we could not observe this association by microscopy in wild-type cells, such contacts were nevertheless detected in the present Hi-C data for 10 telomeres (out of 32) in wild-type cells with low rDNA activity.

These observations show that Sir3 influences the overall genome organization not only at the level of subtelomeric regions, but also more broadly.

It is noteworthy that Sir3 recruitment at the rDNA was first reported as a hallmark of aged cells [START_REF] Kennedy | Redistribution of Silencing Proteins from Telomeres to the Nucleolus Is Associated with Extension of Life Span in S. cerevisiae[END_REF][START_REF] Sinclair | Accelerated Aging and Nucleolar Fragmentation in Yeast sgs1 Mutants[END_REF], possibly owing to a lack of sensitivity to detect Sir3 in the nucleolus of young cells. Sir3 was especially visible in the fragmented nucleoli resulting from the instability of the rDNA in old cells. It will be important to test whether this is related to the ability of Sir3 to compact the rDNA and whether this new property of Sir3 has impacts on the stability of this locus.

Tethering Sir3 is sufficient to bridge chromatin loci.

In addition to the transcontacts between large Sir3 covered regions, such as subtelomeres, and the rDNA, we also observed contacts between Sir3 bound subtelomeric domains and at least 5 discrete, few kb long regions enriched in Sir3 and positioned internally on chromosomal arms [START_REF] Hocher | Expanding heterochromatin reveals discrete subtelomeric domains delimited by chromatin landscape transitions[END_REF][START_REF] Mitsumori | Analysis of novel Sir3 binding regions in Saccharomyces cerevisiae[END_REF][START_REF] Sperling | Histone H3 N-terminus regulates higher order structure of yeast heterochromatin[END_REF][START_REF] Takahashi | Dot1 and Histone H3K79 Methylation in Natural Telomeric and HM Silencing[END_REF]. Although weaker than contacts between subtelomeres, they can counteract the canonical Rabl-like configuration (where telomeres and centromeres are on opposite sides of the nucleus), drawing together the centromere proximal locus of Chromosome IV and its subtelomeres. These internal sites also interact with subtelomeres from other chromosomes in trans-(Figure 5). Albeit these sites do not show specific contacts with telomeres in wild-type cells, we could detect an increased compaction of those loci.

While the functional significance of these internal Sir3 binding sites remains to be elucidated, they correspond to intergenic regions or genes that are not expressed in our culture conditions. Of note, one example is the YAT1 gene that has the highest G+C content of the budding yeast genome (58%) [START_REF] Chavez | Hpr1 Is Preferentially Required for Transcription of Either Long or G+ C-Rich DNA Sequences in Saccharomyces cerevisiae[END_REF]. This is also one of the rare ORF containing a cluster of meiotic double strand break hot spots [START_REF] Pan | A Hierarchical Combination of Factors Shapes the Genome-wide Topography of Yeast Meiotic Recombination Initiation[END_REF]). Furthermore, this gene is prone to form R-loops when expressed under the control of the strong GAL1 promoter [START_REF] Bonnet | Introns Protect Eukaryotic Genomes from Transcription-Associated Genetic Instability[END_REF]). This could be related to our previous findings that Sir3 is recruited at sites of chromatin stress, favoring their perinuclear anchoring [START_REF] Dubarry | Tight protein-DNA interactions favor gene silencing[END_REF]. As for the rDNA, it will be important to test whether Sir3 contributes to the genetic stability of these loci. Finally, we showed that the artificial tethering of Sir3 to two lacO arrays is sufficient to increase their trans-association, independently of Sir3 interaction with its known partners (Rap1, and Sir4), Sir2 activity or chromosome context (Figure 6).

Dynamics of Sir3 mediated long-range contacts

Telomere clusters are dynamic. They can split and fuse [START_REF] Hozé | Spatial telomere organization and clustering in yeast Saccharomyces cerevisiae nucleus is generated by a random dynamics of aggregation-dissociation[END_REF][START_REF] Schober | Controlled exchange of chromosomal arms reveals principles driving telomere interactions in yeast[END_REF], and dissolve during mitosis to reform in G1 phase. We observed that Sir3 overexpression prevents this dissolution probably by stabilizing telomere-telomere interactions. We noticed that telomere clusters are more visible by microscopy as well as by Hi-C in slow growing cells possibly due to the time required for telomeres to encounter one another again after mitosis. This is also true for other interactions between Sir3 bound regions, such as the rDNA, or discrete internal or synthetic sites. We propose three non-exclusive explanations for this observation. First, some chromatin movements or constraints linked to genome activity could destabilize or prevent Sir3 mediated contacts. Indeed, rDNA-telomere contacts are limited when the rDNA is transcriptionally active, possibly owing to a stronger anchoring at the nuclear periphery when the rDNA is active. Alternatively, the high density of ongoing transcripts could prevent telomeres to contact the rDNA through steric hindrance. Finally, long-range contacts may be limited by the time required for Sir3 bound loci to meet in the nuclear space.

Mechanisms of Sir3 mediated trans-interactions.

Recent work [START_REF] Gibson | Organization of Chromatin by Intrinsic and Regulated Phase Separation[END_REF] suggests that properties inherent to chromatin, including nucleosomal spacing and histone acetylation status, could promote phase separation phenomena within the nucleoplasm. Reconstituted chromatin undergoes liquid-liquid phase separation (LLPS) in physiologic salt, forming droplets that can be dissolved by acetylating histone tails. Given the presence of the histone deacetylase Sir2 in subtelomeric regions, telomere clustering could have been proposed to result from the LLPS of non-acetylaled chromatin. However, we showed that cells expressing the sir3-A2Q allele can form telomere hyperclusters even in the absence of Sir2. Furthermore, the artificial tethering of Sir3 to two distant euchromatic loci is sufficient to bridge them independently of Sir2 activity. These results indicate that the clustering of Sir3 bound loci does not depend on their chromatin status but rather is mediated by direct Sir3-Sir3 interactions. Consistent with this hypothesis, Sir3 carries a very well-characterized dimerization domain on its C-terminal part, the wH domain [START_REF] Oppikofer | Dimerization of Sir3 via its C-terminal winged helix domain is essential for yeast heterochromatin formation[END_REF]). In addition, Sir3 C-terminal domain can interact with a more internal part of Sir3 [START_REF] King | Domain Structure and Protein Interactions of the Silent Information Regulator Sir3 Revealed by Screening a Nested Deletion Library of Protein Fragments[END_REF]. The importance of the dimerization properties of Sir3 has been mainly discussed for its contribution to Sir3 spreading in cis-, and its consequence on chromatin compaction and accessibility [START_REF] Oppikofer | Dimerization of Sir3 via its C-terminal winged helix domain is essential for yeast heterochromatin formation[END_REF][START_REF] Behrouzi | Heterochromatin assembly by interrupted Sir3 bridges across neighboring nucleosomes[END_REF][START_REF] Swygert | Solution-state conformation and stoichiometry of yeast Sir3 heterochromatin fibres[END_REF][START_REF] Swygert | SIR proteins create compact heterochromatin fibers[END_REF]. However, these specific Sir3-Sir3 interactions could also build up molecular bridges between Sir3-bound regions in trans-. Sir3 can bridge mononucleosomes or nucleosomal arrays in vitro [START_REF] Georgel | Sir3-dependent assembly of supramolecular chromatin structures in vitro[END_REF][START_REF] Mcbryant | The Silent Information Regulator 3 Protein, SIR3p, Binds to Chromatin Fibers and Assembles a Hypercondensed Chromatin Architecture in the Presence of Salt[END_REF][START_REF] Behrouzi | Heterochromatin assembly by interrupted Sir3 bridges across neighboring nucleosomes[END_REF], and Sir3 ability to bridge mononucleosomes requires its wH dimerization domain. We thus propose, that specific Sir3-Sir3 interactions between arrays of Sir3 molecules associated with subtelomeres, the rDNA, or internal regions would form a dynamic, gel like, network structure rather than a liquid droplet as previously proposed for other type of nuclear foci [START_REF] Sawyer | Phase separated microenvironments inside the cell nucleus are linked to disease and regulate epigenetic state, transcription and RNA processing[END_REF]. Further work is needed to decipher the physical nature of these foci. Although heterochromatin foci in mouse cells were first described as liquid droplets [START_REF] Strom | Phase separation drives heterochromatin domain formation[END_REF], a recent study indicates that these foci rather resemble collapsed polymer globules [START_REF] Erdel | Mouse Heterochromatin Adopts Digital Compaction States without Showing Hallmarks of HP1-Driven Liquid-Liquid Phase Separation[END_REF]. Whether specific heterochromatin factors are responsible for this collapse is not known, but HP1, the functional homolog of Sir3 does not seem required [START_REF] Erdel | Mouse Heterochromatin Adopts Digital Compaction States without Showing Hallmarks of HP1-Driven Liquid-Liquid Phase Separation[END_REF][START_REF] Peters | Loss of the Suv39h Histone Methyltransferases Impairs Mammalian Heterochromatin and Genome Stability[END_REF][START_REF] Mateos-Langerak | Pericentromeric Heterochromatin Domains Are Maintained without Accumulation of HP1□D[END_REF].

Independently of the exact mechanism by which Sir3 promotes long range interactions, it provides a means to cluster SIR nucleation domains into a sub-volume of the nucleus, thus increasing the local concentration of the SIR complex. This will in turn favor the spreading of the complex along the chromatin fiber, thus reinforcing the clustering of these regions. As proposed earlier [START_REF] Meister | Building silent compartments at the nuclear periphery: a recurrent theme[END_REF], this positive feedback will lead to the sequestration of the SIR complex favoring the silencing in these subnuclear compartments while preventing its action elsewhere in the genome.

We show that Sir3 plays an important, direct role in the colocalization of multiple chromatin loci in budding yeast. How these long-range interactions are regulated in response to environmental cues, eventually reshaping chromosome folding to a larger extent and playing new roles in genetic regulation for instance, has to be further investigated.

Methods

Media and Growth conditions

Yeast cells were grown either in rich medium (YPD) or in enriched complete synthetic medium (2× final concentration of CSM, MP Biomedicals) supplemented with 2% glucose or 2% galactose (wt/vol). All the strains were grown at 30°C with shaking at 250 rpm. For galactose induction, cells were grown overnight in YPGal medium (yeast extract, peptone, 2% galactose wt/vol) for pre-culture and diluted the next day in the same medium for the experiment. For Sir2 inhibition, splitomycin was added directly to the culture at a final concentration of 62.5 µM for 4 hours. G2/M synchronization was done as in [START_REF] Lazar-Stefanita | Cohesins and condensins orchestrate the 4D dynamics of yeast chromosomes during the cell cycle[END_REF]. Synchronization at the G2/M transition was achieved by restarting G1 cells in YPD at 30°C for 1 h, followed by the addition of nocodazole (Calbiochem; 15 µg/ml) and incubation for another 2 h at 30°C.

Strains

The strains used in this study are listed in Supplemental Table S1 and Supplemental Table S3. They are all derivatives of W303 [START_REF] Thomas | Elevated recombination rates in transcriptionally active DNA[END_REF] except the ones used for the Hi-C experiment (BY4741, Euroscarf). Gene deletions, insertions of alternative promoters and gene tagging were performed by PCR-based gene targeting. Plasmids pAT234 is an integrative plasmid expressing GFP-LacI-Sir3 under the HIS3 promoter. This plasmid is derived from a plasmid expressing GFP-LacI-NLS under the HIS3 promoter (pAFS135; [START_REF] Straight | Time-Lapse Microscopy Reveals Unique Roles for Kinesins during Anaphase in Budding Yeast[END_REF] and was built in two steps. First, the STOP codon of GFP-LacI-NLS TAA was mutated to AGA by direct mutagenesis using primer pair am449 (5' AAAGAAGAAGAGAAAGGTTGCCAGATCTAGAGCGGCCGCCACCGCGGTGG 3') /am450 (5' CCACCGCGGTGGCGGCCGCTCTAGATCTGGCAACCTTTCTCTTCTTCTTT 3') yielding pAT233. The full length SIR3 gene was amplified by PCR using primer pair am446 (5' ATAAGAATGCGGCCGCaATGGCTAAAACATTGAAAG 3') /am448 (5' AGTCGAGCTCTCAAATGCAGTCCATATTTTTG 3'). The resulting PCR product was digested NotI/sacI and cloned into pAT233 digested NotI/sacI to generate the plasmid encoding the GFP-LacI-Sir3 protein.

Chromatin Immunoprecipitation experiments A total of 20 OD600nm equivalent of cells were fixed in 20 mL with 0.9 % formaldehyde for 15 min at 30°C, quenched with 0.125 M glycine for 5 min and washed twice in cold TBS 1´ pH 7.6. Pellets were suspended in 1mL TBS 1´, centrifuged and frozen in liquid nitrogen for -80°C storage. All following steps were done at 4°C unless indicated. Pellets were resuspended in 500 µL of lysis buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA pH8, 16.7 mM Tris pH8, 167 mM NaCl, 0.5 % BSA, 0.02 g/L tRNA and 2.5 µL of protease inhibitor from Sigma-Aldrich P1860) and mechanically lysed using a Fastprep instrument (MP Biomedicals) with 0.5mm zirconium beads (Biospec Products): intensity 6, 3 cycles of 30 s with 3 min incubation on ice in between cycles. 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 16000 g. 10 µL were kept to be used as Input DNA. Cleared lysate was incubated overnight with 1 µL of polyclonal antibody anti-Sir3 (Agro-bio). 50 µL of magnetic beads protein A (NEB) were added to the mixture and incubated for 4 h at 4°C on a rotating wheel. Magnetic beads were washed sequentially with lysis buffer, twice with RIPA buffer (0.1% SDS, 10mM Tris pH7.6, 1mM 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 1/10 with elution buffer (50 mM Tris, 10 mM EDTA pH8, 1% SDS) and beads were re-suspended in 100 µL of elution buffer. A reversal cross-linking was performed by heating samples overnight at 65°C. Proteins were digested with Proteinase K (0.4 mg/ml) in the presence of glycogen and the remaining DNA was purified on QIAquick PCR purification columns. Finally, samples were treated with 29 µg/mL RNase A for 30 min at 37°C and used for quantitative PCR.

ChIP quantification by quantitative PCR

Quantitative PCR was performed on 1/40 of the immunoprecipitated DNA and 1/3200 of the input DNA. Sequences of interest were amplified using the SYBR Green PCR Master Mix (Applied Biosystems) and the primers listed in Supplemental table S2. PCR reactions were conducted at 95°C for 10 min followed by 40 cycles at 95°C for 15 s and 60°C for 30 s on a real-time quantitative PCR system (7900HT Fast Real-Time PCR; Applied Biosystems). Each real-time PCR reaction was performed in triplicate. The signal from a given region was normalized to the one from the OLI1 control locus in immunoprecipitated and input DNA samples. Plots represent the mean value obtained for at least three independent experiments; error bars correspond to SEM.

Microscopy

Set of images from any given figure panels were acquired using identical acquisition parameters. For all fluorescent images, the axial (z) step is 200 nm and images shown are a maximum intensity projection of z-stack images with the exception of Figure 4C and Supplemental Fig S4D that are presenting only a slice of the z-stack. Images were acquired on two different systems, either a widefield microscopy system or a spinning disk system, both of them being driven by MetaMorph software (Molecular Devices). Images of panel 3D, S3D, S6B were acquired using a wide-field microscopy system based on an inverted microscope (TE2000; Nikon) equipped with a 100´/1.4 NA immersion objective, a charge-coupled device (CCD) camera (Coolsnap HQ2; Photometrics). A xenon arc lamp (Lambda LS; Sutter Instrument Co.) was used to illuminate the samples. A dual-view micro-imager device, described in [START_REF] Guidi | Spatial reorganization of telomeres in long-lived quiescent cells[END_REF], allows the simultaneous measurement of two-color information on the same sensor. Images of panels 1A, 4A, 4C, S4A and S4D were acquired with the same microscope, using a Cmos camera and another illumination system the Spectra X light engine lamp (Lumencor, Inc). This system allows the fast acquisition of dual-color images when used in combination with a double filter. Images of panels 3A, 4B, 6A, S4B, S4C were acquired on a spinning-disk confocal microscope (Revolution XD Confocal System; ANDOR) equipped with a spinning-disk unit (CSU-X1; Yokogawa), a microscope (Ti 2000; Nikon) with a 100´/1.4 NA oil immersion objective, and an EM CCD camera (iXON DU-885; ANDOR). Images shown in panel 4C and S4D were deconvolved (see below). Images of panel 3A were acquired with an upgraded version of the system allowing the simultaneous acquisition of the GFP and RFP channels using a Tu-Cam module (Andor).

Microscopy data processing Deconvolution of images acquired with the wide-field microscopy system was made using the Meinel algorithm in Metamorph (8 iterations; sigma = 0.8; frequency 3; MDS Analytical Technologies).

Hi-C library generation Hi-C libraries were generated as described in [START_REF] Lazar-Stefanita | Cohesins and condensins orchestrate the 4D dynamics of yeast chromosomes during the cell cycle[END_REF], using a DpnII four-cutter enzyme. The Hi-C libraries were sequenced on an Illumina NextSeq 500 apparatus (2´75 highthroughput kits). The PE reads were filtered and aligned according to the protocol described in [START_REF] Cournac | Normalization of a chromosomal contact map[END_REF]. Sequencing libraries are publicly available on the NCBI database (accession number PRJNA642827).

Visualization of contact maps Sparse matrices were binned at 5 or 50 kb pixel size as indicated in figure legends. Normalization, when applied, used the sequential normalization procedure (SCN; [START_REF] Cournac | Normalization of a chromosomal contact map[END_REF])) and rasterized using a color-scale.

Ratio maps and serpentine binning

The log2 ratio of pairs of raw contact maps, binned at 50 kb, was computed. The resulting matrix was normalized by subtracting the median of its values. The result was rasterized using a colorscale. Serpentine binning was applied for some of the ratio plots. Serpentine is an algorithm developed to overcome the limits of standard binning with respect to read coverage. When using Serpentine, the bin sizes are chosen non-uniformly on the maps as a function of the sequencing coverage (see [START_REF] Baudry | Serpentine: a flexible 2D binning method for differential Hi-C analysis[END_REF] for validation and details on the program). Filtering was applied to remove speckles to avoid artefacts. The Serpentine ratio allows emphasizing contact patterns otherwise drown in the sampling noise of Hi-C data. Serpentine binning was run independently on contact maps of single or couple of chromosomes, binned at 1250 bp resolution, with the following parameters: threshold=70 and minthreshold=7, as discussed in [START_REF] Baudry | Serpentine: a flexible 2D binning method for differential Hi-C analysis[END_REF]. Resulting ratio maps were then normalized by subtracting the mean value of their pixels and rasterized using a colorscale centered in zero.

Contact probability as a function of genomic distance

To compute the intra-chromosomal P(s) plots, pair of reads aligned in intra-chromosomal positions were partitioned by chromosome arms. Reads oriented towards different directions or separated by < 1.5 kb were discarded to discard self-circularizing events. For each chromosome, read pairs were logbinned as a function of their genomic distance s (in kb), according to the following formula: 𝑏𝑖𝑛 = floor(log(𝑠) / log(1.1)). The P(s) plot is the histogram computed on the sum of read pairs for each bin. This sum is weighted by the bin size 1.1 (1+bin) (because of the log-binning), as well as the difference between the length of the chromosome and the genomic distance s. The difference acts as a proxy for the number of possible events.

Inter-subtelomere and Inter-centromere cumulative contact maps Inter-subtelomere/centromere contacts where obtained by selecting all the centromere/telomere centered trans-interactions in normalized contact maps as a set of small sub-matrices. A mean interaction profile is then obtained by taking the mean of all these sub-matrixes, reoriented consistently, excluding all the unmapped regions. Subtelomeric regions are aligned and centred around the X-core element.

Inter-subtelomere and Inter-centromere contacts as a function of the distance to telomeres

The profile of co-alignment has been obtained by selecting the row/column as the basis of interaction between the respectively the X-core element or the centromere, and the subtelomere/pericentromere region, in the respective cumulative contact map. The error bar reflects the standard error on the mean, as a consequence of the variability of interaction between each telomere-telomere or centromere-centromere intra couple.

Ratio of the Inter-subtelomere contacts as a function of the distance to telomeres The contacts are obtained by comparing the maps obtained in exponential phase of the wild type, the Sir3 overexpression and the Sir3-A2Q overexpression strains, to the maps obtained in the sir3∆ mutant.

For each telomere we obtained, in tabular form, the interactions between the bin containing the Xcore element with the sub-telomeres of all the other chromosomes. Chromosome III was excluded from the analysis. The table is then reordered as a function of the distance between subtelomere location and the X-core element of the corresponding telomere. The values obtained the aforementioned way in the considered condition are then divided by the values obtained the same way in the sir3∆ mutant. The mean ratio-table, obtained over all X-core elements and subtelomeres, was then computed. Discarding infinite and NaN values. The resulting table represents the Intersubtelomere contacts as a function of the distance from telomeres for the considered condition.

Detection of ESD boundaries

Sir3 binds to telomeres and subtelomeres in the wild type and overexpressed strains. We obtained the location of ESD boundaries from the z-transformed Sir3 ChIP binding profiles from [START_REF] Hocher | Expanding heterochromatin reveals discrete subtelomeric domains delimited by chromatin landscape transitions[END_REF]. Those profiles show z-values higher than 1 at the extremities of chromosomes. We obtained z-values by binning the ChIP data in 5 kb bins, removing bins without data, then calculating the median and the median-absolute-deviation over all chromosomes. The z-values is then computed by the following formula: Log[0.67599 (𝑥 -𝑚𝑒𝑑𝑖𝑎𝑛) / (𝑀𝐴𝐷)]. For each chromosome, the two ESD boundaries were chosen as the coordinate of the lowest/higher bin in the chromosome with z-values less than 1 in ChIP signal of the overexpressed Sir3 mutant.

Cumulated ratio-matrices at the ESD boundary

The cumulated ratio-matrices were obtained starting from two raw contact maps binned at 5 kb, here named the biological condition and the control. For each couple of ESD boundaries belonging to different chromosomes, excluding Chromosome III, for each matrix, a submatrix was obtained such that the pixel accounting for the direct ESD-ESD interaction is placed at the center. Each of those submatrices is then reoriented such that the telomere-telomere boundary is placed toward the lower right corner, all interactions beyond the telomere-telomere boundary (such as interaction belonging to adjacent chromosomes) are discarded by setting the values to NaN. The ratio submatrices are obtained by dividing the biological condition over the control and taking its logarithm in base 2. Ratiosubmatrices are then normalized by subtracting to all of them the same value, computed by the mean of all the pixel in all the ratio-submatrices, excluding NaNs and Infinite values. Finally, the cumulated ratio-matrix is the mean of all ratio-submatrices.

4C extraction profiles

From Hi-C matrices, is it possible to extract interactions between a single locus on the genome, with the rest of the genome, by slicing the full rows of the matrix corresponding to the bin containing the locus of interest. If the locus spans multiple bins, we sliced multiple rows and then reduced the signal to 1 dimension by taking the sum of the values for each column. In the case of the 4C of the rDNA locus, in order to obtain maximum specificity, the raw data was realigned on a genome with the rDNA removed from the sequence of Chromosome XII and placed on an individual chromosome, stripped of any repeated sequences. The 4C data was then obtained by slicing the full rows corresponding to that individual chromosome, and summing the values of each columns, on the matrix obtained using this special genome as a reference. To detect significant interactions, we plot in green color the z-transform of the 4C signal. The procedure is computed on the logarithm in base 10 of the 4C signal. The median 𝜇 and the median absolute deviation (𝑀𝐴𝐷) was computed. The standard deviation was estimated using the following formula: 𝜎 = 𝑀𝐴𝐷 / 0.675. The z-transformed 4C is obtained by the following formula: 𝑧 = (𝑥 -𝜇)/𝜎. Interactions are considered significant, and plotted in red color, according to the following condition: 𝑧 > 2.5. The S. cerevisiae rDNA locus is composed of ~150 tandem copies of a 9.1 kb repeating unit, each encoding two transcribed region RDN5 and RDNA37 (comprising RDN18, RDN5.8 and RDNA25 genes). The graph represents Sir3 occupancy along the rDNA locus probed by ChIP-qPCR using an anti-Sir3 antibody [START_REF] Ruault | Clustering heterochromatin: Sir3 promotes telomere clustering independently of silencing in yeast[END_REF] in WT (yAT232), GAL1p-SIR3 (yAT208), GAL1p-Sir3-A2Q (yAT1205), and GAL1p-SIR3 sir2∆ (yAT772) strains. Primer pair 1 amplifies a region of the RDN25 locus, primer pair 2 amplifies a region in NTS1 (Non-Transcribed Spacer 1) region, primer pair 3 amplifies a region in NTS2 and primer 4 amplifies a region in the ETS1 (External Transcribed Spacer 1) region (see Supplemental table S2 for primer sequences). Strains were grown in YPGal for 48 hours. The bar graph represents the Sir3 enrichment over the mitochondrial locus OLI1. Error bars show s.e.m of three independent experiments, each analyzed in triplicate qPCRs. (C) 4C extraction profiles representing contact maps between the rDNA repeats and the rest of the genome, profiles are shown for all strains in both exponential and overnight cultures. Red highlighted loci correspond to bins with z-value greater than 2.5. (D) Representative fluorescent images of a double tagged strain Rap1-GFP / Sik1-mRFP in strains expressing endogenous level of Sir3 (yAT340), high levels of Sir3 (yAT341) or high levels of the separation of function mutant Sir3-A2Q (yAT1198) after an overnight culture in CSM 2% galactose. Magnification of representative nuclei (1 and 2) are presented at the bottom of the panel. (E) Distance between the brightest Rap1-GFP cluster and the nucleolus center is plotted for a wild-type (yAT340, n=581), a strain overexpressing Sir3 (yAT341, n=627) and a strain overexpressing Sir3-A2Q (yAT1198, n=590) using the Nucloc software [START_REF] Berger | High-resolution statistical mapping reveals gene territories in live yeast[END_REF]. Cells were grown in CSM 2% galactose overnight before imaging. Scale bar is 1 µm in all panels. Representative fluorescent images of Rap1-GFP / Net1-mRFP strains expressing either endogenous level of Sir3 (yAT3729), high levels of Sir3 (yAT3730) or high levels of the Sir3-A2Q mutant (yAT3733). Cells were grown in CSM with 2% galactose and imaged after an overnight culture. (B) Representative fluorescent images of a double tagged strain Net1-GFP / Sik1-mRFP in strains expressing endogenous level of Sir3 (yAT1004), high levels of Sir3 (yAT1008) or high levels of the Sir3-A2Q mutant (yAT1541). Cells were grown in CSM 2% galactose and imaged after an overnight culture. In the bottom panels, close-up of selected nuclei from the field are shown (GFP-channel, RFP channel and merge). (C) Representative fluorescent deconvolved images of triple tagged strains, individual and merge channels are shown. Cells were grown overnight in CSM 2% glucose and are all expressing high Sir3 levels (Sir3 under the control of the constitutive GPD promoter). From the Top to the Bottom: Net1-BFP2 Rap1-GFP Sir3-mCherry (yAT3901), Sik1-BFP2 Net1-GFP sir3-mCherry (yAT2213) and Sik1-BFP2 Rap1-GFP Sir3-mCherry (yAT3666). (D) Schema representing the nuclear organization of a wild-type, a strain overexpressing the Sir3-A2Q separation of function mutant and a strain overexpressing the wild-type Sir3. Scale bar is 1 µm in all panels.

  Figure 4C upper panel and Supplemental Fig S4D) with no overlap between these two signals. Furthermore, the Sir3 signal overlapping with Net1 was always facing the Sik1 signal (i.e. the nucleolus), while the Sir3 signal colocalizing with Rap1 was found at the opposite side of the nucleolus (Figure 4C lower panel and Supplemental Fig S4D). Therefore, the subtelomeres (enriched in Sir3 and deprived of Rap1) are in contacts with the Sir3-bound rDNA, while the extremity of the telomeres (enriched with Rap1) protrudes towards the nuclear interior. These results led us to propose the model presented in Figure 4D: in strains overexpressing Sir3, Sir3

Figure Legends: Figure 1 :

 1 Figure Legends: Figure 1: Sir3 impacts genome organization. (A) Representative fluorescent images of the telomereassociated protein Rap1 tagged with GFP in exponentially growing strains expressing different Sir3 levels: wild-type (yAT2583), no Sir3 (yAT2584), high Sir3 levels (yAT2476), or high Sir3-A2Q levels (yAT2822). The 2 panels on the far right: representative mitotic cells (M) in the wild-type strain or in the strain overexpressing Sir3. (B) Normalized chromosome contact maps (bin: 50 kb) of cells expressing various levels of Sir3 synchronized either in G1 or G2/M. (C) Ratio plots of pairs of contact maps of four representative chromosomes. Blue: enrichment of contacts in wt. Red: enrichment in mutants.

Figure 2 :

 2 Figure 2: Sir3 spreading coincides with telomere-telomere contacts. (A) Contact probability as a function of genomic distance Pc(s) (log scale) for different strains either in G1 or G2. (B) cumulated normalized contacts over 40 kb subtelomeric windows (0 = X-core elements). (C) Sir3 enrichment over 40 kb subtelomeric windows (data from Hocher et al., 2018). (D) Pile-up contact maps ratio for various pairs of strains of subtelomeric windows centered on the extended silent domains (ESD; dotted line) determined in the SIR3 OE condition. The windows extend over 50 kb in both direction from the ESD. (E) Representative interchromosomal ratio maps of the same pairs of strains as in (D) processed through Serpentine binning. Sir3 enrichment observed by ChIP (Hocher et al, 2018) in the indicated conditions is plotted along the top and right axis. (F) Schemas illustrating the behavior of telomeres and subtelomeric regions in the SIR3 OE, WT and sir3-A2Q OE conditions.

Figure 3 :

 3 Figure 3: Sir3 but not the silencing deficient mutant, Sir3-A2Q, associates with the rDNA and promotes rDNA-telomere contacts. (A) Representative fluorescent images of a double tagged Sir3-yeGFP / Net1-TagRFP-T strain (yAT2803). Cells were grown in CSM 2% glucose and imaged in exponential phase. (B)The S. cerevisiae rDNA locus is composed of ~150 tandem copies of a 9.1 kb repeating unit, each encoding two transcribed region RDN5 and RDNA37 (comprising RDN18, RDN5.8 and RDNA25 genes). The graph represents Sir3 occupancy along the rDNA locus probed by ChIP-qPCR using an anti-Sir3 antibody[START_REF] Ruault | Clustering heterochromatin: Sir3 promotes telomere clustering independently of silencing in yeast[END_REF] in WT (yAT232), GAL1p-SIR3 (yAT208), GAL1p-Sir3-A2Q (yAT1205), and GAL1p-SIR3 sir2∆ (yAT772) strains. Primer pair 1 amplifies a region of the RDN25 locus, primer pair 2 amplifies a region in NTS1 (Non-Transcribed Spacer 1) region, primer pair 3 amplifies a region in NTS2 and primer 4 amplifies a region in the ETS1 (External Transcribed Spacer 1) region (see Supplemental table S2 for primer sequences). Strains were grown in YPGal for 48 hours. The bar graph represents the Sir3 enrichment over the mitochondrial locus OLI1. Error bars show s.e.m of three independent experiments, each analyzed in triplicate qPCRs. (C) 4C extraction profiles representing contact maps between the rDNA repeats and the rest of the genome, profiles are shown for all strains in both exponential and overnight cultures. Red highlighted loci correspond to bins with z-value greater than 2.5. (D) Representative fluorescent images of a double tagged strain Rap1-GFP / Sik1-mRFP in strains expressing endogenous level of Sir3 (yAT340), high levels of Sir3 (yAT341) or high levels of the separation of function mutant Sir3-A2Q (yAT1198) after an overnight culture in CSM 2% galactose. Magnification of representative nuclei (1 and 2) are presented at the bottom of the panel. (E) Distance between the brightest Rap1-GFP cluster and the nucleolus center is plotted for a wild-type (yAT340, n=581), a strain overexpressing Sir3 (yAT341, n=627) and a strain overexpressing Sir3-A2Q (yAT1198, n=590) using the Nucloc software[START_REF] Berger | High-resolution statistical mapping reveals gene territories in live yeast[END_REF]. Cells were grown in CSM 2% galactose overnight before imaging. Scale bar is 1 µm in all panels.

Figure 4 :

 4 Figure 4: Sir3 overexpression impacts the rDNA spatial organization and its compaction. (A)Representative fluorescent images of Rap1-GFP / Net1-mRFP strains expressing either endogenous level of Sir3 (yAT3729), high levels of Sir3 (yAT3730) or high levels of the Sir3-A2Q mutant (yAT3733). Cells were grown in CSM with 2% galactose and imaged after an overnight culture. (B) Representative fluorescent images of a double tagged strain Net1-GFP / Sik1-mRFP in strains expressing endogenous level of Sir3 (yAT1004), high levels of Sir3 (yAT1008) or high levels of the Sir3-A2Q mutant (yAT1541). Cells were grown in CSM 2% galactose and imaged after an overnight culture. In the bottom panels,

Figure 5 :

 5 Figure 5: Sir3 bound regions coincide with long-range contacts. (A) Intrachromosomal ratio plots of Chromosome I, VI and IV-L contact maps generated in sir3-A2Q OE and SIR3 OE conditions, and processed by Serpentine binning. The ChIP deposition profile of Sir3 in OE conditions are plotted along the top and right axis. Intrachromosomal loci enriched in Sir3 are indicated by the closest gene name. (B) Serpentine binning plots recapitulating the enrichment in contacts in SIR3 OE conditions with respect to sir3-A2Q OE conditions, showing Chromosomes I, IV, V and VI interacting with the rest of the genome. (C) Schematic representation of the behavior of internal Sir3 binding sites in different conditions.

Figure 6 :

 6 Figure 6: Arrays of Sir3 are sufficient to promote trans-interactions. (A) Schema describing the strains used in the assay. sir3∆ rap1-17 strains carrying a LacOp array at the LYS2 locus on Chromosome II and a LacOp array at the LEU2 locus on Chromosome III are expressing either GFP-LacI or GFP-LacI-Sir3. Merge image of a transmitted-light image and the GFP channel fluorescent image of a strain expressing the GFP-LacI-Sir3 construct (yAT1470). (B) Graphic showing the percentage of cells with 1 spot in different strains and conditions: GFP-LacI (yAT1476), GFP-LacI-Sir3 (yAT1470) in the presence or in the absence of splitomycin (a Sir2 inhibitor) and GFP-LacI-Sir3 sir4∆ (yAT1864). Cells were grown in CSM 2% glucose to mid exponential phase of growth before being imaged. (C) Schema representing the nuclear organization of strains expressing either the GFP-LacI (left) or the GFP-LacI-Sir3 (right).

Acknowledgements

The authors thank Pr. Masayasu Nomura for sharing strains, Mickael Garnier for his help on image analysis, Julien Mozziconacci, the members of the Taddei laboratory for helpful discussions and Susmita Sridhar for her help in editing the manuscript. A.T. team was financially supported by funding from the Labex DEEP (ANR-11-LABEX-0044 DEEP and ANR-10-IDEX-0001-02 PSL), from the ANR DNA-Life (ANR-15-CE12-0007), Fondation pour la Recherche Médicale (DEP20151234398), and CNRS grant 80prime PhONeS. The authors greatly acknowledge the PICT-IBiSA@Pasteur Imaging Facility of the Institut Curie, member of the France Bioimaging National Infrastructure (ANR-10-INBS-04). V.F.S. is the recipient of a Roux-Cantarini Pasteur fellowship. L.L.S. was supported in part by a fellowship from the Fondation pour la Recherche Médicale. This research was supported by funding to R.K. from the European Research Council under the Horizon 2020 Program (ERC grant agreement 771813).

Data access

The Hi-C library data generated in this study have been submitted to the NCBI BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/) under accession number PRJNA642827

Competing interest statement

The authors declare that they have no conflict of interest.

Author contributions M.R. and A.H. generated strains. M.R. performed and analyzed the microscopy experiments. L.L.S performed the Hi-C experiments. A.H. performed and analyzed ChIP on chip experiments. I.L. carried out FACs and ChIP-qPCR experiments. V.F.S conducted the bioinformatics analyses. A.T., R.K., M.R. and A.H. contributed to the design of the experiments. A.T., R.K., V.F.S., M.R. contributed to the interpretation of the data, the drafting of the figures and the writing/revision of the manuscript.

Supplemental information

The silencing factor Sir3 is a molecular bridge that sticks distant loci together

Supplemental Figure S3

Supplemental Figure S3. (A-C) Functional assessment of Sir3-Cterminal tagged proteins. (A) Telomeric silencing assay at telVR::ADE2. To assess the functionality of Sir3-Cterminal tagged proteins, wild-type (WT; yAT71), sir3∆ (yAT1196), SIR3-mCherry (yAT52), and sir3-yeGFP (yAT4064) cells were streaked on a YPD plate. After 4 days, the plates were stored for 4 days at 4°C before taking the pictures of the plates. The color of the colonies is indicative of the state of silencing of the ADE2 reporter gene at telVR: the ADE2 gene is expressed in white colonies and repressed in pink colonies. (B) Telomeric silencing assay at telVIIL::URA3. The same strains than in (A) were grown in YPD, cultures were normalized to 1.2 ´ 10 7 cells / ml and fivefold serial dilutions were plated either onto CSM, URA-drop out or 5-FOA plates. Growth on URA-plates and decreased growth on 5-FOA plates reflect a disruption of telomeric silencing. (C) The graphs represent Sir3 occupancy along subtelomere VIR and the rDNA locus probed by ChIP-qPCR using an anti-Sir3 antibody [START_REF] Ruault | Clustering heterochromatin: Sir3 promotes telomere clustering independently of silencing in yeast[END_REF] in WT (yAT1684), sir3∆ (yAT1710), SIR3-mCherry (yAT3653), and SIR3-yeGFP (yAT2803) strains. The bar graph represents the Sir3 enrichment over OGG1. (D-F) Association of the telomere hypercluster and the rDNA in strains overexpressing Sir3 is regulated by the physiology of the cell and the size of the rDNA array. (D) Representative fluorescent images of a double tagged strain Rap1-GFP / Sik1-mRFP overexpressing Sir3 under the GPD promoter (yAT1046) in different physiological conditions: after an overnight culture and 3h, 6h and 9h after dilution in fresh medium. Cells were grown in CSM 2% glucose. Scale bar is 1 µm. (E) Distance between the brightest Rap1-GFP cluster and the nucleolus center is plotted for a wild-type (yAT1782) and a strain overexpressing Sir3 (yAT1827) measured in different conditions: after an overnight culture (n=757 for yAT1782, n=596 for yAT1827), in early (n=652 for yAT1782, n=546 for yAT1827) and late exponential phase (n=739 for yAT1782, n=513 for yAT1827). Cells were grown in CSM 2% galactose. Imaged were analyzed using the Nucloc software [START_REF] Berger | High-resolution statistical mapping reveals gene territories in live yeast[END_REF]. (F) Distance between the brightest Rap1-GFP cluster and the nucleolus center is plotted for a wild-type (yAT340, n=581, same data than in Figure 3E), for a long rDNA strain overexpressing Sir3 (190 repeats, yAT1778, n= 588) and for a short rDNA strain overexpressing Sir3 (25 repeats, yAT1780, n= 367). Cells were grown in CSM 2% galactose. Data were acquired after an overnight culture and analyzed using the Nucloc software [START_REF] Berger | High-resolution statistical mapping reveals gene territories in live yeast[END_REF].

Supplemental Figure S4

Supplemental Figure S4: rDNA spatial reorganization upon Sir3 overexpression is independent of the formation of the telomere hypercluster and is regulated by the physiology of the cell. (A) Representative fluorescent images of Net1-GFP in wild-type strain (yAT1004) and in a strain expressing high Sir3 levels (yAT1724). Images were taken in different physiological conditions: after an overnight culture and 5h and 8h after dilution in fresh medium. (B) Representative fluorescent images of a double tagged strain Rap1-GFP / Sik1-mRFP in a wild-type strain (yAT3729), in a strain expressing high Sir3 levels (yAT3730), in a sir4∆ (yAT3765) and in a sir4∆ expressing high Sir3 levels (yAT3743). Cells were grown in synthetic CSM 2% galactose and imaged after an overnight culture. (C) Representative fluorescent images of a double tagged strain Net1-GFP / Sik1-mRFP in wild-type strain (yAT1004), in a strain expressing high Sir3 levels (yAT1724), in a sir4∆ (yAT1723) or in a sir4∆ strain expressing high Sir3 levels (yAT2124). Cells were grown in CSM 2% glucose and imaged after an overnight culture. (A) Growth assay, telomeric silencing assay at telVIIL::URA3 were carried out with the following strains: wild-type (yAT69), sir3∆ (yAT1010), pHIS3-GFP-Sir3 (yAT1011), pHIS3-GFP-SIR3 sir3∆ (yAT1676), pHIS3-GFP-LacI-Sir3 (yAT821) and pHIS3-GFP-LacI-Sir3 sir3∆ (yAT921). To monitor telomeric silencing at telVIIL::URA3, strains were grown in YPD overnight and plated in 5-fold serial dilutions starting at OD600nm= 1 (corresponding to 1 ´ 10 7 cells/ml) onto appropriate plates: complete CSM without uracil as a growth control plate and onto a 5-FOA plate to assess telomeric silencing. Decreased growth on the 5-FOA plate reflects the disruption of telomeric silencing (expression of the URA3 gene at telo VIIL). (B) Representative images (Transmitted-light image and the GFP channel fluorescent image) of a strain expressing the pHIS3-GFP-LacI-Sir3 construct (yAT788) grown in complete synthetic medium (exponential phase).

Materials and Methods for Supplemental Figure 3 and6 Silencing assays For telomeric silencing assays, cultures were grown in liquid YPD and plated in five-fold serial dilutions starting at OD600nm =1 (1.2 ´ 10 7 cells /ml) onto appropriate plates. 5-fluoroorotic acid (5-FOA; Zymo Research) plates were prepared by adding 5-FOA to a final concentration of 0.1% to supplemented synthetic medium. S1. Yeast strains used in this study.

Supplemental Table

W303-1 derived strains : MATa ade2-1 can1-100 leu2-3,112 his3-11,15 trp1-1 ura3-1 (W303-1) yAT208 MATa ade2-1::ADE2 adh4::URA3-4xUASG-(C1-3A)n ppr1∆::HIS3 rap1::GFP-RAP1(LEU2) sir3::pGAL1-SIR3(KanMX) yAT232 MATa ade2-1::ADE2 adh4::URA3-4xUASG-(C1-3A)n ppr1∆::HIS3 rap1::RAP1-GFP(LEU2) yAT340 MATa ade2-1::ADE2 rap1::GFP-RAP1(LEU2) sik1::SIK1-mRFP(KanMX) yAT341 MATa ade2-1::ADE2 rap1::GFP-RAP1(LEU2) sik1::SIK1-mRFP(KanMX) sir3::pGAL1-SIR3(KanMX) yAT772 MATa ade2-1::ADE2 rap1::RAP1-GFP(LEU2) sir2∆::KanMX sir3::pGAL1-SIR3(NAT) yAT1004 MATa ade2-1::ADE2 net1::NET1-GFP(HIS3MX) sik1::SIK1-mRFP(KanMX) yAT1008 MATa ade2-1::ADE2 net1::NET1-GFP(HIS3MX) sik1::SIK1-mRFP(KanMX) sir3::pGAL1-SIR3(NAT) yAT1198 MATa ade2-1::ADE2 rap1::GFP-RAP1(LEU2) sik1::SIK1-mRFP(KanMX) sir3::pGal1-sir3-A2Q(NAT) yAT1205 MATa ade2-1::ADE2 adh4::URA3-4xUASG-(C1-3A)n ppr1∆::HIS3 rap1::RAP1-GFP(LEU2) sir3::pGal1-sir3-A2Q(KanMX) yAT1470 MATa ade2-1::ADE2 adh4::URA3 his3::GFP-LacI-SIR3(HIS3) leu2::LacOp(LEU2) lys2::LacOp(TRP1) rap1-17 sir3∆::kanMX yAT1476 MATa ade2-1::ADE2 his3::GFP-LacI(HIS3) leu2::LacOp(LEU2) lys2::LacOp(TRP1) rap1-17 sir3∆::kanMX yAT1541 MATa ade2-1::ADE2 net1::NET1-GFP(HIS3MX) sik1::SIK1-mRFP(KanMX) sir3::pGAL1-sir3-A2Q(NAT) yAT1864 MATa ade2-1::ADE2 his3::GFP-LacI-SIR3(HIS3) leu2::LacOp(LEU2) lys2::LacOp(TRP1) rap1-17 sir3∆::