The Polycomb repressive complexes PRC1 and PRC2 maintain embryonic stem cell (ESC) pluripotency by silencing lineage-specifying developmental regulator genes1. Emerging evidence suggests that Polycomb complexes act through controlling spatial genome organization2,3,4,5,6,7,8,9. We show that PRC1 functions as a master regulator of mouse ESC genome architecture by organizing genes in three-dimensional interaction networks. The strongest spatial network is composed of the four Hox gene clusters and early developmental transcription factor genes, the majority of which contact poised enhancers. Removal of Polycomb repression leads to disruption of promoter-promoter contacts in the Hox gene network. In contrast, promoter-enhancer contacts are maintained in the absence of Polycomb repression, with accompanying widespread acquisition of active chromatin signatures at network enhancers and pronounced transcriptional upregulation of network genes. Thus, PRC1 physically constrains developmental transcription factor genes and their enhancers in a silenced but poised spatial network. We propose that the selective release of genes from this spatial network underlies cell fate specification during early embryonic development.
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We thank members of the Elderkin, Fraser and Luscombe groups for discussions and J. Houseley and P. Rugg-Gunn for commenting on the manuscript. We thank F. Krueger for help with data processing and formatting. We thank R.J. Klose and N. Brockdorff for sequencing. We thank D. Bolland, J. Martins and A. Corcoran for help and advice with the three-dimensional DNA FISH and MetaCyte data analyses. This work was funded by the Wellcome Trust (WT085102MA) (S.E.), the Biotechnology and Biological Science Research Council, the Medical Research Council UK (P.F.) and the European Union Framework Programme 7 Epigenesys Network of Excellence (N.M.L.).
The authors declare no competing financial interests.
Integrated supplementary information
(a) Contact enrichment for promoters occupied by the indicated chromatin proteins, for promoter-promoter contacts >10 Mb apart. (b) Circos plot of significant cis and trans Promoter CHi-C contacts between Hox cluster gene promoters in WT ESCs. Inner circle, chromosomes; outer circle, gene names; edges, significant promoter contacts weighted by read count. (c) Genome-wide virtual 4C promoter-promoter contacts using the Hoxa cluster as a viewpoint, generated from WT ESC Promoter CHi-C data. Significant promoter-promoter contacts >10 Mb apart are marked with arrows. The table indicates the number of raw interactions originating from promoter fragments in each Hox cluster and the number of raw interactions (i) within a Hox cluster, (ii) in cis but outside the cluster or (iii) in trans. (d) Gene Ontology analysis (DAVID) showing biological processes enriched among Hox network genes compared to all genes. (e) Protein domain enrichment analysis (DAVID and PROSITE) among all Hox network genes (left) and Hox cluster–contacting genes (right) compared to all genes. (f) RING1B peak enrichment at promoters. P values were calculated by Wilcoxon rank-sum tests.
(a) Top left, immunoblot for RING1B, NANOG and OCT4 in total cell extracts from WT, RING1A-KO and RING1A/B-dKO ESCs. Top middle, immunoblot for H2AK119ub1 in histone extracts from WT, RING1A-KO and RING1A/B-dKO ESCs and Coomassie Brilliant Blue (CBB). Top right, alkaline phosphatase staining of RING1A-KO and RING1A/B-dKO cells 48 h after treatment with 800 nM tamoxifen. Bottom, immunofluorescence for RING1B (green) and OCT4 (red) in WT, RING1A-KO and RING1A/B-dKO ESCs. (b,c) Hox network Circos contact map in RING1A-KO (b) and RING1A/B-dKO (c) ESCs. Inner circle, chromosomes; outer circle, gene names; red dots, Hox network RING1B-bound promoters; edges, significant promoter contacts (orange, weighted by read count) and non-significant contacts (grey, minimum of three reads). (d–f) CHi-C promoter-promoter contacts within the Hoxb, Hoxc and Hoxd clusters for WT, RING1A-KO and RING1A/B-dKO ESCs, displayed using the WashU Epigenome Browser30. Upper tracks show genomic location, RefSeq genes and WT RING1B ChIP-seq signal17.
(a,b) Double-label 3D DNA FISH in WT, RING1A-KO and RING1A/B-dKO ESCs for (a) the Hoxb cluster (green), Lhx1 (red) and Gm11443 (red) and (b) the Hoxd cluster (green), Lmx1b (red) and Gm13481 (red). Upper panels show the genomic locations of the gene loci analyzed by DNA FISH. Middle panels show representative double-label 3D DNA FISH images. Scale bar, 5 µm. Below are the cumulative frequency distribution plots, the percentage of alleles colocalizing at increasing distance cutoffs and the percentage of interprobe distances below the specified cutoff, set at the distance that included the lowest quintile of measurements for Hox network member alleles in WT or RING1A-KO ESCs. P values were calculated by Mann-Whitney test (comparison of interprobe distances within the same cell) and Kruskall-Wallis/Dunn’s multiple-comparisons test (comparison of interprobe distances between RING1A-KO and RING1A/B-dKO cells).
(a) 3C-PCR validation of Hoxa cis contacts, as depicted in Figure 2, using a second biological replicate. (b,c) 3C-PCR validation of Hoxb (b) and Hoxc (c) cis contacts for regions identified by Promoter CHi-C as making PRC1-dependent contacts with Hoxb (b) (Lhx1 and Cbx8/4) and Hoxc (c) (Wnt7b) and control regions identified as non-contacting regions for Hoxb (b) (Hlf and Gna13) and Hoxc (c) (Cntn1). Left, genomic locations of gene loci analyzed by 3C-PCR, RING1B ChIP-seq signal from WT ESCs17 and virtual 4C (v4C) cis contacts using the Hoxb (b) and Hoxc (c) clusters as viewpoints, generated from Promoter CHi-C data from WT, RING1A-KO and RING1A/B-dKO ESCs. Right, 3C-PCR results for selected regions for two independent biological replicates from WT, RING1A-KO and RING1A/B-dKO cells.
(a,b) 3C-PCR validation of Hoxd (a) and Hist1 (b) cluster cis contacts for regions identified by Promoter CHi-C as making PRC1-dependent contacts with Hoxd (a) (Lmx1b and Pax6) and PRC1-independent contacts with Hist1h2ae (b) (Hist1h3e and Hist1h4i) and control regions identified as non-contacting regions for Hoxd (a) (Gtdc1 and Rag1) and Hist1h2ae (b) (Vmn1r198). Top, genomic locations of the gene loci analyzed by 3C-PCR, RING1B ChIP-seq signal from WT ESCs17 and virtual 4C (v4C) cis contacts, using the Hoxd cluster (a) and Hist1h2ae (b) as viewpoints, generated from Promoter CHi-C data from WT, RING1A-KO and RING1A/B-dKO ESCs. Below, 3C-PCR results for selected regions for two independent biological replicates from WT, RING1A-KO and RING1A/B-dKO cells.
Supplementary Figure 6 Polycomb promoter categories and the contribution of PRC2 to Hox network contacts.
(a) Left, Venn diagram showing the number of promoters in the PRC2-only, PRC1/PRC2 and PRC1-only categories. Bars show the percentage of promoters in each category that also overlap an H3K27me3 peak (dark gray), and the actual number of promoters are reported. Right, H3K27me3 peak fold enrichment at PRC2-only, PRC1/PRC2 and PRC1-only promoters (considering only those promoters in each category that overlap an H3K27me3 peak). (b) RING1B (left), SUZ12 (middle) and EZH2 (right) peak fold enrichment at PRC1-only, PRC2-only or PRC1/PRC2-occupied promoters as appropriate. P values were calculated by Wilcoxon rank-sum test. (c) Left, immunoblot for EED, EZH2, OCT4 and NANOG in total cell extracts from Eed+/+ and Eed−/– ESCs. Middle, immunoblot for H3K27me3 in histone extracts from Eed+/+ and Eed−/– ESCs. Right, alkaline phosphatase staining of Eed+/+ and Eed−/– ESCs. (d–g) 3C-PCR validation of Hoxb (d), Hoxc (e), Hoxd (f) and Hist1 (g) cis contacts for two independent biological replicates from Eed+/+ and Eed−/– ESCs.
Supplementary Figure 7 Effect of RING1A and RING1B double-knockout on the spatial contacts of PRC1-independent genes.
(a) 3D FISH validation of OCT4/SOX2/NANOG promoter-promoter networks. Left, genomic locations of OSN and control gene loci analyzed by 3D DNA FISH, RING1B ChIP-seq signal from WT ESCs17 and virtual 4C (v4C) contacts, generated from Promoter CHi-C data, using Ranbp10 as a viewpoint. Right top, representative double-label 3D DNA FISH images from WT, RING1A-KO and RING1A/B-dKO cells. Scale bar, 5 µm. Right middle, the cumulative frequency distribution plots show the percentage of alleles colocalizing at increasing distance cutoffs. Lower right, percentage of interprobe distances below the specified cutoff, set at the distance that included the lowest quintile of measurements for OCT4/SOX2/NANOG network member alleles in WT or RING1A-KO ESCs. P values were calculated by Mann-Whitney test (comparison of interprobe distances within the same cell) and Kruskall-Wallis/Dunn’s multiple-comparisons test (comparison of interprobe distances between RING1A-KO and RING1A/B-dKO cells). (b) Contact enrichment for non-PRC1/PRC2 promoters that are adjacent to PRC1/PRC2-bound promoters and an equal number of randomly selected non-PRC1/PRC2 promoters.
Supplementary Figure 8 RING1B promoter-enhancer contacts and enhancer status in RING1A-KO and RING1A/B-dKO cells.
(a) Stacked bar chart displaying the average number of significant enhancer contacts per promoter for poised, active and intermediate enhancers (categories as depicted in Fig. 4a). (b) Proportion of promoters contacting one or more intermediate or active enhancers in WT ESCs (gray) and maintaining one or more poised enhancer contacts in RING1A-KO (blue) and RING1A/B-dKO (green) cells. P values were calculated by Fisher’s exact test (WT ESC proportions). (c) Expression status of promoters that contact PRC1/PRC2-bound promoters in WT ESCs (left) and that form new contacts with PRC1/PRC2-bound promoters in RING1A/B-dKO cells (right). (d) Proportion of promoters that gain contacts with active enhancers in RING1A/B-dKO cells. (e) Gene expression changes in RING1A/B-dKO cells relative to RING1A-KO cells for RING1B-bound promoters that gain contacts with active enhancers. P values were calculated by Wilcoxon rank-sum test. (f) Heat map of input-normalized log2 (RPKM) values in RING1A-KO and RING1A/B-dKO cells for H3K4me1, H3K27me3 and H3K27ac at all enhancers defined in WT ESCs. Each line in the heat map represents an individual enhancer, grouped by WT enhancer status and ordered within each group by H3K4me1 values in RING1A-KO cells. The poised enhancer section of the heat map is shown at higher magnification. (g) Changes in H3K4me1, H3K27me3 and H3K27ac occupancy at intermediate and active enhancers that maintain contacts with RING1B-bound promoters in RING1A/B-dKO cells. (h) Changes in H3K27ac at intermediate enhancers that maintain contacts with RING1B-occupied non-Hox network or Hox network promoters in RING1A/B-dKO cells.
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Supplementary Figures 1–8. (PDF 2281 kb)
Promoter fragment baits in different categories. (XLSX 1374 kb)
Next-generation sequencing statistics for promoter CHi-C, nuclear RNA-seq and ChIP-seq. (XLSX 17 kb)
Publically available data sets used. (XLSX 10 kb)
Enhancer fragment baits in different enhancer classes. (XLSX 6818 kb)
BACs and primer sequences used for 3C-PCR and 3D DNA FISH. (XLSX 11 kb)
Interprobe distances for 3D DNA FISH. (XLSX 168 kb)
Full-length gels for 3C analysis in the main figures. (PDF 46791 kb)
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Schoenfelder, S., Sugar, R., Dimond, A. et al. Polycomb repressive complex PRC1 spatially constrains the mouse embryonic stem cell genome. Nat Genet 47, 1179–1186 (2015). https://doi.org/10.1038/ng.3393
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