Volume 32, Issue 4, Pages 503-518 (November 2008) Ezh1 and Ezh2 Maintain Repressive Chromatin through Different Mechanisms  Raphael Margueron, Guohong Li, Kavitha Sarma, Alexandre Blais, Jiri Zavadil, Christopher L. Woodcock, Brian D. Dynlacht, Danny Reinberg  Molecular Cell  Volume 32, Issue 4, Pages 503-518 (November 2008) DOI: 10.1016/j.molcel.2008.11.004 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 Ezh1 Is a Nuclear Protein Expressed in Adult Mouse Tissue and Partners with Suz12, Eed, and RbAP46/48 (A) Western blot of Ezh1, Ezh2, and HDAC1 in various cell lines as indicated (top). Nuclear extract (10 μg) was loaded for Ezh2 and HDAC1 and 40 μg for Ezh1. Equal amounts of F9 cell fractions (cytoplasmic, nuclear fraction, chromatin soluble, and insoluble) were loaded. α-tubulin and histone H3 served as cytoplasmic and nuclear markers, respectively (bottom). (B) Six-month-old adult mouse tissue sections stained with H&E or IHC with the antibodies indicated (left). Tissue extracts were prepared concomitantly, and proteins were detected by western blot using the same amount of extract in all cases (right). (C) Immunoprecipitation from F9 high-salt nuclear extract using antibody against Ezh1, Ezh2, Suz12, and Eed and probed with the same set of antibodies and anti-YY1. IgG served as control. (D) High-salt nuclear extract from F9 cells was loaded onto a DE52 column; proteins were eluted with BC350 and then run on a Superose 6 sizing column. Every other fraction was loaded. Molecular weights are indicated on top. Complexes containing Ezh proteins are indicated by red arrows. Molecular Cell 2008 32, 503-518DOI: (10.1016/j.molcel.2008.11.004) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 Reconstitution and Characterization of PRC2-Ezh1 (A) Immunoprecipitated PRC2-Ezh1 was loaded onto a Superose 6 sizing column and the eluted fractions analyzed by SDS-PAGE followed by western blot and silver staining. (B) Histone methyltransferase (HMT) assay using purified PRC2-Ezh2 or PRC2-Ezh1 with various substrates as indicated on top. ntNuc (native nucleosomes), rOct (recombinant octamers), and rNuc (recombinant nucleosomes). (C) HMT assay with PRC2-Ezh1 and PRC2-Ezh2 using peptides either unmethylated, mono-, di-, or trimethylated on lysine 27. (D) Quantitative HMT assay. Equal amounts of PRC2-Ezh1 and PRC2-Ezh2 were loaded, as evidenced by Suz12 levels in the western blot. (E) RNAi-mediated knockdown of Ezh1 and of Ezh2 in NIH 3T3 cells. Nuclear extracts were prepared 72 hr after transfection and analyzed by western blot. Molecular Cell 2008 32, 503-518DOI: (10.1016/j.molcel.2008.11.004) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 PRC2-Ezh1 and PRC2-Ezh2 Share Target Genes (A) Chromatin immunoprecipitation (ChIP) was performed in different cell lines expressing Gal4-Ezh1, Gal4-Ezh2, or Gal4-SET under a doxycycline-regulated promoter in 293 T-Rex cells. Ezh1, Ezh2, and Suz12 recruitment at a stably integrated luciferase reporter construct and at endogenous MYT1were analyzed 24 hr after doxycycline addition. (B) ChIP-chip experiments were performed in F9 cells using a mouse promoter array covering −5.5 kb to 2.5 kb of the entire mouse genome. (Left, top) Comparison of Ezh1 and Ezh2 targets. (Left, bottom) Comparative gene ontology for Ezh1 and Ezh2 target genes. (Right) Binding patterns of Ezh1 and Ezh2 at three different promoters. Foxf1a and Cyp26a1 are detected as Ezh1 and Ezh2 target genes. Adcy5 is considered an Ezh2-specific target gene. (C) ChIP experiments performed in F9 cells. Chromatin was first immunoprecipitated with IgG, or with Ezh1 or Ezh2 antibodies and then with the antibodies indicated on top. (D) F9 cells were treated with retinoic acid or vehicle for 16 hr, and ChIP was performed at known RAR and Ezh2 target genes (Cyp26a1 and HoxA1), Ezh2 target gene (Foxf1a), or control (CcnD1). Molecular Cell 2008 32, 503-518DOI: (10.1016/j.molcel.2008.11.004) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 Ezh1/2 Regulation in Aging Mouse Kidney (A) Tissue sections from mouse females harvested from birth to 9 months and stained with H&E or by IHC using the antibodies indicated. (B) Tissue extracts were performed concomitantly with the IHC. The same amount of extracts was loaded in all cases. (C) ChIP performed from newborn and 9-month-old female tissues. After ChIP, elution and input were amplified by LM-PCR. Equal amounts of DNA were used for qPCR. Log values of the enrichment are the average of two independent experiments. (D) Nuclear extracts from NIH 3T3 cells grown either asynchronously or under conditions of serum starvation (media containing 0.5% bovine serum for the indicated period of time) were loaded for western blot analysis using 10 μg for Ezh2 detection and 40 μg for Ezh1 detection. (E) Equal amounts of whole-cell/tissue extracts from mouse renal adenocarcinomal RAG cells or kidney from 6-month-old females were loaded side by side. Molecular Cell 2008 32, 503-518DOI: (10.1016/j.molcel.2008.11.004) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 PRC2-Ezh1 Represses Transcription Independently of Histone Methylation (A) (Top) Luciferase activity in cell lines stably transfected with a luciferase reporter and expressing the indicated Gal4 fusion proteins in a doxycycline dependent manner. (Bottom) Expression control for the Gal4 fusion proteins. Luciferase values are the mean ± SD (standard deviation) of three independent biological replicates. (B) ChIPs were performed with or without Gal4-Ezh1 expression. Histone methylation enrichment and Bmi-1 recruitment at the transgene were analyzed by qPCR. qPCR values are the mean ± SD of three independent biological replicates. (C) In vitro transcription assay performed on naked DNA or chromatinized templates using Gal4-VP16 as activator. Similar amounts of PRC2-Ezh1 and PRC2-Ezh2 were titrated in the assay. A representative result of three independent experiments is shown. (D) “Bypass” in vitro transcription assay was performed as indicated in the scheme at the top. PRC2-Ezh1/2 was added as indicated (1, 2). A representative result of three independent experiments is shown. Molecular Cell 2008 32, 503-518DOI: (10.1016/j.molcel.2008.11.004) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 PRC2-Ezh1 Compacts Chromatin and Binds to Tailless Chromatin, but Not to DNA Chromatin reconstituted with (A) hyperacetylated native octamers or (B) bacterially expressed and purified recombinant octamers was incubated with PRC2-Ezh1 or PRC2-Ezh2 in the presence or absence of SAM as indicated. The complexes were loaded on a 10%–30% sucrose gradient, and peak fractions (highlighted in red) were analyzed by EM. Pictures of representative molecules are shown. Compaction events were close to 100% in the presence of PRC2-Ezh1. (C) As in (B), but using recombinant tailless chromatin or naked DNA with PRC2-Ezh1. Molecular Cell 2008 32, 503-518DOI: (10.1016/j.molcel.2008.11.004) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 7 All Components of PRC2-Ezh1 Are Required for Full Compaction with One PRC2-Ezh1 Complex Bringing Together Three to Four Nucleosomes (A) Individual PRC2-Ezh1 components or partially reconstituted PRC2-Ezh1 complexes were purified and analyzed for their ability to shift recombinant chromatin on sucrose gradients (left). Peak fractions (highlighted in red) were analyzed by EM (right). (B) Native hyperacetylated chromatin was incubated with a limiting amount of PRC2-Ezh1 (roughly 35% of the arrays displayed compaction) and analyzed by EM (dark-field method). A control and three representative compacted arrays are shown. (C) (Top) Native hyperacetylated chromatin was reconstituted with a limiting amount of histones (an average of eight nucleosomes per array) and analyzed by EM (dark-field method). A control and three representative compacted arrays are shown. (Bottom) Quantification of the number of free nucleosomes per array. (D and E) Chromatin accessibility of an integrated Gal4-luciferase reporter (D) and of an endogenous PRC2 target gene (E). ([D], top) Representation of the transgene, with arrows indicating the primers used. ([E], top) ChIP as described in Figure 3A, and the MYT1 gene was analyzed by PCR. Isolated nuclei were treated with increasing amounts of DNase I, and protection was quantified by qPCR. Results are given as percentages of control after normalization to an undigested region of the control SYN1 gene and are the mean ± SD (standard deviation) of three independent biological replicates. Molecular Cell 2008 32, 503-518DOI: (10.1016/j.molcel.2008.11.004) Copyright © 2008 Elsevier Inc. Terms and Conditions