Molecular Cell Article

A Role for Mammalian Sin3 in Permanent Silencing

Chris van Oevelen,1 Jinhua Wang,1 Patrik Asp,1 Qin Yan,2,3 William G. Kaelin, Jr.,2,3 Yuval Kluger,1,* and Brian David Dynlacht1,* 1New York University School of Medicine, NYU Cancer Institute, 522 1st Avenue, New York, NY 10016, USA 2Howard Hughes Medical Institute 3Department of Medical Oncology Dana Farber Cancer Institute and Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA *Correspondence: [email protected] (B.D.D.), [email protected] (Y.K.) DOI 10.1016/j.molcel.2008.10.015

SUMMARY substoichiometric regulatory , including Swi/Snf-remod- eling proteins, retinoblastoma (RB)-binding 2 (RBP2), and The multisubunit Sin3 complex regu- other proteins (Hayakawa et al., 2007; Nagl et al., 2007; Sif et al., lates gene through deacetylation of nu- 2001). Interestingly, RBP2 was recently shown to be a demethy- cleosomes. However, the full range of Sin3 activities lase specific for di- and trimethylated lysine 4 of histone H3 and targets is not well understood. Here, we have (Christensen et al., 2007; Klose et al., 2007). Thus, the Sin3 investigated genome-wide binding of mouse Sin3 complex provides a versatile platform for chromatin modifying and RBP2 as well as histone modifications and nucle- and remodeling activities. Sin3/Rpd3 corepressor complexes are recruited to promoter osome positioning as a function of myogenic differ- regions via sequence-specific repressors such as Ume6 or entiation. Remarkably, we find that Sin3 complexes Mad in yeast and mammalian cells, respectively, resulting in spread immediately downstream of the transcription localized deacetylation of histones within promoter regions and start site on repressed and transcribed during transcriptional silencing (Ayer et al., 1995; Kadosh and Struhl, differentiation. We show that RBP2 is part of a Sin3 1997; Schreiber-Agus et al., 1995). Interestingly, in addition to complex and that on a subset of E2F4 target genes, its well-established role in promoter binding and gene repres- the coordinated activity of Sin3 and RBP2 leads to sion, distinct Sin3/Rpd3 complexes have recently been identi- deacetylation, demethylation, and repositioning of fied in both budding and fission yeast and shown to deacetylate nucleosomes. Our work provides evidence for coor- nucleosomes within the coding regions of active and repressed dinated binding of Sin3, chromatin modifications, genes, preventing spurious forward and antisense transcription and chromatin remodeling within discrete regulatory (Carrozza et al., 2005; Keogh et al., 2005; Li et al., 2007; Nicolas et al., 2007). However, the relationship between yeast and mam- regions, suggesting a model in which spreading of malian Sin3 complexes has not been completely resolved, and Sin3 binding is ultimately linked to permanent gene whether a mammalian complex(es) exhibits regulatory activities silencing on a subset of E2F4 target genes. (besides histone deacetylation) analogous to those in yeast is not known. INTRODUCTION Expression of cell-cycle genes is regulated by the reversible recruitment of the E2F/DP family of transcription factors and as- Chromatin structure and compaction can be altered by a combi- sociated chromatin-remodeling enzymes during discrete stages nation of posttranslational histone modifications and the specific of the mammalian cell cycle (Blais and Dynlacht, 2007; Frolov and positioning of nucleosomes. Histone acetyltransferases (HATs) Dyson, 2004). In quiescent or early G1 cells, E2F4/DP and asso- and deacetylases (HDACs), recruited by sequence-specific acti- ciated retinoblastoma tumor suppressor protein (pRb) family vators and repressors, respectively, antagonistically regulate the members p107 and p130 bind to the promoters of cell-cycle- acetylation of lysines on amino-terminal histone tails and directly regulated genes, in some instances recruiting the Sin3/HDAC link histone modifications with (Grunstein, complex and repressing transcription (Rayman et al., 2002). As 1997). Mammalian HDAC1 is a deacetylase that is highly homol- cells progress into S phase, E2F4 and Sin3 dissociate from ogous to yeast Rpd3 (Ekwall, 2005). Studies in yeast and mam- genes, leading to increased histone H3 and H4 acetylation and malian cells showed that HDAC1/Rpd3 is an enzymatic compo- gene expression (Balciunaite et al., 2005; Rayman et al., 2002; nent of multiprotein complexes containing the Sin3 corepressor Takahashi et al., 2000). This strongly suggests a model in which protein. In mammalian cells, the Sin3 core complex consists of E2F4/pocket protein complexes periodically and reversibly at least eight subunits (Alland et al., 2002; Hassig et al., 1997; recruit Sin3 to cell-cycle-regulated genes during cell-cycle Laherty et al., 1997; Zhang et al., 1997). However, there is consid- progression. erable disagreement regarding a ‘‘holo-Sin3’’ complex, most We have also postulated a role for E2F4 and HDACs in terminal likely due to transient associations with, and heterogeneity of, differentiation of skeletal muscle cells, wherein cell-cycle genes

Molecular Cell 32, 359–370, November 7, 2008 ª2008 Elsevier Inc. 359 Molecular Cell Sin3 Mediates Repression during Differentiation

are deacetylated during cell-cycle arrest and are subsequently refer to genes bound by Sin3A, Sin3B, or both as Sin3 targets for permanently silenced in differentiated myotubes through simplicity, unless otherwise noted. pRb-mediated methylation of histone H3 lysine 27 (Blais et al., To functionally annotate our set of Sin3 target genes and to 2007). However, the molecular mechanisms by which genes test whether genes bound by E2F4 and Sin3, or Sin3 only, play are permanently repressed in a stepwise fashion during differen- different physiological roles, we clustered them using the Gene tiation are not understood, and a general description of changes Ontology (GO) program DAVID (Dennis et al., 2003) and exten- at the level of chromatin remodeling is completely lacking. sive manual curation (Figures 1D–1F and Table S1). Generally, In order to gain insight into the molecular mechanisms of Sin3 target genes were enriched in categories related to cell- permanent gene repression in differentiated cells, we performed cycle regulation, transcription, mitochondrial and cellular protein extensive genome-wide transcription factor binding analyses synthesis, lipid metabolism, stress response, and RNA metabo- in which we coupled chromatin immunoprecipitation (ChIP) lism, a result reminiscent of depletion and knockout experiments with promoter microarrays (ChIP-on-chip). Here, we performed conducted in yeast, Drosophila, and mammalian cells (Dannen- large-scale binding analyses for Sin3A, Sin3B, RBP2, and berg et al., 2005; Kurdistani et al., 2002; Pile et al., 2003; Robert E2F4, and in parallel, we analyzed histone H3 acetylation and et al., 2004). When compared to genes bound by Sin3 alone, methylation levels, nucleosome positioning, and gene expres- Sin3/E2F4 targets were more strongly enriched in several func- sion in growing and differentiated skeletal muscle cells. Our tional categories, particularly those related to cell-cycle regula- data suggest that Sin3 and RBP2 play a concerted role in repres- tion, DNA repair, and DNA damage checkpoint (Figures 1D and sing expression of a subset of E2F4 target genes through the 1E). This strongly suggests that Sin3/E2F4 complexes represent modification and repositioning of nucleosomes in differentiated a major corepressor of cell-cycle genes during differentiation. cells. Genes bound exclusively by Sin3 were more highly enriched in processes related to mitochondrial and cellular protein synthe- RESULTS sis, differentiation and development, and lipid metabolism. Inter- estingly, we also found a subcluster of Sin3-only targets involved Recruitment of Sin3 Complexes to Diverse in Wnt signaling. Thus, genes bound by Sin3 and E2F4 function Sets of Target Genes within distinct cellular pathways as compared to genes that are Our previous studies indicated that repression of E2F target bound by Sin3 only. genes in differentiated myotubes proceeds through a succession of steps in which promoters of cell-cycle genes are deacetylated Sin3 Binds Activated and Repressed Genes and subsequently methylated on histone H3 (Blais et al., 2007). at Distinct Positions We asked how the first step, namely, histone deacetylation, In mammalian cells, Sin3 is recruited to promoter regions by was achieved in growing C2C12 myoblasts and cells differenti- sequence-specific repressors to silence gene transcription ated to produce myotubes through mitogen depletion. Using (Silverstein and Ekwall, 2005). The identification of genes that quantitative chromatin immunoprecipitation (qChIP), we showed differentially recruit Sin3 complexes (in the presence or absence that the Sin3 corepressor is recruited to E2F4 target genes in of E2F4) prompted us to test whether these complexes differ terminally differentiated myotubes (Figure 1A). with respect to binding site preferences (Figure 2A, left panel). To ask whether Sin3 plays a global role in transcriptional Surprisingly, we found that Sin3 was bound primarily to regions regulation during differentiation or if it is restricted to E2F4 target downstream of the TSS rather than promoters. Interestingly, we genes in myotubes, we performed genome-wide factor location also observed that E2F4 binding in the presence of Sin3 is analysis with antibodies specific for mouse Sin3A and Sin3B (see centered about the TSS, whereas E2F4 binding in the absence Figure S1A available online). We performed genome-wide ChIP- of Sin3 is slightly biased toward the upstream region. on-chip experiments with promoter arrays that contained ap- Next, we analyzed whether genes bound by both E2F4 and proximately 17,000 known mouse genes centered on the region Sin3 differ with respect to expression levels when compared to from À5.5 to +2.5 kb relative to the TSS at 250 bp resolution. genes bound by E2F4 or Sin3 alone (Figure 2A, right panel). Analysis of at least two independent experiments for each factor Genes bound by E2F4 and Sin3 are more highly repressed (aver-

(Figure S3) identified 707 target genes for Sin3A, 686 target age log2 Mt/Mb of À0.90) compared to genes bound by E2F4 genes for Sin3B, and 1058 target genes for E2F4 (Table S1). A (average log2 Mt/Mb of À0.49). On average, genes bound by comparison of Sin3 and E2F4 target genes indicated limited Sin3 alone did not show a change in expression, although Sin3 overlap (Figure 1B). The vast majority of Sin3 target genes binds to a subcluster of highly expressed genes (Figure 2A, right (80% for Sin3A and 62% for Sin3B) were not bound by E2F4. panel, and Figure 2B). Thus, Sin3 preferentially binds to the Using qChIP analysis, we confirmed that this population of Sin3 downstream regions of both repressed and activated genes. In target genes did not bind E2F4 in myotubes (Figure 1C). In addi- budding yeast, a Sin3/Rpd3 complex was also shown to bind tion, we verified Sin3A and Sin3B binding to a select group of within coding regions of transcribed and infrequently transcribed Sin3 targets by qChIP and found that many genes were bound genes (Carrozza et al., 2005; Keogh et al., 2005; Kurdistani et al., by both Sin3A and Sin3B (Figure S1C). Although we and others 2002; Li et al., 2007). To test whether the positional bias for mam- have uncovered functional distinctions between the two iso- malian Sin3 binding reflects differences in expression levels, we forms (David et al., 2008; and data not shown), our goal here plotted the location of Sin3 binding (based on our promoter array was to capture as many Sin3-binding events as possible, without data) as a function of gene expression during differentiation respect to isoform differences, and therefore we will collectively (Figure 2B) (Blais et al., 2007). Sin3 (Sin3A and Sin3B) binding

360 Molecular Cell 32, 359–370, November 7, 2008 ª2008 Elsevier Inc. Molecular Cell Sin3 Mediates Repression during Differentiation

Figure 1. Identification and Characterization of Sin3 Targets in Differentiated C2C12 Cells (A) Analysis of Sin3 and E2F4 binding to selected E2F4 target genes in myoblasts (Mb) and differentiated myotubes (Mt) by qChIP. IP signal was defined as the ratio of IP/Input for specific versus a control amplicon (Gapdh). The average of three independent experiments is shown. Error bars represent standard deviation. (B) Venn diagrams showing overlap between E2F4 and Sin3 target genes identified in differentiated myotubes using genome-wide ChIP-on-chip. Target genes were deduced from at least two independent experiments (Figure S3). (C) Verification of E2F4 binding to selected Sin3/E2F4 and Sin3 target genes by qChIP in growing myoblasts (Mb) and differentiated myotubes (Mt) as in (A). (D) Distribution of GO annotations for Sin3 targets. In some instances, a gene is assigned to more than one category. The percentage refers to the numberof bound genes within a particular category in relation to the total number of bound genes that have a GO annotation. The histogram depicts the distribution of GO categories among genes bound by Sin3 and E2F4 versus genes bound by Sin3 only. Percentages for the histogram were calculated as described above. (E) The percentage of cell-cycle-related genes that fall into the indicated groups bound by E2F4 is shown. E, genes bound by E2F4; EA/B, genes bound by E2F4 and Sin3A or Sin3B; EAB, genes bound by E2F4, Sin3A, and Sin3B. (F) Sin3 binds to a cadre of genes involved in transcriptional regulation. was restricted to regions immediately downstream of the TSS majority (58%) of these repressed genes (data not shown). These (median distance from TSS: +182 bp) on genes that were observations fail to support the prevailing notion that mammalian repressed (Figure 2B). In contrast, Sin3 spreads further into Sin3 is generally recruited to proximal promoters via sequence- downstream regions on genes that are not repressed, and this specific repressor proteins. Further, binding of Sin3 complexes observed positional bias was even more pronounced on genes is not restricted to genes that are repressed. upregulated in differentiated C2C12 myotubes (median distance from TSS: +568 bp). Surprisingly, our analysis also clearly Sin3 Complexes Spread over Regions showed that the majority of Sin3 target genes were not re- Downstream of the TSS pressed: we found that only 20% of Sin3 target genes were re- To define Sin3- and E2F4-binding events more precisely, we pressed (log2 Mt/Mb < À0.58) in differentiated versus growing designed high-density tiling arrays (Supplemental Data) and C2C12 cells (Figure 2B), and E2F4 targets accounted for the performed ChIP-on-chip analysis on both factors (Table S2).

Molecular Cell 32, 359–370, November 7, 2008 ª2008 Elsevier Inc. 361 Molecular Cell Sin3 Mediates Repression during Differentiation

Figure 2. Selective Targeting of Sin3 to Transcribed Regions of Differentially Expressed Groups of Genes (A) Distribution of E2F4, Sin3A, and Sin3B binding (left panel) and expression (right panel) of distinct E2F4 and Sin3 target genes, deduced from promoter array data. Groups represent genes bound by E2F4 (E) or Sin3 (S) only or by both Sin3 and E2F4 (SE). Mb, myoblasts; Mt, myotubes. (B) Location of Sin3 binding (central probe) relative to the TSS was plotted as a function of relative expression during differentiation (Blais and Dynlacht, 2007).

Activated, log2 Mt/Mb > 0.58; repressed, Mt/Mb < À0.58. (C) Heat maps of E2F4, Sin3A, and Sin3B binding deduced from tiling array data. Genes were grouped as described for (A). Probes associated with factor binding are shown in green. A binding event (peak) was defined by a minimum of three probes with log2 (IP/Input) > 1, maximally spaced by 80 bp (Figure S3). The results of three independent experiments are shown. (D) E2F4-, Sin3A-, and Sin3B-binding profiles on genes grouped (as [A]) according to factor occupancy. For each group, genes were aligned with respect to their TSS. Probes associated with a binding event (as determined by our peak finding algorithms) were set to 1, while all others were set to 0. The total number of binding events for all genes per group per probe was calculated, normalized for maximum binding, and plotted relative to the TSS. (E) Binding profiles for E2F3 and E2F4 on genes bound by E2F4 (Cdc2a) or by E2F4 and Sin3 (Mybl2).

We observed multiple, overlapping E2F4- and Sin3-binding stream regions in the absence of E2F4. In contrast, E2F4 binding events downstream of the TSS per gene, strongly suggesting in the absence of Sin3 is restricted to sites near the TSS and did coordinated recruitment, or spreading, of Sin3/E2F4 corepressor not show a bias for downstream regions (Figures 2C and 2D and complexes (Figures 2C and 2D and Figure S4A). We also ob- Figure S4B). Taken together, this suggests that E2F4 and Sin3 served a small subpopulation of E2F4-binding events specifically mutually influence each other’s binding position and behave in the upstream promoter region that is apparently not associated like a complex on chromatin. To further verify our conclusions re- with Sin3 binding. In agreement with the genome-wide promoter garding spreading, we performed ChIP-on-chip with antibodies array analysis, our high-density tiling array data confirmed that against another E2F family member, E2F3, as this factor is also Sin3 binding is influenced by E2F4 binding: on E2F4 targets, present on a subset of E2F4 target genes in differentiated cells Sin3 is restricted to positions immediately downstream of the (Figure 2E and data not shown). Here, we observed sharp distinc- TSS, while Sin3 exhibited more scattered binding within down- tions in binding profiles. For example, we observed that E2F3

362 Molecular Cell 32, 359–370, November 7, 2008 ª2008 Elsevier Inc. Molecular Cell Sin3 Mediates Repression during Differentiation

binds within a narrow window upstream of the TSS of the Cdc2a 2007). Therefore, we tested whether RBP2 was recruited to Sin3 gene, reminiscent of E2F4 binding in the absence of Sin3. targets in differentiated myotubes using our high-density tiling Furthermore, although both E2F3 and E2F4 are recruited to the arrays. Our analysis showed that the majority of Sin3 target Mybl2 gene, binding of E2F4 (in the presence Sin3) can be readily genes (58%) were bound by RBP2 (Table S2). We confirmed distinguished from that of E2F3. These data strengthen our RBP2 binding to selected Sin3/E2F4 target genes by qChIP (Fig- findings regarding specific spreading of Sin3/E2F4 downstream ure S1D). Interestingly, comparison of Sin3B and RBP2-binding of the TSS. profiles on genes bound by Sin3/E2F4 and RBP2 indicated In subsequent studies, we focused our work primarily on Sin3/ a very striking positional overlap immediately downstream of E2F4 target genes to gain insight into the role of these factors in the TSS (Figure 3E), strongly suggesting coordinated binding silencing gene expression. of Sin3 and RBP2 within this region. To further verify this Sin3- RBP2 interaction, we asked whether Sin3 associates with E2F4 and Sin3 Are Coordinately Recruited RBP2 in myotube extracts. Indeed, coimmunoprecipitation as a Corepressor Complex experiments confirmed that RBP2 is part of a Sin3 complex in Using genome-wide and high-density tiling arrays, we observed differentiated cells (Figure 3F). Interestingly, when we immuno- a striking overlap between E2F4 and Sin3 binding primarily precipitated Sin3B, but not Sin3A, RBP2 coprecipitated. In the downstream of the TSS, strongly suggesting that E2F4 and Sin3 reciprocal experiment, when we immunoprecipitated RBP2, are recruited as a complex. To further demonstrate a functional Sin3B was detected. In agreement with previous reports, we interaction, we transfected myotubes with siRNAs to deplete did not observe an association between either RBP2 or Sin3 both isoforms of Sin3 (Figure 3A) or DP1 (Figure S1B) and and E2F4, suggesting either that this interaction occurs only on assessed recruitment of E2F4 and Sin3B. All E2F transcription chromatin (Figures 3B and 3C) or that the interaction is not stable factors (with the exception of E2F7 and E2F8) bind to DNA as under our immunoprecipitation conditions (Hayakawa et al., obligate heterodimers with the DP family of proteins (Blais and 2007; Silverstein and Ekwall, 2005). Dynlacht, 2004). Since DP1 is known to exclusively pair with Next we asked whether Sin3/RBP2 binding overlaps with HDAC E2Fs, we reasoned that depletion of this factor would be the and demethylase activity within this downstream region on a sub- most robust and effective way to eliminate both E2F4 activity set of E2F4 target genes using our tiling arrays (see Figure S3C). and prevent promoter occupancy by compensatory repressor We observed a profound decrease in histone H3 acetylation E2Fs, since it is well known that the remaining E2F family mem- (H3ac) and histone H3 lysine 4 trimethylation (H3K4-3M) levels im- bers can readily replace depleted factors at promoters. Indeed, mediately downstream of the TSS in differentiated myotubes as depletion of DP1 resulted in a reduction of E2F4 binding (Fig- compared to proliferating myoblasts (Figures 3G and 3H). Further, ure S1B). More importantly, Sin3B binding was preferentially we asked whether the recruitment and activities of Sin3 and RBP2 and markedly reduced in downstream regions of several genes, are also temporally coordinated during differentiation. In order to including Kif20a and Mcm5, upon depletion of DP1 (Figure 3B). address this question, we isolated chromatin from cells during As we have depleted all E2F activity present on our target genes, a differentiation time course and analyzed E2F4, Sin3, and RBP2 loss of other E2F family members, besides E2F4, might contrib- binding to an E2F4 target gene, Kif20a. Each factor exhibited sub- ute to reduced Sin3B binding. Thus, to further strengthen our stantial or maximal binding to the Kif20a gene at the earliest stages observation about a functional E2F4-Sin3 complex, we depleted of differentiation, when cells are transiently arrested by contact Sin3A and Sin3B and tested for E2F4 binding. Interestingly, inhibition (represented by t = 0) (Figure 4A), although the gene depletion of Sin3 also resulted in reduced binding of E2F4 to was not fully repressed at this stage (Figure 4B). This phenomenon the Kif20a, Mcm5, and Mybl2 genes, again preferentially within was similarly observed for several other E2F4 targets genes (data the downstream region, but not on a set of genes bound by not shown). Interestingly, H3Ac and H3K4-3M levels remain high E2F4 only (Figure 3C). Thus, in agreement with our location anal- at this stage, but 48 after induction of differentiation, both marks ysis, Sin3 and E2F4 appear to functionally interact on chromatin showed a precipitous decrease specifically within the region as a complex, and stable association of this complex with target immediately downstream of the TSS (Figure 4C). Previously, we genes in differentiated cells is mediated by both factors. had shown that the acquisition of the H3 lysine 27 di-/trimethyla- Interestingly, we did not find a correlation between binding of tion mark (H3K27-2/3M) is associated with repression of E2F4 Sin3/E2F4 complexes and H3K36 trimethylation, a modification genes in differentiated cells (Blais et al., 2007). Therefore, we that mediates recruitment of Sin3 complexes to coding regions tested H3K27-2/3M levels during differentiation. Interestingly, in budding yeast (Carrozza et al., 2005; Keogh et al., 2005; Nic- H3K27-2/3M levels on the Kif20a gene peaked 48–96 hr after olas et al., 2007), as maximal Sin3B binding and peak levels of induction of differentiation, most prominently within the region H3K36 trimethylation were offset on the Kif20a gene (Figure 3D). immediately downstream of the TSS (Figure 4). Although we cannot rule out a role for the H3K36 mark in recruit- To directly assess a functional role for Sin3 complexes in local- ment of Sin3 complexes, these data suggest that it may not be ized histone deacetylation and demethylation, we tested the obligatory for genes repressed by Sin3/E2F4 complexes. impact of ablating both isoforms by transfecting myotubes with specific and nonspecific (scrambled) siRNAs (Figure 3A). Since Coordinated Activity of Sin3 and RBP2 RBP2 (Figure 3F) and HDAC1/2 are part of a Sin3 complex during Differentiation (Hayakawa et al., 2007; Silverstein and Ekwall, 2005), we rea- Recently, the Sin3 complex has also been linked to demethylase soned that depletion of Sin3 activity should result in changes in activity by virtue of its association with RBP2 (Hayakawa et al., histone H3 acetylation and histone H3 lysine 4 trimethylation

Molecular Cell 32, 359–370, November 7, 2008 ª2008 Elsevier Inc. 363 Molecular Cell Sin3 Mediates Repression during Differentiation

Figure 3. Sin3/E2F4-RBP2 Functions as a Complex on Target Genes (A) Representative western blot indicates knockdown of Sin3A and Sin3B after transfection of myotubes with indicated siRNAs. Myotubes were transfected 48 hr after induction of differentiation and were isolated 72 hr after transfection. (B) Histogram depicting Sin3B binding assessed by qChIP of selected genes in differentiated myotubes transfected with a nonspecific control (NS) and DP1 siRNAs. Relative binding is expressed as a ratio of signals obtained in specific versus NS siRNA transfected cells. The average of three independent experiments is shown. Error bars represent standard deviation. (C) As in (B), except that cells were transfected with control and Sin3A/Sin3B (Sin3) siRNAs and E2F4 binding was measured. (D) H3K36 trimethylation levels and Sin3B binding were examined on the Kif20a gene using qChIP. Enrichment was calculated as percentage of input and normalized to maximum binding levels. The results of three independent experiments are shown. (E) Binding profiles for RBP2 and Sin3B binding to genes bound by Sin3, E2F4, and RBP2, as described in Figure 2D. Only genes bound by all three factors were examined. (F) Endogenous Sin3B and RBP2 interact in vivo. Sin3 and RBP2 were immunoprecipitated using whole-cell extracts of differentiated C2C12 cells (myotubes) with the respective antibodies and probed for either RBP2 or Sin3. (G and H) (G) Genome-wide histone H3 acetylation (H3Ac) and (H) histone H3 lysine 4 trimethylation (H3K4-3M) profiles of genes bound by Sin3, E2F4, and RBP2 in myoblasts and differentiated myotubes were obtained using tiling arrays. Genes were aligned relative to the TSS, and average histone H3Ac or H3K4-3M values per probe were calculated. The results of two independent experiments are shown. on genes that recruit this Sin3 complex. Depletion of Sin3 in acetylation levels are a consequence of enhanced transcription differentiated C2C12 myotubes resulted in robust localized in- after depletion of Sin3 (see Figure 7A), we note that increased creases in H3Ac and H3K4-3M levels on genes bound by E2F4 acetylation levels were most obvious near Sin3-binding sites and Sin3 (Figures 4D and 4E). In striking contrast, we did not ob- immediately downstream of the TSS on multiple genes, and we serve changes in H3Ac on genes bound by Sin3 or E2F4 only or did not observe reacetylation of transcribed regions further on a control gene not bound by either factor (Hoxa9), nor did we downstream on the same genes (Figure 4D and data not shown). observe changes in H3K4-3M on genes bound by E2F4 only. Al- Furthermore, we reasoned that ablation of E2F activity should though we cannot formally rule out the possibility that elevated promote alterations similar to those observed upon depletion of

364 Molecular Cell 32, 359–370, November 7, 2008 ª2008 Elsevier Inc. Molecular Cell Sin3 Mediates Repression during Differentiation

Figure 4. Coordinated Removal of ‘‘Active’’ Chromatin Marks by Sin3 and RBP2 Leads to Repression (A) Analysis of Sin3, E2F4, and RBP2 binding to a selected E2F4 target gene (Kif20a) during a differentiation time course as in Figure 1A. Cycling myoblasts (Mb), arrested myoblasts (Mb, t = 0), and myotubes after 48 or 96 hr after induction of differentiation (Mt, t = 48 or t = 96, fully differentiated myotubes) were examined. (B) Gene expression analysis using quantitative real-time RT-PCR for a differentiation time course, as described in (A). (C) Histone H3 acetylation (H3Ac, left panel), histone H3 lysine 4 trimethylation (H3K4-3M, middle panel), and histone H3 lysine 27 di-/trimethylation (H3K27-2/3M, right panel) levels were measured by qChIP during a differentiation time course as in (A). Enrichment by IP was measured as a function of input signal. (D and E) Histograms depicting H3Ac (D) and H3K4-3M (E) levels, assessed by qChIP on selected genes in differentiated myotubes transfected with a nonspecific control (NS) or Sin3A/Sin3B (Sin3) siRNA. See Figure 3B for details. (F) H3Ac levels were measured after knockdown of DP1 as described in (D) and (E). In all panels, averages of at least three independent experiments are shown and error bars represent standard deviation.

Sin3 if there were a physical (and functional) link between E2F4 showed a significant (p < 0.03) enhancement of nucleosomes and a Sin3-RBP2 complex. Indeed, depletion of DP1 resulted immediately downstream of the TSS in differentiated myotubes in increased H3Ac on genes bound by Sin3 and E2F4, but not as compared to cycling myoblasts (Figures 5A and 5B and Fig- on control genes bound by E2F4 or Sin3 only (Figure 4F). ures S5A and S6C). In sharp contrast, we did not observe any sig- These data strongly suggest that Sin3 complexes, comprised nificant differences in nucleosome density over identical regions of Sin3, HDAC, and RBP2, are recruited by E2F4, resulting in of genes bound only by Sin3 (Figure 5A, center panel; Figure 5C, highly specific modification of nucleosomal targets immediately far right panel; and Figure S5B) or E2F4 (Figure 5A, second to right downstream of the TSS. panel; and Figure 5C, left three panels). We considered the possibility that differences in nucleosome Sin3 Impacts Local Chromatin Structure occupancy within this downstream region simply reflected Next, we asked whether genes bound by E2F4 and Sin3 exhibit markedly different gene expression levels in each group of differences in nucleosome occupancy (see Figure S3C), since Sin3 targets. To test this possibility, we first compared nucleo- acetylation of nucleosomes is tightly linked to chromatin remod- some density profiles of genes with low, intermediate, and high eling in certain settings (Gregory et al., 2001; Neely and Work- expression levels in differentiated myotubes, irrespective of man, 2002). Remarkably, genes bound by both Sin3 and E2F4 Sin3 and E2F4 binding (Figure 6A). For this comparison, average

Molecular Cell 32, 359–370, November 7, 2008 ª2008 Elsevier Inc. 365 Molecular Cell Sin3 Mediates Repression during Differentiation

Figure 5. High-Resolution Nucleosome Density Profiles of Sin3 Target Genes (A) Average nucleosome density profiles for genes bound by Sin3 and E2F4 (left panel), Sin3 only (middle panel), and E2F4 only (right panel) in myoblasts and differentiated myotubes deduced from our tiling arrays (see Figure 2C). Genes were aligned relative to the TSS, and average MNase values per probe were cal- culated for each group. The results of at least two (myotubes) or three (myoblasts) experiments are shown. Mb, myoblasts; Mt, myotubes. (B) Nucleosome density profiles for individual genes bound by Sin3 and E2F4 in myoblasts and myotubes. MNase ratio is given by the moving average (window size 140 bp, step size five probes). (C) Genes bound by E2F4 only (three left panels) or a gene bound by Sin3 only (Psmd14, far right panel) was analyzed as described in (B). expression levels were clearly different, yet we did not observe sufficient to generate a nucleosome density profile similar to distinct, associated nucleosome density profiles. Second, we genes bound by both E2F4 and Sin3 (Figure 6B, right panel). compared genes bound by E2F4 and Sin3 with a set of control Moreover, ChIP-on-chip analysis of Sin3 binding and nucleo- genes (i.e., those not bound by E2F4 or Sin3; Table S2) that some densities indicated a partial overlap of Sin3 binding and exhibit a similar expression profile (wherein the distribution of enhanced nucleosome densities (Figure 6C), providing an expla- absolute expression values is not different between Sin3/E2F4 nation for the underlying alterations of nucleosome positioning in and control groups; Figure S5C) in differentiated myotubes and the immediate vicinity of Sin3 binding. found that genes bound by E2F4 and Sin3 displayed significantly enhanced nucleosome density at positions immediately down- Sin3 Is Essential for the Permanent Repression of Genes stream of the TSS (Figure 6B, left panel). Thus, we conclude by Maintaining Chromatin Structure that in differentiated myotubes, nucleosome density patterns To examine a direct role for Sin3 on localized nucleosome accu- are not simply a reflection of transcription rates; rather, they mulation, chromatin derived from myotubes transfected with are shaped in part by the presence of E2F4 and Sin3. To further either control or specific Sin3 siRNAs was immunoprecipitated dissect the influence of E2F4 and Sin3 on chromatin structure, with antibodies against histone H3, and enriched DNA was ana- we compared nucleosome-binding profiles of genes bound by lyzed using quantitative PCR. Histone H3 levels in control sam- E2F4 and Sin3 versus genes bound exclusively by E2F4. Impor- ples mirrored nucleosome density profiles obtained on the Kif20a tantly, this analysis indicated that E2F4 binding alone was not gene using our tiling arrays (Figure 6D and Figure 5B, second

366 Molecular Cell 32, 359–370, November 7, 2008 ª2008 Elsevier Inc. Molecular Cell Sin3 Mediates Repression during Differentiation

Figure 6. Sin3 Complex Directly Impacts Nucleosome Structure Downstream of the TSS (A) Average nucleosome density profiles for genes that were classified according to absolute expression levels (based on Affymetrix gene expression arrays) in differentiated myotubes (Blais et al., 2007): low (average, 100; range 1–200), intermediate (average, 485; range 201–1000), and high (average, 4024; range 1000+) abundance transcripts. (B) Average nucleosome density profiles for genes bound by Sin3 and E2F4, a control group of genes (65 genes not bound by Sin3/E2F4 but similarly expressed; see Table S2), or genes bound by E2F4 only in differentiated myotubes, with median log2 (Mt/Mb) expression values for each group being À1.4, À1.6, and À0.54, respectively. (C) Binding profiles of Sin3A and Sin3B relative to nucleosome density profiles of the same genes. Binding and MNase ratios were normalized for maximum levels. (D) Histogram depicting H3 levels on distinct regions of an individual Sin3/E2F4 target gene in myotubes transfected with a nonspecific control (NS) or Sin3 silencing (Sin3) siRNA. The asterisk indicates a histone H3 signal significantly lower than the NS control (p < 0.01 by t test). The average of at least three independent experiments is shown. Error bars represent standard deviation. (E) Analyses were performed as in (D) except that histone H3 signal is expressed relative to the signal obtained in cells transfected with NS control. from left panel). Remarkably, cells depleted of Sin3A and Sin3B Next, we sought to determine whether Sin3 binding and its showed specific and highly significant (p < 0.01) decreases in effect on downstream chromatin structure are critical for gene histone H3 levels downstream of the TSS. Importantly, we did expression. To address this question, differentiated myotubes not observe any significant decreases in histone H3 levels either were transfected with control or specific siRNAs targeting upstream or far downstream of the TSS (Figure 6D). Analysis of Sin3A and Sin3B (Figure 3A), and differences in RNA abundance additional Sin3/E2F4 target genes showed consistent diminution were analyzed using quantitative RT-PCR. We found that ex- of histone H3 levels immediately downstream of the TSS upon pression of genes targeted by both E2F4 and Sin3 was markedly Sin3 ablation (Figure 6E). In contrast, genes bound exclusively enhanced upon depletion of Sin3, indicative of derepression by Sin3 or E2F4 and an unrelated control gene not bound by (Figure 7A). Similar results were obtained using a different com- either Sin3 or E2F4 (Hoxa9) did not exhibit significant reductions bination of specific siRNAs targeting Sin3 (data not shown). in histone H3 levels at corresponding locations. In striking contrast, we observed diminished or unaltered

Molecular Cell 32, 359–370, November 7, 2008 ª2008 Elsevier Inc. 367 Molecular Cell Sin3 Mediates Repression during Differentiation

Figure 7. Expression of Sin3 Target Genes after Knockdown of Sin3 (A) Gene expression analysis using quantitative real-time RT-PCR of selected genes after ablation of Sin3. Myotubes were transfected 48 hr after induction of differentiation and were isolated 96 hr after transfection. Expression levels of selected genes in Sin3 siRNA-transfected cells were compared to nonspecific control (NS) trans- fected cells. The average of three independent ex- periments is shown. Error bars represent standard deviation. (B) Model detailing Sin3 function in differentiated muscle cells on a subset of E2F4 target genes. Me, methylation of H3K4 (green) and H3K27 (red); Ac, acetylation. See text for details.

sites. In addition, although we cannot formally rule out the possibility that Sin3 associates with other repressors within the same region, extensive computa- tional analysis with search algorithms de- signed to identify enriched motifs failed to identify enrichment of known Sin3-asso- ciated repressor proteins (Supplemental Data and Table S3). (2) The activities of expression of several genes that bind Sin3 only. These data high- Sin3 and RBP2 result in the coordinated removal of acetylation light the functional distinctions, including differences in nucleo- and methylation marks, and this coincides with the timely acqui- some organization and gene expression, between Sin3-only sition of the repressive H3K27 methylation mark and altered nu- and Sin3/E2F4 target genes. cleosome architecture. (3) Ablation of Sin3 activity leads to loss of nucleosome density, and this coincides with reacetylation, DISCUSSION reflecting hyperacetylation and subsequent re-expression of cell-cycle-regulated genes in differentiated cells. (4) Consistent Our studies provide evidence that Sin3 may play an essential role with a role in permanent gene silencing, our recent conditional in heterochromatin formation and permanent gene repression by knockout studies in mice suggest both overlapping and redun- means of promoter binding and spreading, localized deacetyla- dant roles for Sin3 isoforms in maintenance of the differentiated tion and demethylation of one or more proximal nucleosomes, state (C.v.O, G. David, and B.D.D., unpublished data). and subsequent repositioning of nucleosomes on a subset of How Sin3 stably associates with this downstream region is E2F4-regulated genes. Thus, the Sin3/E2F4 complex provides not known. Recruitment of the Sin3 complex creates localized a paradigm for the coordinated modification of chromatin ulti- regions of histone H3 hypoacetylation and methylation in a coor- mately leading to the formation of heterochromatin and perma- dinated manner (Figure 4). Interestingly, in vitro experiments nent gene silencing in differentiated cells. suggest that the Sin3 complex shows a preference for the hypo- acetylated amino-terminal tail of histone H3, resulting in strong, A Model for Stable Gene Repression in Terminally repressor-independent anchoring of the Sin3 complex (Vermeu- Differentiated Cells len et al., 2006). Here, we have observed a mutual dependency on A mammalian Sin3B complex is reversibly recruited to cell-cycle E2F4 and Sin3 for the stable association of Sin3/E2F4 com- genes in quiescent and early G1 cells (Balciunaite et al., 2005; plexes. Thus, we propose that in differentiated myotubes, Sin3 Rayman et al., 2002), and the activity of Sin3B appears essential is stably associated with downstream promoter regions by for transient cell-cycle withdrawal (David et al., 2008). Our E2F4 and interaction with hypoacetylated histone tails. Other detailed analysis suggests a model in which Sin3 is also a critical chromatin modifications, in particular H3K36 methylation, could component for the permanent repression of cell-cycle genes possibly also play a role in directing mammalian Sin3 recruitment during differentiation (Figure 7B). Several observations lend cre- to target genes. dence to this model. (1) In differentiated cells, distinct Sin3/E2F4 complexes (and on a subset of Sin3/E2F4 target genes, RBP2) A Role for Sin3 Complex in Chromatin Remodeling bind to downstream regions and locally alter both histone mod- Our nucleosome density experiments and gene expression stud- ifications and chromatin structure. Moreover, our genome-wide ies suggest that binding of Sin3 to cell-cycle-regulated genes has location, factor ablation, and bioinformatics analyses show a a profound effect on chromatin structure and gene expression clear functional relationship between Sin3 and E2F4 at target (Figures 5–7A). How do Sin3/E2F4 complexes alter chromatin

368 Molecular Cell 32, 359–370, November 7, 2008 ª2008 Elsevier Inc. Molecular Cell Sin3 Mediates Repression during Differentiation

structure? It is known that Sin3 and E2F/pocket protein protein, cells were resuspended in Trizol and processed according to the complexes interact with a variety of factors involved in chromatin manufacturer’s instructions. siRNA sequences appear in the Supplemental function. For example, the Sin3 complex was shown to interact Experimental Procedures section. with Brg1, the catalytic subunit of the Swi/Snf chromatin-remod- eling enzyme (Nagl et al., 2007; Sif et al., 2001). Given this con- RT-PCR RT-PCR was used to analyze gene expression after knockdown of Sin3. nection, it is reasonable to speculate that, in the proper setting, RT-PCR was performed using 250 ng of total RNA per reverse transcriptase the recruitment and activity of Sin3-HDAC and Swi/Snf (or func- reaction (Invitrogen). cDNA was diluted and quantified twice by real-time tionally related remodeling enzymes) are tightly coordinated to PCR using the SYBR Green method and the results expressed relative to promote nucleosome repositioning. the nonspecific control siRNA. Nucleosome positioning within promoter regions plays a critical role in regulating gene expression by limiting the access Coimmunoprecipitation of transcription factors. The presence of Sin3/E2F4 complexes Cells were lysed in lysis buffer E (50 mM Tris$HCl [pH 7.9], 300 mM NaCl, could therefore physically block access of the preinitiation com- 0.1 mM EDTA, 1 mM DTT, 0.5% NP-40) supplemented with complete protease inhibitor cocktail (Roche Molecular Biochemicals). The cell lysates were di- plex and recruitment of factors to promoters at least in part by in- luted with equal volumes of NETN buffer (20 mM Tris$HCl [pH 7.9], 100 mM creasing the density of nucleosomes locally. Such regions could NaCl, 1 mM EDTA, 0.5% NP-40) supplemented with protease inhibitors and represent localized, facultative heterochromatin in differentiated immunoprecipitated with the specified antibodies. skeletal muscle cells, as we have previously observed the pres- ence of the repressive H3K27 methylation mark on a subset of ACCESSION NUMBERS E2F4/pRb target genes (Blais et al., 2007), many of which have been shown to bind Sin3 in this study. Our work also highlights Microarray data are available from http://www.ncbi.nlm.nih.gov/geo/ under an especially important role for nucleosomes immediately down- accession number GSE13247. stream of the TSS. Further work will be required to elucidate the importance of TSS-proximal nucleosomes implicated in this SUPPLEMENTAL DATA study, to define the chromatin marks associated with this region, to determine whether higher-order chromatin compaction can The Supplemental Data include Supplemental Experimental Procedures, Supplemental References, and six figures and can be found with this article occur, and to examine whether PolII binding is blocked. online at http://www.molecule.org/supplemental/S1097-2765(08)00731-4.

EXPERIMENTAL PROCEDURES ACKNOWLEDGMENTS

Cell Culture We are grateful to members of the Dynlacht lab and to G. David for comments The C2C12 murine myoblast cell line (obtained from ATCC) was cultured as on the manuscript and helpful suggestions during the course of this study. We described (Blais et al., 2007). Briefly, cells were grown in DMEM supplemented thank A. Bhattacharjee and G. Das for assistance with experiments. This work with 10% fetal bovine serum. Differentiated cells were obtained by culturing was supported by National Institutes of Health (NIH) grant CA077245 (to confluent C2C12 cells in DMEM supplemented with 2% horse serum. Differen- B.D.D.), NIH grant CA076120 (to W.G.K.), Susan Komen Postdoctoral Fellow- tiated cells were separated from undifferentiated cells using diluted trypsin. ship (to C.v.O.), and Department of Defense Breast Cancer Research Program Concept Award W81XWH-07-1-0581 (to Q.Y.). W.G.K. is an Investigator of the Antibodies Howard Hughes Medical Institute. Antibodies against E2F3 (sc-878), E2F4 (sc-1082), Sin3A (sc-767, sc-994), and Sin3B (sc-768) were obtained from Santa Cruz. Antibody against DP1 was Received: May 5, 2008 obtained from BD PharMingen (556462). Antibodies against RBP2 (1416 and Revised: August 19, 2008 2470) were described previously (Benevolenskaya et al., 2005; Klose et al., Accepted: October 17, 2008 2007). Antibodies against histone H3 (ab1791), histone H3K4-3M (ab8580), Published: November 6, 2008 histone H3K36-3M (ab9050), and anti-H3Ac (K9Ac + K14Ac, 06-599) were obtained from Abcam and Millipore. Anti-H3K27-2/3M (clone 7B11; gift of REFERENCES D. Reinberg) has been characterized previously (Sarma et al., 2004). Alland, L., David, G., Shen-Li, H., Potes, J., Muhle, R., Lee, H.C., Hou, H., Jr., ChIP, ChIP-on-chip, and Data Analysis Chen, K., and DePinho, R.A. (2002). Identification of mammalian Sds3 as an Cells were crosslinked, harvested, and lysed as described previously (Takaha- integral component of the Sin3/histone deacetylase corepressor complex. shi et al., 2000). For conventional ChIP, nuclei were collected by centrifugation, Mol. Cell. Biol. 22, 2743–2750. resuspended in sonication buffer (1 mM EDTA, 0.5 mM EGTA, 10 mM Tris [pH Ayer, D.E., Lawrence, Q.A., and Eisenman, R.N. (1995). Mad-Max transcrip- 8], 0.5% N-lauroyl-sarcosine and protease inhibitors), and sonicated on ice to tional repression is mediated by ternary complex formation with mammalian an average length of 350 bp. To enrich for histone H3 or acetylated H3 after homologs of yeast repressor Sin3. Cell 80, 767–776. knockdown of Sin3, 1 and 5 mg of chromatin was used per immunoprecipita- tion, respectively. Mononucleosome preparation, ChIP-on-chip experiments, Balciunaite, E., Spektor, A., Lents, N.H., Cam, H., Te Riele, H., Scime, A., and data analysis are detailed in the Supplemental Experimental Procedures. Rudnicki, M.A., Young, R., and Dynlacht, B.D. (2005). Pocket protein com- plexes are recruited to distinct targets in quiescent and proliferating cells. Mol. Cell. Biol. 25, 8166–8178. RNAi Cells were grown to confluence, induced to differentiate for 48 hr, and trans- Benevolenskaya, E.V., Murray, H.L., Branton, P., Young, R.A., and Kaelin, fected in triplicate with specific siRNA duplexes (Dharmacon) or a scrambled W.G., Jr. (2005). Binding of pRB to the PHD protein RBP2 promotes cellular nonspecific control using siMPORTER (Millipore), according to the manufac- differentiation. Mol. Cell 18, 623–635. turer’s instructions. Differentiated myotubes were harvested at 72 (ChIP) or Blais, A., and Dynlacht, B.D. (2004). Hitting their targets: an emerging picture of 96 hr (RT-PCR) after transfection using diluted trypsin. To isolate RNA and E2F and cell cycle control. Curr. Opin. Genet. Dev. 14, 527–532.

Molecular Cell 32, 359–370, November 7, 2008 ª2008 Elsevier Inc. 369 Molecular Cell Sin3 Mediates Repression during Differentiation

Blais, A., and Dynlacht, B.D. (2007). E2F-associated chromatin modifiers and Kurdistani, S.K., Robyr, D., Tavazoie, S., and Grunstein, M. (2002). Genome- cell cycle control. Curr. Opin. Cell Biol. 19, 658–662. wide binding map of the histone deacetylase Rpd3 in yeast. Nat. Genet. 31, Blais, A., van Oevelen, C.J., Margueron, R., Acosta-Alvear, D., and Dynlacht, 248–254. B.D. (2007). Retinoblastoma tumor suppressor protein-dependent methylation Laherty, C.D., Yang, W.M., Sun, J.M., Davie, J.R., Seto, E., and Eisenman, of histone H3 lysine 27 is associated with irreversible cell cycle exit. J. Cell Biol. R.N. (1997). Histone deacetylases associated with the mSin3 corepressor me- 179, 1399–1412. diate mad transcriptional repression. Cell 89, 349–356. Carrozza, M.J., Li, B., Florens, L., Suganuma, T., Swanson, S.K., Lee, K.K., Li, B., Gogol, M., Carey, M., Pattenden, S.G., Seidel, C., and Workman, J.L. Shia, W.J., Anderson, S., Yates, J., Washburn, M.P., and Workman, J.L. (2007). Infrequently transcribed long genes depend on the Set2/Rpd3S (2005). Histone H3 methylation by Set2 directs deacetylation of coding regions pathway for accurate transcription. Genes Dev. 21, 1422–1430. by Rpd3S to suppress spurious intragenic transcription. Cell 123, 581–592. Nagl, N.G., Jr., Wang, X., Patsialou, A., Van Scoy, M., and Moran, E. (2007). Christensen, J., Agger, K., Cloos, P.A., Pasini, D., Rose, S., Sennels, L., Distinct mammalian SWI/SNF chromatin remodeling complexes with oppos- Rappsilber, J., Hansen, K.H., Salcini, A.E., and Helin, K. (2007). RBP2 belongs ing roles in cell-cycle control. EMBO J. 26, 752–763. to a family of demethylases, specific for tri-and dimethylated lysine 4 on Neely, K.E., and Workman, J.L. (2002). Histone acetylation and chromatin histone 3. Cell 128, 1063–1076. remodeling: which comes first? Mol. Genet. Metab. 76, 1–5. Dannenberg, J.H., David, G., Zhong, S., van der Torre, J., Wong, W.H., and Nicolas, E., Yamada, T., Cam, H.P., Fitzgerald, P.C., Kobayashi, R., and Depinho, R.A. (2005). mSin3A corepressor regulates diverse transcriptional Grewal, S.I. (2007). Distinct roles of HDAC complexes in promoter silencing, networks governing normal and neoplastic growth and survival. Genes Dev. antisense suppression and DNA damage protection. Nat. Struct. Mol. Biol. 19, 1581–1595. 14, 372–380. David, G., Grandinetti, K.B., Finnerty, P.M., Simpson, N., Chu, G.C., and Pile, L.A., Spellman, P.T., Katzenberger, R.J., and Wassarman, D.A. (2003). Depinho, R.A. (2008). Specific requirement of the chromatin modifier mSin3B The SIN3 deacetylase complex represses genes encoding mitochondrial in cell cycle exit and cellular differentiation. Proc. Natl. Acad. Sci. USA 105, proteins: implications for the regulation of energy metabolism. J. Biol. Chem. 4168–4172. 278, 37840–37848. Dennis, G., Jr., Sherman, B.T., Hosack, D.A., Yang, J., Gao, W., Lane, H.C., Rayman, J.B., Takahashi, Y., Indjeian, V.B., Dannenberg, J.H., Catchpole, S., and Lempicki, R.A. (2003). DAVID: Database for Annotation, Visualization, Watson, R.J., te Riele, H., and Dynlacht, B.D. (2002). E2F mediates cell cycle- and Integrated Discovery. Genome Biol. 4, 3. Published online April 3, 2003. dependent transcriptional repression in vivo by recruitment of an HDAC1/ 10.1186/gb-2003-4-5-p3. mSin3B corepressor complex. Genes Dev. 16, 933–947. Ekwall, K. (2005). Genome-wide analysis of HDAC function. Trends Genet. 21, Robert, F., Pokholok, D.K., Hannett, N.M., Rinaldi, N.J., Chandy, M., Rolfe, A., 608–615. Workman, J.L., Gifford, D.K., and Young, R.A. (2004). Global position and recruitment of HATs and HDACs in the yeast genome. Mol. Cell 16, 199–209. Frolov, M.V., and Dyson, N.J. (2004). Molecular mechanisms of E2F-depen- dent activation and pRB-mediated repression. J. Cell Sci. 117, 2173–2181. Sarma, K., Nishioka, K., and Reinberg, D. (2004). Tips in analyzing antibodies directed against specific histone tail modifications. Methods Enzymol. 376, Gregory, P.D., Wagner, K., and Horz, W. (2001). Histone acetylation and 255–269. chromatin remodeling. Exp. Cell Res. 265, 195–202. Schreiber-Agus, N., Chin, L., Chen, K., Torres, R., Rao, G., Guida, P., Grunstein, M. (1997). Histone acetylation in chromatin structure and transcrip- Skoultchi, A.I., and DePinho, R.A. (1995). An amino-terminal domain of Mxi1 tion. Nature 389, 349–352. mediates anti-Myc oncogenic activity and interacts with a homolog of the Hassig, C.A., Fleischer, T.C., Billin, A.N., Schreiber, S.L., and Ayer, D.E. (1997). yeast transcriptional repressor SIN3. Cell 80, 777–786. Histone deacetylase activity is required for full transcriptional repression by Sif, S., Saurin, A.J., Imbalzano, A.N., and Kingston, R.E. (2001). Purification mSin3A. Cell 89, 341–347. and characterization of mSin3A-containing Brg1 and hBrm chromatin remod- Hayakawa, T., Ohtani, Y., Hayakawa, N., Shinmyozu, K., Saito, M., Ishikawa, eling complexes. Genes Dev. 15, 603–618. F., and Nakayama, J. (2007). RBP2 is an MRG15 complex component and Silverstein, R.A., and Ekwall, K. (2005). Sin3: a flexible regulator of global gene down-regulates intragenic histone H3 lysine 4 methylation. Genes Cells 12, expression and genome stability. Curr. Genet. 47, 1–17. 811–826. Takahashi, Y., Rayman, J.B., and Dynlacht, B.D. (2000). Analysis of promoter Kadosh, D., and Struhl, K. (1997). Repression by Ume6 involves recruitment of binding by the E2F and pRB families in vivo: distinct E2F proteins mediate a complex containing Sin3 corepressor and Rpd3 histone deacetylase to activation and repression. Genes Dev. 14, 804–816. target promoters. Cell 89, 365–371. Vermeulen, M., Walter, W., Le Guezennec, X., Kim, J., Edayathumangalam, Keogh, M.C., Kurdistani, S.K., Morris, S.A., Ahn, S.H., Podolny, V., Collins, R.S., Lasonder, E., Luger, K., Roeder, R.G., Logie, C., Berger, S.L., and Stun- S.R., Schuldiner, M., Chin, K., Punna, T., Thompson, N.J., et al. (2005). Cotran- nenberg, H.G. (2006). A feed-forward repression mechanism anchors the Sin3/ scriptional set2 methylation of histone H3 lysine 36 recruits a repressive Rpd3 histone deacetylase and N-CoR/SMRT on chromatin. Mol. Cell. complex. Cell 123, 593–605. Biol. 26, 5226–5236. Klose, R.J., Yan, Q., Tothova, Z., Yamane, K., Erdjument-Bromage, H., Zhang, Y., Iratni, R., Erdjument-Bromage, H., Tempst, P., and Reinberg, D. Tempst, P., Gilliland, D.G., Zhang, Y., and Kaelin, W.G., Jr. (2007). The retino- (1997). Histone deacetylases and SAP18, a novel polypeptide, are compo- blastoma binding protein RBP2 is an H3K4 demethylase. Cell 128, 889–900. nents of a human Sin3 complex. Cell 89, 357–364.

370 Molecular Cell 32, 359–370, November 7, 2008 ª2008 Elsevier Inc.