PRC2.1- and PRC2.2-specific accessory proteins drive recruitment of different forms of canonical PRC1

Abstract

Polycomb repressive complex 2 (PRC2) mediates H3K27me3 deposition, which is thought to recruit canonical PRC1 (cPRC1) via chromodomain-containing CBX proteins to promote stable repression of developmental genes. PRC2 forms two major subcomplexes, PRC2.1 and PRC2.2, but their specific roles remain unclear. Through genetic knockout (KO) and replacement of PRC2 subcomplex-specific subunits in naïve and primed pluripotent cells, we uncover distinct roles for PRC2.1 and PRC2.2 in mediating the recruitment of different forms of cPRC1. PRC2.1 catalyzes the majority of H3K27me3 at Polycomb target genes and is sufficient to promote recruitment of CBX2/4-cPRC1 but not CBX7-cPRC1. Conversely, while PRC2.2 is poor at catalyzing H3K27me3, we find that its accessory protein JARID2 is essential for recruitment of CBX7-cPRC1 and the consequent 3D chromatin interactions at Polycomb target genes. We therefore define distinct contributions of PRC2.1- and PRC2.2-specific accessory proteins to Polycomb-mediated repression and uncover a new mechanism for cPRC1 recruitment.

Keywords: CBX2; CBX4; CBX7; H3K27me3; JARID2; PRC1; PRC2.1; PRC2.2; Polycomb; Polycomb-like protein.

Graphical abstract

Figure 1

Co-recruitment and co-displacement of PRC2.1 and PRC2.2 during ESC to EpiLC differentiation (A) Top: schematic of differentiation model. Bottom: bar plots showing the expression of ESC marker genes Prdm14 and Klf4, and EpiLC marker genes Fgf5 and Dnmt3b by qPCR (n = 2) and RNA-seq (n = 3). Error bars represent SD. (B) Left: heatmap representing fold change in SUZ12 binding at PRC2 target promoters in ESC versus EpiLC cells. Indicated are three categories of PRC2 targets—displaced SUZ12 (log₂FC < −1 and p value < 0.05; n = 78), maintained SUZ12 (n = 2,175), and recruited SUZ12 in EpiLC cells (log₂FC > 1 and p value < 0.05; n = 398). Right: tornado plots showing enrichments of indicated antibodies at displaced, maintained, and recruited promoters in ESCs and EpiLCs. (C) Genome browser tracks showing ChIP-Rx for the indicated antibodies and RNA-seq profiles in ESC and EpiLC cells at Epcam (displaced), Sox8 (maintained), and Tbx3 (recruited). (D) Boxplots presenting mRNA abundance of displaced, maintained, and recruited PRC2 target genes. ^∗∗∗p value < 0.001. See also Figure S1 and Table S1.

Figure 2

PRC2.1 drives H3K27me3 deposition while PRC2.2 drives CBX7-cPRC1 recruitment to Polycomb target genes (A) Schematic of ESC lines used. (B) Tornado and average plots showing ChIP-Rx enrichments for the indicated antibodies at recruited Polycomb target genes (n = 398 sites) in WT and mutant EpiLCs. (C) Genome browser tracks showing ChIP-Rx profiles of the indicated antibodies in WT and mutant EpiLCs at the maintained Six3 and Six2 gene loci. (D) Line plots representing ChIP-Rx enrichment of indicated antibodies in WT and mutant EpiLCs, relative to their respective levels in WT EpiLCs. (E) Western blot of the indicated antibodies on cytoplasm, nucleosol, or chromatin fractions of the indicated cell lines. See also Figure S2.

Figure 3

JARID2 promotes CBX7-cPRC1 while MTF2 promotes CBX4-cPRC1 recruitment to Polycomb target genes in ESCs (A) Schematic of PRC2.1 or PRC2.2 rescue strategy in QKO ESCs. (B) Western blot analyses of the indicated antibodies on total protein extracts from QKO ESC rescue lines, described in (A). (C) Average and tornado plots showing ChIP-Rx enrichments of indicated antibodies at maintained Polycomb targets (n = 2,175) in the relevant cell lines. (D) Genome browser tracks showing ChIP-Rx enrichments of indicated antibodies in the relevant cells at the extended HoxA locus. (E) Average plot showing ChIP-Rx and ChIP-seq enrichments of indicated antibodies at maintained Polycomb target genes (n = 2,175) in the relevant cell lines. (F) Genome browser tracks showing ChIP-Rx and ChIP-seq enrichments of indicated antibodies in the relevant cell lines at the Pitx1 locus. See also Figure S3.

Figure 4

CBX7-cPRC1 requires JARID2 and low levels of H3K27me3 to bind to Polycomb target genes (A) Top: schematic of experimental design. Bottom: western blot analyses for the indicated antibodies on total protein extracts. (B) Average and tornado plots showing ChIP-Rx enrichments of indicated antibodies at maintained Polycomb targets (n = 2,175) in tazemetostat-treated or DMSO control ESCs. (C) Tornado plots showing ChIP-Rx enrichments of indicated antibodies in tazemetostat-treated or DMSO control ESCs at maintained Polycomb targets (n = 2,175), grouped into quintiles based on CBX7 abundance difference between DMSO and tazemetostat-treated ESCs. (D) Genome browser tracks of representative genes from quintile 1 (Nat8I) and quintile 5 (Spata3), showing ChIP-Rx of indicated antibodies in tazemetostat-treated or DMSO control ESCs. (E) Average and tornado plots showing ChIP-seq and ChIP-Rx enrichments of indicated antibodies in WT, Ezh1/2-dKO, and Ezh1 KO/EZH2-Y726D at maintained Polycomb targets (n = 2,175) grouped into quintiles, as described in (C). (F) Genome browser tracks of indicated antibodies in Ezh1/2-dKO and Ezh1 KO/EZH2-Y726D at representative genes from Q1 (Nat8I) and Q5 (Spata3). See also Figure S4.

Figure 5

Contrasting PRC2.1 and PRC2.2 binding profiles consistent with independent recruitment mechanisms (A) Genome browser tracks showing SUZ12 and CBX7 ChIP-Rx binding in the relevant cells at the HoxC locus. The red region represents recruited genes, whereas the gray region represents a group of maintained genes. (B) Genome browser tracks of the indicated antibodies in the indicated cell lines. Bio-CAP tracks generated on wild-type ESCs, taken from GSE43512. (C) Left: schematic of assay design. Right: boxplot representing the distance between the SUZ12 peak center of WT, J2KO, and TKO EpiLCs and the center of the CGIs. ^∗∗∗p value < 0.001. (D) Genome browser tracks of the indicated antibodies in the indicated cell lines at the extended HoxC locus. (E) Genome browser tracks of the indicated antibodies in the indicated cell lines, as well as Bio-CAP, the extended HoxC locus. (F) Boxplot representing the distance between the center of SUZ12 peaks in WT, and in QKO + FLAG-MTF2 or QKO + FLAG-JARID2. See also Figure S5.

Figure 6

DNA and histone modification binding activities of MTF2 and JARID2 facilitate the respective chromatin binding of PRC2.1 and PRC2.2 (A) Schematic of wild-type or mutant MTF2 rescue strategy. (B) Western blot analyses using the indicated antibodies of total protein extracts in the indicated ESC lines. (C) Boxplot of SUZ12 ChIP-Rx read counts in the indicated ESC lines at all PRC2-bound promoters. ^∗∗p value < 0.01. (D) Genome browser tracks showing SUZ12 ChIP-Rx profile on the HoxA locus in the indicated cell lines. Note the y axis values are adjusted to facilitate the visualization of the tracks. (E) Schematic of wild-type or truncated JARID2 rescue strategy. (F) Western blot analyses using the indicated antibodies on total protein extracts in the indicated ESC lines. (G) Genome browser tracks showing SUZ12 ChIP-Rx profile in the indicated cell lines at representative UIM-dependent (top) and UIM-independent (bottom) gene loci. (H) Left: heatmap representing the fold change of SUZ12 binding at PRC2 target promoters in QKO + JARID2-WT or QKO + JARID2-ΔUIM. Right: average plots showing ChIP-Rx normalized read densities for SUZ12 and CBX7 at the UIM-dependent (n = 300) and UIM-independent (n = 300) regions, in QKO and JARID2 rescue ESCs. See also Figure S6.

Figure 7

PRC2.1-deposited H3K27me3 and PRC2.2-JARID2-recruited CBX7-cPRC1 cooperate to mediate Polycomb target repression (A) Genome browser tracks showing 4C-seq analyses of the indicated cell lines using the Tbx3 gene promoter as the viewpoint bait. SUZ12 and H3K27ac ChIP-Rx profiles are shown below. SUZ12-bound sites (S1, S2, and S3) are highlighted in blue, while the H3K27ac-enriched enhancers (E1, E2, and E3) are highlighted in pink. (B) Boxplots representing the 4C-seq densities at S1, S2, and S3 and E1, E2, and E3 in the indicated cell lines. ^∗∗p value < 0.01. (C) Genome browser tracks showing 4C-seq analyses of the indicated cell lines using the Tbx3 gene promoter as the viewpoint bait. ChIP-Rx profiles of the indicated antibodies in the relevant cell lines are also shown. (D) Relative mRNA abundance of Tbx3 in WT ESCs, and WT and Pcgf2/4 KO EpiLCs. Error bars represent SD (n = 3). (E) Bar plots representing the fold change of Tbx3 expression between WT and PcG mutant EpiLCs. ^∗∗∗p value < 0.001. Error bars represent SD (n = 3). (F) Bar plots representing the number of DESeq2-identified differentially expressed recruited Polycomb target genes in the indicated EpiLCs (n = 3). (G) Boxplots showing RNA-seq log₂-fold change of WT compared with the respective KO EpiLCs. The gray dashed line indicates the y axis at 0. ^∗p value < 0.05 and ^∗∗p value < 0.01. Error bars represent SD (n = 3). See also Figure S7.