Genomewide analysis of PRC1 and PRC2 occupancy identifies two classes of bivalent domains

Abstract

In embryonic stem (ES) cells, bivalent chromatin domains with overlapping repressive (H3 lysine 27 tri-methylation) and activating (H3 lysine 4 tri-methylation) histone modifications mark the promoters of more than 2,000 genes. To gain insight into the structure and function of bivalent domains, we mapped key histone modifications and subunits of Polycomb-repressive complexes 1 and 2 (PRC1 and PRC2) genomewide in human and mouse ES cells by chromatin immunoprecipitation, followed by ultra high-throughput sequencing. We find that bivalent domains can be segregated into two classes -- the first occupied by both PRC2 and PRC1 (PRC1-positive) and the second specifically bound by PRC2 (PRC2-only). PRC1-positive bivalent domains appear functionally distinct as they more efficiently retain lysine 27 tri-methylation upon differentiation, show stringent conservation of chromatin state, and associate with an overwhelming number of developmental regulator gene promoters. We also used computational genomics to search for sequence determinants of Polycomb binding. This analysis revealed that the genomewide locations of PRC2 and PRC1 can be largely predicted from the locations, sizes, and underlying motif contents of CpG islands. We propose that large CpG islands depleted of activating motifs confer epigenetic memory by recruiting the full repertoire of Polycomb complexes in pluripotent cells.

Figure 1. Conservation of chromatin state in mouse and human ES cells.

(A) ChIP-Seq signals for H3K4me3 (green), H3K27me3 (red) and H3K36me3 (blue) are plotted across 120 kb of orthologous sequence in mouse and human ES cells. (B) The proportion of promoters that have a given chromatin state in human ES cells is indicated contingent on their state in mouse ES cells. (C) ChIP-Seq signals are shown for developmental regulator loci with divergent chromatin state in mouse and human ES cells. The divergent states correspond to known differences between the two pluripotency models (see text).

Figure 2. PcG complex occupancy at bivalent domains.

(A) ChIP-Seq signals are shown for H3K4me3, H3K27me3 and PRC2 subunits, Suz12 and Ezh2, at a representative panel of bivalent gene promoters. (B) ChIP-Seq signal for the PRC1 subunit Ring1B at these loci. (C) Venn diagram illustrating overlap between promoters marked by H3K27me3, PRC2 and Ring1B. (D) ChIP-qPCR data for Ring1B at bivalent promoters classified by ChIP-Seq as ‘Ring1B-positive’ or ‘Ring1B-negative’. Error bars show standard deviation.

Figure 3. PRC1-positive bivalent domains are functionally distinct.

(A) Box plot shows 25^th, 50^th and 75^th percentile Ring1B ChIP-Seq signals for Ring1B-positive bivalent promoters, Ring1B-negative bivalent promoters, and for H3K4me3 only promoters. (B) Plot illustrates fraction of genes up-regulated (red) or down-regulated (blue) in PRC1-deficient ES cells for the indicated gene sets (see text for details on Ring1A/B dKO ES cell model). De-repression is evident for a significantly greater proportion of PRC1-positive bivalent promoters (p-value by Fisher's exact test). (C) The proportion of bivalent mouse promoters for which the human ortholog also carries H3K27me3 is indicated, contingent on Ring1B status in mouse ES cells. (D) The proportion of bivalent promoters for which H3K27me3 is retained in ES cell-derived neural progenitors (‘NPCs’), contingent on Ring1B status in mouse ES cells. (E) Gene Ontology categories over-represented in PRC1-positive or PRC1-negative bivalent gene sets.

Figure 4. CG-density and DNA motif occurrences predict genomewide PcG complex localization.

(A) Proportion of CpG islands with a given chromatin state in mouse ES cells. More than 97% of Ezh2 sites in mouse ES cells correspond to CpG islands or other highly CG-rich sequences. A systematic screen reveals sets of DNA motifs over-represented in (B) Ezh2-positive CpG islands or (C) Ezh2-negative CpG islands (enrichment in parentheses). (D) Expression levels of implicated TFs in mouse ES cells. Motifs enriched in Ezh2-positive CpG islands correspond to repressors or to TFs that are not expressed. Motifs enriched in Ezh2-negative CpG islands correspond to highly expressed activators. (E) Ezh2 ChIP-Seq signals for CpG islands predicted as PRC2-positive or PRC2-negative based on motif occurrences. (F) H3K27me3 ChIP-Seq signals for human ES cells for CpG islands predicted to be PRC2-positive or PRC2-negative based on occurrences of the motifs originally identified in mouse.