A Role for Mammalian Sin3 in Permanent Gene Silencing Chris van Oevelen, Jinhua Wang, Patrik Asp, Qin Yan, William G. Kaelin, Yuval Kluger, Brian David Dynlacht  Molecular Cell  Volume 32, Issue 3, Pages 359-370 (November 2008) DOI: 10.1016/j.molcel.2008.10.015 Copyright © 2008 Elsevier Inc. Terms and Conditions

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 number of 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. Molecular Cell 2008 32, 359-370DOI: (10.1016/j.molcel.2008.10.015) Copyright © 2008 Elsevier Inc. Terms and Conditions

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 < −0.58. (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). Molecular Cell 2008 32, 359-370DOI: (10.1016/j.molcel.2008.10.015) Copyright © 2008 Elsevier Inc. Terms and Conditions

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. Molecular Cell 2008 32, 359-370DOI: (10.1016/j.molcel.2008.10.015) Copyright © 2008 Elsevier Inc. Terms and Conditions

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. Molecular Cell 2008 32, 359-370DOI: (10.1016/j.molcel.2008.10.015) Copyright © 2008 Elsevier Inc. Terms and Conditions

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 calculated 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). Molecular Cell 2008 32, 359-370DOI: (10.1016/j.molcel.2008.10.015) Copyright © 2008 Elsevier Inc. Terms and Conditions

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 −1.4, −1.6, and −0.54, 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. Molecular Cell 2008 32, 359-370DOI: (10.1016/j.molcel.2008.10.015) Copyright © 2008 Elsevier Inc. Terms and Conditions

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) transfected cells. The average of three independent experiments 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. Molecular Cell 2008 32, 359-370DOI: (10.1016/j.molcel.2008.10.015) Copyright © 2008 Elsevier Inc. Terms and Conditions