Molecular Cell Article A Role for Mammalian Sin3 in Permanent Gene 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 proteins, including Swi/Snf-remod- eling proteins, retinoblastoma (RB)-binding protein 2 (RBP2), and The multisubunit Sin3 corepressor complex regu- other proteins (Hayakawa et al., 2007; Nagl et al., 2007; Sif et al., lates gene transcription 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 genes 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 gene expression (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