Volume 55, Issue 3, Pages 347-360 (August 2014) Gene Silencing Triggers Polycomb Repressive Complex 2 Recruitment to CpG Islands Genome Wide  Eva Madi Riising, Itys Comet, Benjamin Leblanc, Xudong Wu, Jens Vilstrup Johansen, Kristian Helin  Molecular Cell  Volume 55, Issue 3, Pages 347-360 (August 2014) DOI: 10.1016/j.molcel.2014.06.005 Copyright © 2014 Elsevier Inc. Terms and Conditions

Molecular Cell 2014 55, 347-360DOI: (10.1016/j.molcel.2014.06.005) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 PRC2 Is Not Required for Gene Expression Homeostasis in mESCs (A) Categorization of expression changes for either all genes or PRC2 target genes upon KO of different PcG genes in mESCs grown in 2i conditions. (B) Distributions of log2 ratio of gene expression. Same data sets are analyzed here and in (A1)–(A4). (C) Expression fold changes of 12 different PRC2 target genes analyzed by RT-qPCR upon either transient KD or permanent KO of different PcG genes in mESCs grown in serum conditions. Black segments indicate median values. Statistical significances between WT and PcG-depleted cells (Student’s t test) are indicated. See also Figure S1 and Table S1. Molecular Cell 2014 55, 347-360DOI: (10.1016/j.molcel.2014.06.005) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 PRC2 Is Not Required for Gene Expression Changes during Differentiation of mESCs but Is Required for Their Maintenance (A) Gene expression in WT and Suz12−/− mESCs during 3 days of differentiation. Expression levels are indicated as log10 of reads per kilobases per million mapped reads (RPKM). The top graph shows expression distributions for either all genes or changing genes only, together with the colors scale used for the heatmap. Heatmap columns correspond to differentiation time points for each genotype, and a hierarchical clustering of these eight conditions is indicated above. Heatmap rows correspond to genes detected in at least one condition, and their expression change category (i.e., 0 hr versus 72 hr) is annotated on the right. Grey segments on the left and right of the heatmap indicate genes that are Suz12 targets at 0 hr only and 72 hr only, respectively. (B) Categorization of expression changes for either all genes or PRC2 target genes upon Suz12 KO in mESCs grown in serum conditions or after 72 hr of differentiation. (C) Time course analysis of the expression fold change of genes that are de novo PRC2 targets after 72 hr of differentiation. This analysis was performed on genes categorized as downregulated (i.e., 0 hr versus 72 hr) in WT or Suz12−/− cells and Suz12 targets at 72 hr only (top panel), and on a control population of genes (bottom panel). Boxes indicate the median value, 2nd and 3rd quartiles of the percentage distribution. Whiskers indicate 10th and 90th percentiles. (D) Time course analysis by RT-qPCR of the effect of Suz12 KO on the expression of three genes that become PRC2 targets during long-term differentiation in embryoid body (EB) formation assays. Data are represented as mean ±SD. See also Figure S2 and Tables S2 and S3. Molecular Cell 2014 55, 347-360DOI: (10.1016/j.molcel.2014.06.005) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 Inhibition of Transcription Leads to De novo PRC2 Recruitment Genome Wide (A) Western blot analysis of the effect of DRB or Triptolide on RNAPII and PRC2, using antibodies against all forms of RNAPII (N-20) and RNAPII phosphorylated at serine 2 (Ser2-P) or at serine 5 (Ser5-P), Suz12, H3K27me3, and Vinculin as loading control. The phosphorylated form of RNAPII is indicated by an asterisk. (B) Time course analysis of the effect of DRB and Triptolide on transcription by RT-qPCR analysis of unspliced RNA for four genes expressed in mESCs. Data are represented as mean ±SD. (C) Time course analysis of the effect of DRB and Triptolide on PRC2 recruitment by ChIP-qPCR analysis of Suz12 binding on three promoters that are not PcG targets in mESCs. IgG is used as negative control for ChIP. Data are represented as mean ±SD. (D) Example of tracks (Mycn locus) from ChIP-seq analysis of the effect of DRB and Triptolide on Suz12, H3K27me3, and RNAPII binding. Enrichment levels are expressed in read per million mapped reads (RPM). Genome scale, release and annotations are indicated. (E) Number of distinct and overlapping Suz12 peaks before and after inhibition of transcription. See also Figure S3. Molecular Cell 2014 55, 347-360DOI: (10.1016/j.molcel.2014.06.005) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 Drug-Induced Suz12 Binding in mESCs Occurs at the CGIs of Genes that Are PRC2 Targets in Differentiated Tissues (A and B) Percentage of endogenous (orange) or drug-induced Suz12 peaks after either 12 hr DRB (blue) or 9 hr Triptolide (green) treatment that overlap with genes (A) or CGIs (B). (C) Percentage of drug-induced Suz12 target genes in mESCs that are endogenous PRC2 targets in other mouse tissues (see Supplemental Experimental Procedures). (D) Relative contribution of each tissue represented in the mouse PRC2 target database in the identification of drug-induced target genes. (E) Time course analysis of Suz12 binding and H3K27me3 deposition fold changes on nontarget CGIs (top panels) and over control regions (bottom panels) during either DRB or Triptolide treatment. Boxplots are formatted as in Figure 2C. Data are represented as mean ±SD. See also Table S4. Molecular Cell 2014 55, 347-360DOI: (10.1016/j.molcel.2014.06.005) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 5 Transcription Regulates PRC2 Recruitment to Target Genes (A) A 4kb element from the CGI-containing c-Jun promoter was integrated upstream of a luciferase reporter (luc+) in an engineered FRT mESC line (Figures S4A and S4B). Position of insert-specific primers for ChIP-qPCR analysis is indicated in red. (B) Luciferase assay and RT-qPCR analysis of the expression of the c-Jun gene or the luciferase reporter driven by the 4 kb c-Jun element, before (ESC) and after (Diff.) differentiation. Luciferase counts were normalized to total protein amount. (C) ChIP-qPCR analysis of Suz12 binding on both endogenous c-Jun promoter and transgenic 4 kb element before (E) and after (D) 72 hr of differentiation. (D) The 4kb c-Jun element was replaced by a 500 bp subfragment lacking the TSS, at the same genomic insertion site (Figure S4B). (E) Luciferase assay and RT-qPCR analysis as in (B) but with the 500 bp c-Jun element. (F) ChIP-qPCR analysis as in (C) but on integrated 500 bp c-Jun element. (G) The mPGK promoter was inserted upstream of the transgene either lacking (top) or containing (bottom) the 500 bp c-Jun element. Position of mPGK-specific primers for ChIP-qPCR analysis is indicated in red. (H) Luciferase assay with both construct variants presented in (G). Values correspond to the average from seven to eight stable clones for each construct. (I) ChIP-qPCR on the PGK-c-Jun construct. (J) ChIP-qPCR as in (I) but either before or after 7 hr of Triptolide treatment. All FRT cell lines were grown under 2i conditions and induced to differentiate as described in Supplemental Experimental Procedures. See also Figure S4. Molecular Cell 2014 55, 347-360DOI: (10.1016/j.molcel.2014.06.005) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 6 Most CGIs Are either Transcribed or Enriched for PRC2 (A) Setup of transcription inhibition time course analysis by iTC-seq upon DRB or Triptolide treatment of WT mESCs. Sampling time points are indicated on the time line. (B) Example of tracks (Fgf4 locus) from iTC-seq time course analysis. Negative values, due to background subtraction, are plotted in gray. (C) Comparative analysis of Suz12 binding on CGIs and transcription of their overlapping genes. Each parameter was segmented in low and high levels (i.e., 200 RPK for Suz12 binding and 2 RPK for transcription). Inserts show magnifications of the lower values. (D) Distribution of CGIs present in the four categories defined in (C). See also Figure S5. Molecular Cell 2014 55, 347-360DOI: (10.1016/j.molcel.2014.06.005) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 7 PRC2 Is Recruited to Nucleosome Free CGIs (A) CGI category changes upon drug treatments. Heatmap rows correspond to CGIs and columns to cell treatments. (B) Relation between CGIs category changes upon drug treatment and CGI nucleosome content in untreated mESCs. Color code refers to Figure 7A. Boxplots are formatted as in Figure 2C. Statistical significances between tested classes (Student’s t test) are indicated. (C) Average profiles of nucleosome occupancy (in untreated cells) and Suz12 binding on PRC2 target CGIs before and after drug treatments in mESCs. See also Figure S6. Molecular Cell 2014 55, 347-360DOI: (10.1016/j.molcel.2014.06.005) Copyright © 2014 Elsevier Inc. Terms and Conditions