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• W1808593985 abstract "Article18 February 2011free access Dynamics of Sir3 spreading in budding yeast: secondary recruitment sites and euchromatic localization Marta Radman-Livaja Marta Radman-Livaja Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Giulia Ruben Giulia Ruben MCD-Biology, UCSC, Santa Cruz, CA, USA Search for more papers by this author Assaf Weiner Assaf Weiner School of Computer Science and Engineering, Hebrew University, Jerusalem, Israel Alexander Silberman Institute of Life Sciences, Hebrew University, Jerusalem, Israel Search for more papers by this author Nir Friedman Nir Friedman School of Computer Science and Engineering, Hebrew University, Jerusalem, Israel Alexander Silberman Institute of Life Sciences, Hebrew University, Jerusalem, Israel Search for more papers by this author Rohinton Kamakaka Rohinton Kamakaka MCD-Biology, UCSC, Santa Cruz, CA, USA Search for more papers by this author Oliver J Rando Corresponding Author Oliver J Rando Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Marta Radman-Livaja Marta Radman-Livaja Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Giulia Ruben Giulia Ruben MCD-Biology, UCSC, Santa Cruz, CA, USA Search for more papers by this author Assaf Weiner Assaf Weiner School of Computer Science and Engineering, Hebrew University, Jerusalem, Israel Alexander Silberman Institute of Life Sciences, Hebrew University, Jerusalem, Israel Search for more papers by this author Nir Friedman Nir Friedman School of Computer Science and Engineering, Hebrew University, Jerusalem, Israel Alexander Silberman Institute of Life Sciences, Hebrew University, Jerusalem, Israel Search for more papers by this author Rohinton Kamakaka Rohinton Kamakaka MCD-Biology, UCSC, Santa Cruz, CA, USA Search for more papers by this author Oliver J Rando Corresponding Author Oliver J Rando Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Author Information Marta Radman-Livaja1, Giulia Ruben2, Assaf Weiner3,4, Nir Friedman3,4, Rohinton Kamakaka2 and Oliver J Rando 1 1Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA 2MCD-Biology, UCSC, Santa Cruz, CA, USA 3School of Computer Science and Engineering, Hebrew University, Jerusalem, Israel 4Alexander Silberman Institute of Life Sciences, Hebrew University, Jerusalem, Israel *Corresponding author. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA. Tel.: +1 508 856 8879; Fax: +1 508 856 6464; E-mail: [email protected] The EMBO Journal (2011)30:1012-1026https://doi.org/10.1038/emboj.2011.30 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Chromatin domains are believed to spread via a polymerization-like mechanism in which modification of a given nucleosome recruits a modifying complex, which can then modify the next nucleosome in the polymer. In this study, we carry out genome-wide mapping of the Sir3 component of the Sir silencing complex in budding yeast during a time course of establishment of heterochromatin. Sir3 localization patterns do not support a straightforward model for nucleation and polymerization, instead showing strong but spatially delimited binding to silencers, and weaker and more variable Ume6-dependent binding to novel secondary recruitment sites at the seripauperin (PAU) genes. Genome-wide nucleosome mapping revealed that Sir binding to subtelomeric regions was associated with overpackaging of subtelomeric promoters. Sir3 also bound to a surprising number of euchromatic sites, largely at genes expressed at high levels, and was dynamically recruited to GAL genes upon galactose induction. Together, our results indicate that heterochromatin complex localization cannot simply be explained by nucleation and linear polymerization, and show that heterochromatin complexes associate with highly expressed euchromatic genes in many different organisms. Introduction Eukaryotic genomes are packaged into a nucleoprotein complex known as chromatin. Classically, chromatin has been divided into a relatively accessible active compartment termed euchromatin, and an inaccessible compartment termed heterochromatin. In budding yeast, heterochromatin formation requires the silent information regulator (SIR) complex, which is involved in silencing of the silent mating loci, subtelomeric genes and ribosomal RNA gene repeats (Rusche et al, 2003). The now-classic model for Sir complex recruitment is a nucleation–polymerization model. Sir is first recruited to silencers—at the silent mating loci these are the E and I silencers (reviewed in Rusche et al, 2003), while at subtelomeres Sir recruitment occurs via Rap1 binding to the telomeric repeats and ORC and Abf1 binding to the XCS sequence elements (Pryde and Louis, 1997, 1999). After recruitment, Sir spreads via a cycle of histone modification and binding—Sir2 is an H4K16 deacetylase and Sir3 is a K16-sensitive nucleosome binding protein (Hecht et al, 1996; Rusche et al, 2002; Moazed et al, 2004; Norris and Boeke, 2010). The details of Sir complex spreading are unknown, but a number of observations have lead to the idea that Sir spreading can be considered in some ways analogous to polymerization. First, silencing of subtelomeric reporter genes has been observed to decay with increasing distance from the telomere (Renauld et al, 1993; Pryde and Louis, 1999). Second, overexpression of Sir3 leads to increased Sir3 binding at sites relatively distal to the telomere (Hecht et al, 1996), suggesting that Sir protein levels are limiting for the extent of spreading. Third, upon reintroduction of Sir3 into sir3Δ yeast, Sir3 localization extends further into the chromosome at increasing times after Sir3 reintroduction (Katan-Khaykovich and Struhl, 2005). Finally, Sir3 oligomerizes in vitro, and the Sir complex exhibits dramatic structural alterations in the presence of the metabolic by-product of Sir2 action, O-acetyl ADP-ribose, suggesting that chromatin binding is likely to be cooperative in vivo (Liou et al, 2005; Martino et al, 2009). In contrast, in vivo studies of Sir-dependent silencing often reveal evidence for discontinuous silencing (Fourel et al, 1999; Pryde and Louis, 1999; Valenzuela et al, 2008; Zill et al, 2010), which calls linear polymerization models for Sir spreading into question. Here, we investigated the kinetics of Sir complex spreading by inducing expression of an epitope-tagged Sir3 subunit in a sir3Δ mutant, and mapping Sir3 localization patterns genome-wide via deep sequencing. We find that Sir3 localization patterns do not exhibit expected polymerization behaviour. Instead, Sir3 binds to ∼5–6 nucleosomes around silencers rapidly, then slowly associates with novel secondary silencing sites such as the seripauperin (PAU) genes. We show that the PAU genes have Sir recruitment activity, and that Sir binding to the PAU genes is Ume6 dependent. We further find that the Sir complex binds numerous euchromatic sites, largely at highly expressed genes. Binding to highly expressed genes is dynamic, as Sir3 relocalizes to GAL gene bodies upon their induction. Together, these results suggest that the mechanism of heterochromatin spreading cannot be described as a simple linear polymerization reaction, and provide a window into novel heterochromatic loci. Results Bidirectional and ‘cooperative’ Sir3 spreading from the HMR-E silencer sir3Δ mutant yeast containing a galactose-inducible HA-tagged SIR3 construct (Cheng and Gartenberg, 2000) were grown to midlog in YP raffinose media. At varying intervals after induction of Sir3-HA with galactose, cells were crosslinked, and chromatin was digested to mononucleosomes and immunoprecipitated with anti-HA, anti-H4K16ac, anti-H3K4me3 and anti-H3K56ac antibodies. Chromatin immunoprecipitation (ChIP) material was amplified and hybridized to a 20-bp resolution tiled oligonucelotide array covering chromosome III and 223 additional promoters (Yuan et al, 2005) (Figure 1). As expected, Sir3-HA exhibited strong association with known heterochromatic loci, the silent mating loci and subtelomeric regions (Figure 1A). At HMR, Sir3 bound rapidly to an ∼10 nucleosome domain (Figure 1B and C) between the HMR-E silencer and the tRNA-Thr gene previously described as a heterochromatin boundary (Donze et al, 1999). H4K16 and H3K56 deacetylation as well as H3K4me3 demethylation appear to follow the dynamics of Sir3-HA binding, as observed previously (Katan-Khaykovich and Struhl, 2005; Xu et al, 2007), although H3K56 deacetylation was not as complete as were H4K16 deacetylation or H3K4 demethylation. Figure 1.Dynamics of Sir3 binding and histone modifications at HMR. (A) Time course of Sir3 binding to Chr3. Log2 (Sir3/IP input) values were obtained from anti-HA immunoprecipitation from THC70 cells after galactose induction of C-terminal HA-tagged Sir3, followed by microarray hybridization. Lines are results from IP experiments 0, 0.75, 1.5, 2.25, 3 and 4 h after galactose addition. Each column represents one nucleosome (Yuan et al, 2005). (B) Sir3 binding and histone modifications at HMR. Each panel represents a different ChIP-chip dataset, from top to bottom: Anti-HA (Sir3), anti-H4K16ac, anti-H3K56ac and anti-H3K4me3. Each line within each panel is a time point after galactose addition as in (A). (C) Rates of Sir3 binding and histone modification dynamics at HMR. Each data point is the slope of the linear fit to the log2 enrichment ratio time course for each nucleosome (see Supplementary Figure S1). The curves are centred at the HMR-E silencer. Download figure Download PowerPoint Interestingly, we observed Sir3-HA binding at late time points at HMR-adjacent regions beyond the known heterochromatin boundaries. The secondary boundaries for this slower Sir3-HA spread appear to be located near the promoter regions of OCA4 on the centromeric and GIT1 on the telomeric side of HMR. By examining the kinetics of Sir3-HA binding across HMR and adjacent regions, we identify two modes for Sir3-HA spreading. The region spanning from 2 kb upstream to 4 kb downstream of HMR-E is characterized by a uniformly high binding rate (Supplementary Figure S1) most consistent with near-simultaneous binding of Sir3-HA across the region, suggesting either cooperative binding or very rapid polymerization out to boundary elements. Conversely, beyond this region of ‘cooperative’ Sir3-HA binding are ∼2 kb regions with Sir3-HA binding rates that decrease from one nucleosome to the next, which could be explained either by cell-to-cell variability in the level of Sir3-HA expression and resulting breadth of Sir3-HA-bound domains (Xu et al, 2006) or by slow polymerization. Rusché and colleague recently noted the rapidity of Sir3 binding at HMR, which they showed was in contrast to relatively slow Sir3 binding and spreading at TEL6R (Lynch and Rusche, 2009). As these qualitatively distinct Sir3 spreading behaviours are linked to the presence of different silencer sequences, we sought to globally characterize Sir3 spreading behaviour across the entire genome. Dynamics of Sir3 binding at yeast telomeres We therefore extended our results to whole-genome coverage by characterizing the distribution of Sir3 via deep sequencing (Albert et al, 2007; Shivaswamy et al, 2008) of Sir3-HA ChIP at varying times after Sir3-HA induction. Figure 2A shows Sir3-HA binding at all 32 yeast telomeres, with Sir3-HA binding shown in yellow and unmappable repeat regions shown in purple. Telomeres are categorized by three sequence elements internal to the telomeric TG repeats: Y’, XCR (X combinatorial repeats) and XCS (X-core sequence) (Louis, 1995), which are present in four combinations (Figure 2A). Y’ and XCR have been shown to possess insulator activity, and XCS functions as a proto-silencer—it promotes and stabilizes Sir-dependent gene silencing but it is not an autonomous silencer (Fourel et al, 1999; Pryde and Louis, 1999). All telomeres are aligned by the ARS consensus sequence (ACS) within the XCS. Figure 2.Dynamics of Sir3 binding to yeast telomeric regions. (A) Heat map of Sir3-binding dynamics measured by ChIP-seq of galactose induced anti-HA Sir3 IPs. Telomeres are grouped by type: Y’ XCR XCS, Y’ XCS, XCS and XCR XCS. Lines within each telomere panel are results from the galactose induction time course. From top to bottom: 40, 80, 120 and 240 min after galactose addition. Each column is a 30-bp region centred on the ACS element. The value of each data point is the normalized sequence read count number, which is proportional to the amount of Sir3-HA binding. (B) Sir3-HA-binding profiles at TEL6R. (C) Average Sir3-binding profiles for all Y’ XCR XCS telomeres. Data shown are a 150-bp window average. (D) Average Sir3-HA-binding profiles for all XCR XCS telomeres. Download figure Download PowerPoint Several features are apparent in Figure 2A. First, this map is incomplete due to unmappable repetitive segments (purple regions in Figure 2A) such as Y’ elements, and due to sequence differences between our experimental strain and the reference genome at 9L, 10L, 7L, 14R, 1R and 13R (black no-read regions in Figure 2A—see Materials and methods and Supplementary Figure S2). Second, the silent mating loci are apparent in the 3L and 3R maps as prominent Sir3-bound regions ∼10 and ∼25 kb away from the XCS region, respectively. Interestingly, a previously undescribed region similar in Sir-binding profile to the silent mating loci is observed at 15L (see below, Figures 2A and 3). Finally, we note that even after 4 h of Sir3-HA expression, Sir3 does not generally spread further than 5–10 kb downstream of the ACS, with highest and fastest binding concentrated 1–2 kb around the ACS. Figure 2B shows Sir3-HA binding over time at the commonly studied telomere 6R. Figure 3.Seripauperin genes as novel Sir recruitment sites. (A) Sir3-HA-enriched regions (with a read count number >3 at t=240 min after Sir3-HA induction) on all 16 yeast chromosomes are shown in green. PAU gene locations are shown in red/black, the intensity of the red bands is proportional to the Sir3 count number at a PAU gene. Chromosomes are aligned by their left telomeres along the top of the figure. Left and right telomere-proximal PAU gene names are listed on top and bottom of their respective chromosomes. (B) Heat map of Sir3-HA binding to PAU genes. Lines within each PAU gene panel are results from the galactose induction time course. From top to bottom: 40, 80, 120 and 240 min after galactose addition. PAU genes are ordered by distance from the ACS. Data shown are the same as in Figure 2A. PAU genes are aligned by their ATG and the +5 kb mark indicates distance from the ATG. (C–F) Sir3-HA-binding profiles at telomere 8L, 15L, 1L and 4R, as indicated. Locations of PAU genes are depicted as green rectangles. Download figure Download PowerPoint Sir3 ChIP-seq maps and ChIP–qPCR experiments from a wild-type strain obtained using an antibody against Sir3 show a similar pattern of Sir3 binding to those obtained with the Sir3-HA overexpression system, with the most notable difference being a somewhat increased distance of Sir3-HA association from the telomeres in the overexpression system (Supplementary Figures S2 and S3). Interestingly, in wild-type cells some of the binding we observe at secondary recruitment sites seems to be stronger in galactose than in dextrose (see below). What can be inferred about Sir3 spreading from these time courses? We averaged data for the two largest classes of subtelomeres, those with XCR sequences and those with both XCR and Y’ elements (Figure 2C and D, respectively). Interestingly, in both cases we observe two modes of Sir3-HA binding. As at HMR, Sir3-HA binds simultaneously to 1–2 kb around the ACS at XCR telomeres, and this binding is essentially saturated at the earliest time point after Sir3-HA induction. Beyond that region on the centromeric side there is a short but consistent dip in Sir3 binding, followed by decaying Sir3 binding with increasing distance into the chromosome. Unlike the ACS-proximal binding, Sir3 binding in this region increases gradually with time, as noted above for the Sir3 binding beyond the HMR boundaries. Conversely, at telomeres that lack an XCR, Sir3-HA binding does not show any evidence for ‘cooperativity’, as Sir3-HA only binds ∼1–2 nucleosomes around the ACS at the Y’ XCS telomeres 5L and 16R. At the XCS telomere 6R, Sir3-HA binds initially close to the TG repeats at the end of the chromosome and then spreads slowly across the XCS up to 3 kb downstream from the ACS (Figure 2B). While it is tempting to assign the ability of Sir3-HA to bind cooperatively to the presence of the XCR, the sample size of XCR-less telomeres (only four, one of which—1R—has no available data) prevents us from drawing a more definite conclusion. Mutation analysis of XCR sites would shed more light on the role of XCR in Sir3-HA spreading. Seripauperin genes as novel Sir3 recruitment sites Beside XCS regions and the known Sir nucleation sites HML and HMR, new Sir3-binding sites are apparent at several telomeric regions. There is a strong novel binding/nucleation site at 15L about 10 kb from the ACS, and weaker additional sites at 3R, 2R, 8L, 9R, 15R, 7L, 11L and 14R (Figure 2A; Supplementary Figure S4). The observed Sir3 binding was not an artifact of overexpression of Sir3-HA, as we observed the same binding locations, albeit with weaker intensity, when mapping native Sir3 in wild-type yeast grown in dextrose or galactose (Supplementary Figures S2 and S5, see below). To determine whether these loci were associated with the Sir complex or solely with Sir3, we asked whether Sir2 is associated with a subset of these loci (at 8L, 7L and 15L) using a strain carrying HA-tagged Sir2. Consistent with binding of the Sir complex to these regions, we found that these loci associate with both Sir2-HA and Sir3 (Supplementary Figure S6C). Furthermore, Sir3 binding to these loci is Sir2 dependent, as anti-Sir3 ChIP–qPCR in a sir2Δ background gives only non-specific binding signals (Supplementary Figure S6B). These results strongly suggest that Sir3 proteins bound to these sites are part of the Sir complex as previously shown for the canonical subtelomeric heterochromatic sites and the silent mating type loci. What is the nature of these secondary recruitment sites? Interestingly, we found that most of the secondary recruitment sites occur adjacent to members of the repetitive class of little-studied genes known as the seriPAUperin genes (Figure 3). For instance, the locus underlying the TEL15L secondary recruitment site corresponds to PAU20. Our data suggest that PAU genes have three classes of effects on Sir3 spreading based on their distance from the ACS (Figure 3B). One of these, which we termed class II, includes most of the secondary Sir recruitment sites (Figure 3B)—the strongest examples being the clearly separated sites 15L and 8L (Figure 3C and D). Interestingly, we note that many telomeres exhibiting extended spreading behaviours of the Sir complex have PAU genes located very close to the ACS (‘class I’), suggesting that the PAU gene at these telomeres acts as an accessory recruiting site that extends the ‘cooperative’-like Sir3 spreading region (Figure 3B, E and F). Conversely, telomeres that do not contain a PAU gene in close proximity to their ACS typically exhibit much shorter Sir domains or lack ‘cooperative’ Sir spreading around the ACS (telomeres: 5R, 6L, 7R, 8L, 8R, 14L, 3R, 9R, 11R, 15L, 6R, 5L, 16R and 3L; Figure 2A; Supplementary Figure S7). Indeed, PAU genes located >10 kb away from ACS sites (class III in Figure 3B) are not Sir3-binding sites (though they are nonetheless transcribed poorly), suggesting that proximity to silencers might be necessary for PAU genes to promote Sir3 association (see below). Furthermore, PAU genes are derepressed in sir3Δ mutant yeast, and derepression is higher for class I and class II PAU genes than for class III genes, indicating that the Sir complex is involved in seripauperin gene silencing (Supplementary Figure S8). The association of Sir3 and Sir2 with PAU genes and flanking DNA suggested that these loci might directly recruit the Sir complex. We tested two predictions of this hypothesis. First, to determine whether the PAU genes were responsible for Sir3 binding in nearby genomic regions, we examined Sir3 binding adjacent to PAU13 (TEL 8L) in pau13Δ yeast. Consistent with some aspect of the PAU13 locus having a direct role in Sir3 recruitment, we found that deletion of PAU13 at telomere 8L eliminates Sir3 binding in the adjacent genome (Figure 4A and B). As a second test of the role of PAU genes in Sir recruitment, we introduced PAU13 into a sensitized HMR locus lacking the HMR-I silencer (Figure 4C). PAU13 incorporation at this locus increased Sir3 binding to the HMR-E silencer, again suggesting that the PAU genes act as accessory Sir3 recruitment sites (Figure 4D). Figure 4.Effect of pau13Δ and ume6Δ on Sir3 binding. (A) Diagram of PAU gene ORF locations (green rectangles) at telomeres 15L, 8L, 1L and 7L. Ume6 motif sequences (left) and their locations (lavender squares) with respect to PAU genes (green rectangles) at telomeres 15L, 8L, 1L and 7L are also shown. qPCR primer locations are shown as magenta squares. (B) αSir3 ChiP–qPCR for indicated genotypes. Results were normalized to signals from the SSL2 gene locus. Cells were grown in galactose O/N. Data are shown as mean±s.d. (C) Schematic of PAU13 insertion (green rectangle) at HMRa2 (blue rectangle) gene in the pRO869 plasmid (Valenzuela et al, 2009). Quantitative chromatin immunoprecipitation (qChIP) amplicons are shown as magenta rectangles below the diagram. The control amplicons are from TEL 6R, 7.5 and 0.5 kb away from the chromosome end for the negative and the positive control, respectively. (D) qChIP of Sir3 at HMR in GRY662 (hmrΔ;pau13Δ) with or without PAU13 inserted as in (C). ΔCt values were derived from the difference in real-time amplification of equal amounts of immunoprecipitated input DNA, and are normalized to negative control TEL7.5. Data are shown as mean±s.d. for four replicates (two biological replicates). TEL0.5 is used as a positive control. (E) MEME motif search results for a training set with all PAU genes (left) and a training set containing non-PAU gene secondary nucleation sites (right). Note the CCGCC sequence present in both motifs 1 and 2, as well as the Ume6 consensus binding sequence. (F) As in (B), except data are shown as a fraction of the WT-binding signal. Download figure Download PowerPoint How do the PAU genes recruit Sir3? Intriguingly, we found that PAU gene regions with good Sir3 recruitment properties harbour Ume6-like DNA motifs (see Supplementary Figure S9; Figure 4E). Conversely, PAU genes with poor Sir3 recruitment properties all lack the CCGCCCACC sequences observed at strongly Sir-bound PAU genes. Furthermore, some of the observed secondary nucleation sites do not correspond to PAU genes (including those at 7L, 11L, 14R and 15R) (Figure 2A), but these sites also appear to have Ume6-like binding motifs (Figure 4E; Supplementary Figure S9B; see Discussion). As described above, deletion of PAU13 (at 8L) eliminates the Ume6-like binding motif, and eliminates Sir3 binding nearby (Figure 4B). To more specifically determine whether Ume6 is required for Sir3 recruitment to PAU genes, we carried out Sir3 ChIP–qPCR experiments in ume6Δ mutants, finding that Sir3 binding was reduced at PAU gene loci at 8L and 1L, as well as the 7L secondary nucleation site and HMR (Figure 4F; Supplementary Figure S10). Curiously, the deletion of UME6 had no effect on Sir3 binding to PAU20 (15L). Sir3-dependent nucleosome positioning at subtelomeric genes What is the nature of Sir3 ‘cooperative’ binding? As has been proposed earlier, the Sir complex probably promotes secondary structure chromatin folding (Shogren-Knaak et al, 2006; Johnson et al, 2009; Lynch and Rusche, 2009; Sperling and Grunstein, 2009). Chromatin folding into a 30-nm-like fibre or some other higher-order state could account for near-simultaneous association of Sir3 with a dozen nucleosomes. As nucleosomes in a higher-order structure might be expected to exhibit altered spacing, we therefore investigated the effects of Sir3 on nucleosome positioning at telomeres by whole-genome nucleosome mapping (Albert et al, 2007; Kaplan et al, 2008; Shivaswamy et al, 2008; Weiner et al, 2010) in WT and sir3Δ yeast. Overall, we found that nucleosome maps were quite consistent between WT and sir3Δ yeast—the two datasets exhibit a 0.8 correlation. Genes exhibiting the most dramatic chromatin differences between WT and sir3Δ were enriched for genes involved in pheromone signaling and other haploid-specific functions (Figure 5A and B; Supplementary Figure S11). This is consistent with many prior observations that loss of Sir-dependent silencing of the silent mating loci results in haploid yeast that exhibit ‘pseudo-diploid’ gene expression patterns (including downregulation of haploid-specific genes such as pheromone response genes) as a result of expression of the a1/α2 transcription factors. As shown in Figure 5A and B, loss of SIR3 results in significant increases in nucleosome occupancy over the promoters of pheromone response genes, likely as a secondary consequence of decreased TF binding to these promoters or decreased expression of these genes. Figure 5.Nucleosome mapping in sir3Δ cells. (A) Examples of mating-related gene with Sir-dependent chromatin structure. Schematic chromatin structure of the indicated genes for WT and sir3Δ yeast is shown. Ovals indicate nucleosomes and vertical bars indicate TF-binding sites. (B) Mating-related genes generally close in sir3Δ pseudo-diploid yeast. Average TSS-aligned chromatin structure of all genes whose mRNA abundance changes more than four-fold in sir2Δ. (C) Changes in subtelomeric promoter packaging in sir3Δ, as in (A). (D) Subtelomeric genes are underpackaged in sir3Δ. As in (B), for all genes within 15 kb of a telomere. A full-colour version of this figure is available at The EMBO Journal Online. Download figure Download PowerPoint To identify more direct effects of the Sir complex on chromatin structure, we focused on the chromatin structure of subtelomeric genes shown to bind Sir3 above. Average nucleosome occupancy profiles for the promoters of all subtelomeric genes (within 15 kb from chromosome ends) show a wider nucleosome-free region upstream of the transcription start site (TSS) in the sir3Δ strain compared with WT (Figure 5C and D). In other words, the nucleosome immediately upstream of the TSS (−1 nucleosome) is missing on average in the sir3Δ strain, suggesting that the Sir complex has a role in stabilizing nucleosomes at these locations (although an indirect role via effects on RNA polymerase cannot be ruled out). Furthermore, overall nucleosome occupancy over subtelomeric regions appears to be decreased in sir3Δ, consistent with results observed upon Sir relocalization in ageing cells (Dang et al, 2009). The higher nucleosome occupancy and more uniform spacing in WT cells might be expected to stabilize secondary chromatin structure folding in subtelomeres, and to have a role in subtelomeric gene repression. Sir3 binds to actively transcribed euchromatic genes One unanticipated feature of our Sir3 maps was the presence of Sir3 at numerous euchromatic sites—some of these can be seen in Figure 1A. For instance, in addition to telomeric regions, we detected strong Sir3 binding for both Sir3-HA and wild-type Sir3 at the rDNA locus: 20–50 read counts/rDNA repeat (data not shown). This was unexpected given that while Sir2 is known to have a key role in silencing of a subset of rDNA repeats (Straight et al, 1999; Cockell et al, 2000), Sir3 is typically only thought to migrate to the nucleolus in aged cells (Kennedy et al, 1997; Gasser et al, 1998a), which should comprise a small fraction of our actively growing population of yeast. To identify patterns of Sir3 binding to euchromatic genes, we aligned all genes by TSS and carried out K-means clustering with K=4. Three major patterns emerge from this analysis—genes with background levels of Sir3-HA binding (cluster 2), genes with Sir3-HA bound at promoters (clusters 1 and 3) and genes with Sir3-HA relatively uniformly associated with the gene body (cluster 4) (Figure 6A and B). Genes with Sir3-HA bound to their promoters had few features in common, although they were slightly enriched for divergent gene orientations (not shown). We therefore focused on genes where Sir3-HA was bound throughout the coding region (cluster 4). Genes associated with Sir3-HA over coding regions were enriched for gene ontology categories such as ‘translation’ and ‘ribosome biogenesis’, categories of genes that are very highly expressed in actively growing yeast. Indeed, we found that Sir3-HA-bound genes were significa" @default.
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• W1808593985 title "Dynamics of Sir3 spreading in budding yeast: secondary recruitment sites and euchromatic localization" @default.
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• W1808593985 doi "https://doi.org/10.1038/emboj.2011.30" @default.
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