Fig 2: PRC2 sub-complexes differentially contribute to the BAP1-dependent intergenic H3K27me3 deposition(A) Cartoon showing divergent compositions of the PRC2.1 and PRC2.2 complexes and their differing affinities for chromatin features.(B) Western blot analysis with the indicated antibodies on total protein extracts from the Bap1 KO, Aebp2+Jarid2 (A+J) KO, Aebp2+Jarid2+Bap1 (A+J+B) KO, and matching WT ESCs.(C) Western blot analysis with the indicated antibodies on total protein extracts from the indicated ESC lines.(D) Boxplots representing H2AK119ub1 ChIP-seq RPKM levels in the indicated cell lines at intergenic sites (n = 38,068).(E) Boxplots representing H3K27me3 ChIP-seq RPKM levels in the indicated cell lines at intergenic sites (n = 38,068).(F) Boxplots showing the log2 fold change RPKM ratio for H2AK119ub1 or H3K27me3 in the indicated cell lines (A = Aebp2, J = Jarid2, P = Pcl1-3) at intergenic regions (n = 38,068).(G) Genome-wide comparison of ChIP-seq signal using 5 kb windows. Log2 fold change H2AK119ub1 ChIP-seq for the Aebp2/Jarid2/Bap1 KO versus Aebp2/Jarid2 KO and the matching Bap1 KO versus WT comparison (x axis) plotted against log2 fold change of H3K27me3 ChIP-seq (y axis). Each dot represents one 5 kb window.(H) Genome-wide comparison of ChIP-seq signal using 5 kb windows. Log2 fold change H2AK119ub1 ChIP-seq for the Pcl1-3/Bap1 KO versus Pcl1-3 KO and the matching Bap1 KO versus WT comparison (x axis) plotted against log2 fold change of H3K27me3 ChIP-seq (y axis). Each dot represents one 5 kb window.See also Figure S4.

Fig 3: JAZF1-SUZ12 activates polycomb target genes and dysregulates decidualization in primary hEnSC(A) Principal component analysis of RNA-seq data from undifferentiated (day 0) and decidualized (day 8) hEnSCs from three donors expressing GFP, SUZ12, SUZ12?93, JAZF1-SUZ12, or JAZF1.(B) Genes (n = 909) that exhibit significant changes in expression in hEnSCs containing JAZF1-SUZ12 at day 0 or 8. The colors indicate log2 fold change in expression of these genes in hEnSCs from three donors containing GFP, SUZ12, SUZ12?93, JAZF1-SUZ12, or JAZF1 relative to the average expression in hEnSCs containing GFP at day 0, according to the scale on the right.(C) Metagene plot showing average H3K27me3 occupancy in hEnSCs around the transcription start site (TSS) of genes upregulated by JAZF1-SUZ12 (red) versus all genes (gray).(D) Genes (n = 453) significantly upregulated during decidualization of hEnSCs expressing GFP. Left: log2 fold change in expression of these genes in hEnSCs containing GFP or JAZF1-SUZ12 at days 0 and 8 relative to the average expression in hEnSCs containing GFP at day 0. Right: log2 fold change in expression of the same genes in hEnSCs containing JAZF1-SUZ12 at day 8 relative to hEnSCs containing GFP at day 8.(E) Enrichment (-log10 p value) of representative GO terms in sets of genes upregulated during decidualization of cells containing GFP, upregulated by JAZF1-SUZ12 in non-decidualized cells (day 0), downregulated by JAZF1-SUZ12 in decidualized cells (day 8), or a control set of genes that do not change in expression.(F) Top: expression of genes (n = 310) previously identified as upregulated in LG-ESS in hEnSCs containing GFP or JAZF1-SUZ12 relative to the average expression in hEnSCs containing GFP. Bottom: gene set enrichment analysis (GSEA) plotting the position of genes upregulated in LG-ESS in the set of genes ranked by change in expression in cells containing JAZF1-SUZ12 versus GFP. NES, normalized enrichment score.(G) Model. Loss of the SUZ12 N terminus prevents interaction of JAZF1-SUZ12 with EPOP, PALI1, and JARID2, decreasing PRC2 occupancy and reducing H3K27me3 at polycomb target genes. Fusion to JAZF1 induces association with NuA4/TIP60 and increases H4Kac at these sites. These changes are mirrored by ectopic activation of polycomb target genes in hEnSCs, inducing part of the decidualization gene expression program that lacks activation of immune response genes associated with clearance of senescent decidual cells from the endometrium.See also Figure S4 and Table S2.

Fig 4: JARID2 expression is regulated by adipogenesis and JARID2 knockdown affects fat cell phenotype in vitro. A, Expression of JARID2 mRNA in hASC during differentiation (RT-qPCR, n = 3). B, Western blot analysis of JARID2 expression during adipogenesis. Both described isoforms of 140 and 80 kD are indicated. Quantification of the expression of JARID2 isoforms during differentiation (80 kD - C and 140 kD - D, n = 3, paired t-test). C, Representative picture of control and siJARID2-treated cells on day 9 of differentiation. D, Fold change of glycerol concentration in conditional media normalized to cell number (Hoechst) and compared to control on day 9 of differentiation (n = 3, Kruskal-Wallis test). E, Basal and insulin-stimulated glucose uptake on day 13 of differentiation (n = 3, 2-way ANOVA). F. Basal and isoprenaline-stimulated lipolysis measured on day 13 of differentiation (n = 4, 2-way ANOVA), *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig 5: JARID2 interacts with EED and HBL1. (A) Scheme for generation of JARID2-/- H9 hESCs using dual gRNAs and CRISPR/Cas-9. (B) Detection of relative JARID2 mRNA expression in WT and JARID2-/- hESCs using RT-qPCR. (C) Representative images of WT and JARID2-/- hESCs cultured in mTesR1 medium. (D) Detection of percentages of CTNT+ CMs derived from WT and JARID2-/- hESCs using flow cytometry. (E) Quantification of percentage of CTNT+ CMs derived from WT and JARID2-/- hESCs. (F) Two proposed models showing HBL1 interaction with the JARID2 and EED-PRC2 complexes (see text for explanation). (G) Co-IP western blotting to detect the interaction between JARID2 and EED. Left: pull-down of EED protein in WT and HBL1-/- (#22, #150) hiPSCs using a specific JARID2 antibody by Co-IP. Right: pull-down of JARID2 protein in WT and HBL1-/- (#22, #150) hiPSCs using a specific EED antibody by Co-IP. 1% Input is the Co-IP control. (H) ChIP-seq was performed on WT and HBL1-/- hiPSCs using specific anti-JARID2 antibody. (I) Representative genome browser peak tracks of key mesodermal and cardiogenic genes with decreased JARID2 binding (HBL1-/- versus WT hiPSCs). 1% Input is the control. Arrow shows the gene transcription direction. (J) ChIP-qPCR detects JARID2 occupancy on cardiac gene promoters in WT and HBL1-/- hiPSCs. Arrows show the primer sets. Data are mean±s.d. n=3, *P<0.05 [two-tailed unpaired Student's t-test (two groups)]. See also Fig. S5.