Volume 37, Issue 1, Pages 46-56 (January 2010) A Subset of the Histone H3 Lysine 9 Methyltransferases Suv39h1, G9a, GLP, and SETDB1 Participate in a Multimeric Complex  Lauriane Fritsch, Philippe Robin, Jacques R.R. Mathieu, Mouloud Souidi, Hélène Hinaux, Claire Rougeulle, Annick Harel-Bellan, Maya Ameyar-Zazoua, Slimane Ait-Si-Ali  Molecular Cell  Volume 37, Issue 1, Pages 46-56 (January 2010) DOI: 10.1016/j.molcel.2009.12.017 Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 Characterization of Suv39h1, G9a, GLP, and SETBD1 Complexes and Endogenous Interactions (A) Silver staining of Suv39h1 complex. The double immunopurification of eSuv39h1 complex has been performed on either nucleosome-enriched extracts (C) or from nuclear soluble fraction (S). Eluted eSuv39h1 complexes were resolved on a 4%–12% gradient SDS-PAGE gel. Note that we have loaded ten times more eSuv39h1 soluble complex (S) than chromatin-associated complex (C). MW, molecular weight marker, in KDa. (B) Silver staining of eluate from HeLa control cells. (C–E) Silver staining of G9a, GLP, and SETDB1 complexes. Mock, eluate obtained from the HeLa control cell line. S, soluble; C, chromatin-associated. Hist., histones. IP, immunoprecipitation. E, ectopic. (F–H) Endogenous Suv39h1, G9a, GLP, and SETDB1 interact in HeLa cells and in MEFs. Nuclear extracts from HeLa cells (F and G) or from MEFs (H) were used for IP with the indicated antibodies (IP Ab) or normal IgG as a negative control (Ctr). The resulting precipitates were then subjected to western blot analyses (WB) as indicated. WB signals were quantified and presented as percent of total input (H). Molecular Cell 2010 37, 46-56DOI: (10.1016/j.molcel.2009.12.017) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 H3K9 HKMTs Stability Is Affected in Suv39h−/− and in G9a−/− Null Cells (A) Expression of H3K9 HKMTs in Suv39h−/− MEFs. Nuclear extracts from wild-type MEFs (WT MEF) or Suv39h double knockout MEFs (Suv39h−/− MEF) were analyzed by WB using the indicated antibodies (Ab). As a control for Suv39h1 Ab, we ran extracts from HeLa cells stably expressing Flag-HA-tagged Suv39h1 (lane 5, HeLa-Suv39h1), which corresponds to a slower migrating band indicated by an asterisk (∗). 1 and 2, independent experiments. (B) Expression of H3K9 HKMTs in G9a−/− cells. Nuclear extracts from wild-type ES (WT ES) or G9a−/− ESCs (G9a−/− ES) were analyzed by WB using the indicated Ab. (C and D) Rescue of G9a, GLP, and SETDB1 protein levels in Suv39h−/− cells. Suv39h−/− MEFs were infected with retroviral vectors expressing Flag-HA-tagged versions of either a wild-type Suv39h1 (lane 3) or a mutant lacking enzymatic activity (H324K, lane 4), as shown by IF using HA antibody (C). Nuclear extracts from these cells (lanes 3 and 4), from wild-type (WT, lane 1), or from Suv39h−/− MEFs (Ctr, lane 2) were subjected to WB analyses (D). Molecular Cell 2010 37, 46-56DOI: (10.1016/j.molcel.2009.12.017) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 A Subset of Suv39h1, G9a, GLP, and SETBD1 Forms a Multimeric Complex (A) Fractionation of eSuv39h1 chromatin-associated complex. eSuv39h1 complex was treated with ethidium bromide and migrated on a 20%–41% glycerol gradient. Fractions were then manually collected, concentrated, and subjected to WB using the indicated AB (upper) or silver staining (lower). (B) Sequential immunoprecipitation (IP) of the four H3K9 HKMTs. Schematics of the sequential IP protocol (left). eSuv39h1 complex has been subjected to SETDB1 IP using SETDB1 Ab or a control Ab. Eluted proteins were then analyzed by WB (right). IP Ab, Ab used for IP; Ctr, IP negative control. Molecular Cell 2010 37, 46-56DOI: (10.1016/j.molcel.2009.12.017) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 Suv39h1, G9a, GLP, and SETDB1 Interact Directly In Vitro (A) Suv39h1 and SETDB1 interact directly in a GST pull-down assay. Equivalent amounts of in vitro-translated and radiolabeled SETDB1 were incubated with either GST (negative control) or GST-Suv39h1. After a GST pull-down, SETDB1 was detected by autoradiography (lanes 1–3). The mirror experiment was performed using in vitro-translated Suv39h1 and GST-SETDB1 (lanes 4–6). Control experiments were conducted using GST fusion proteins and in vitro-translated and radiolabeled luciferase (Luc) as negative controls (lanes 7–10). Input, 10% of the used radiolabeled protein. (B) G9a interacts directly with Suv39h1, GLP, and SETDB1, but not with luciferase. Equivalent amounts of GST, GST-HP1α, (positive control) or GST-G9a were incubated with in vitro-translated and radiolabeled Suv39h1, SETDB1, GLP, or Luc (10% inputs, lanes 1–4). (C) GLP interacts directly with Suv39h1 and SETDB1. Equivalent amounts of GST, GST-Suv39h1, or GST-SETDB1 were incubated with in vitro-translated and radiolabeled G9a, GLP, or luciferase (Luc) (10% inputs, lanes 1, 5, and 9, respectively). (D) Suv39h1 domains involved in interaction with H3K9 HKMTs. (Upper) Suv39h1 domains. (Lower) Equivalent amounts of GST, GST-Suv39h1, or its deletion mutants were incubated with in vitro-translated and radiolabeled SETDB1, G9a, GLP, or Luc (10% inputs, lanes 1–4). GST fusion protein amounts are shown in Figure S14. Molecular Cell 2010 37, 46-56DOI: (10.1016/j.molcel.2009.12.017) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 Functional Cooperation between Suv39h1, G9a, GLP, and SETDB1 in G9a Targets Gene Silencing (A) Histone H3 lysine 9 (H3K9) modifications at the Oct3/4 promoter in untreated and RA-treated P19 cells. Chromatin fractions from proliferating P19 cells untreated (0) or treated for the indicated times with RA (13 or 38 hr) were immunoprecipitated using Ab against acetylated H3K9 (acetylH3K9) or di- or trimethylated H3K9 (H3K9me2 and H3K9me3 respectively) and analyzed by qPCR. 36B4 gene was used as negative control to normalize ChIP results (mean ± SD, n = 3). (B) Suv39h1, G9a, GLP, and SETDB1 recruitment to Oct3/4 promoter. Chromatin from either proliferating P19 cells untreated (0) or treated with RA (13 hr) was immunoprecipitated using Ab against Suv39h1, G9a, GLP, or SETDB1, or with no antibody (Ctr) and analyzed by quantitative PCR (see the Experimental Procedures for ChIP normalization). Fold enrichments varied from one experiment to another, and a representative experiment is presented. (C–E) Concomitant downregulation of Suv39h, G9a, GLP, and SETDB1 led to Oct3/4 reactivation in myoblasts. C2C12 myoblasts were transfected with the indicated siRNAs. Twenty-four hours posttransfection, Oct3/4 mRNA expression was monitored by qRT-PCR (mean ± SD, n = 3) (C). Q-RT-PCR products were analyzed on an agarose gel in duplicates. (D) siRNA knockdown efficiency was checked by WB (E). Four HKMTs, specific siRNAs for each of the studied four HKMTs; Scr, scrambled siRNA. (F and G) Suv39h1, G9a, GLP, and SETDB1 are recruited to Mage-A2 gene. H3K9 modifications at the Mage-A2 promoter in P19 cells (mean ± SD, n = 3) (F). Suv39h1, G9a, GLP, and SETDB1 recruitment to Mage-A2 promoter in P19 cells (mean ± SD, n = 3) (G). Molecular Cell 2010 37, 46-56DOI: (10.1016/j.molcel.2009.12.017) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 6 G9a Target Genes Are Deregulated in Suv39h−/− Cells, and Major Satellite H3K9 Methylation Profile Is Deregulated in G9a−/− Cells (A and B) G9a target gene expression was monitored using quantitative RT-PCR in Suv39h−/− MEFs (A), G9a−/− ESCs (B), and their corresponding wild-type controls. Results (mean values, n = 3) are shown as the ratio between values from KO cells to wild-type controls. Cyclophilin A gene was used as negative control for RNA quantity. (C and D) H3K9 methylation (H3K9me) levels (1, mono; 2, dimethylation; 3, trimethylation) at G9a target genes in Suv39h−/− MEFs (C), G9a−/− ESCs (D). We quantified copy numbers (mean values, n = 3) of the indicated promoters. (E) Suv39h1, G9a, GLP, and SETDB1 recruitment to major satelitte repeats in P19 cells. Chromatin from proliferating P19 cells was immunoprecipitated using Suv39h1, G9a, GLP, or SETDB1 Ab, or with no Ab (Ctr). Enrichment (mean ± SD, n = 3) was calculated as described in the Experimental Procedures. (F and G) Histone H3K9 methylation at major satellite repeats in Suv39h−/− MEFs (E), G9a−/− ESCs (mean values, n = 3) (F). Molecular Cell 2010 37, 46-56DOI: (10.1016/j.molcel.2009.12.017) Copyright © 2010 Elsevier Inc. Terms and Conditions