Research ArticleGENETICS

Parallel PRC2/cPRC1 and vPRC1 pathways silence lineage-specific genes and maintain self-renewal in mouse embryonic stem cells

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Science Advances  01 Apr 2020:
Vol. 6, no. 14, eaax5692
DOI: 10.1126/sciadv.aax5692
  • Fig. 1 cPRC1 and vPRC1 partially overlap with PRC2 at silent CGI TSSs in mESCs.

    (A) Schematic of core PRC1 and PRC2 complexes and their catalytic activities. Targeting of Cbx7-containing cPRC1 relies on PRC2-mediated H3K27me3. Rybp-containing vPRC1 targeting is H3K27me3 independent. (B) Meta plots and heat maps of Eed, Suz12, H3K27me3 (K27me3), Cbx7, Rnf2, Rybp, and H2AK119ub1 (K119ub) centered around the TSS of CGI promoters (±5 or ±10 kb) show relative enrichment in wild-type mESCs. TSSs are classified by k-means clustering into “shared” (965 TSSs, violet) and “variant-specific” (7333 TSSs, green) and “noBioCap” (19,712 TSSs, not shown). TSSs were grouped by enrichment of cPRC1 (Rnf2, Cbx7), vPRC1 (Rnf2, Rybp), and PRC2 (Eed) subunits, and unmethylated CGIs (BioCap). RNA shows heat map of gene expression measured by QuantSeq. Colored scales (bottom) show dynamic range of ChIP-seq and QuantSeq signals. (C) Genomic screenshots of PcG proteins at representative “shared” and “variant-specific” Polycomb target genes in wild-type mESCs. (D) Meta plots and heat maps compare Rnf2 and H2AK119ub1 enrichments at “shared” and “variant-specific” genes in wild-type, Eed-null, and Rybp-null mESCs. (E) Density scatterplots compare ChIP-seq signals of Rnf2 (±5 kb around TSS) and H2AK119ub1 (±10 kb around TSS) in wild-type and Eed-null mESCs (left) and wild-type and Rybp-null mESCs (right). r, Pearson’s correlation coefficient; “shared” TSSs, violet; “variant-specific” TSSs, green. RPKM, reads per kilobase of transcript per million mapped reads. (F) Box plots show gene expression changes relative to wild type in Eed-null mESCs and Rybp-null mESCs at “shared” (violet) and “variant-specific” (green) genes.

  • Fig. 2 Combined depletion of Rybp and Eed or Suz12 results in loss of mESC self-renewal.

    (A) Schematic representation of degron mESC lines. In Eed-null mESCs, AID-3×FLAG-GFP was inserted biallelic at the 3′ end of the endogenous Rybp ORF (left). In Rybp-null mESCs, AID-3×FLAG-GFP was inserted biallelic at the 3′ end of the endogenous Suz12 ORF (middle). In Ring1-null mESCs, AID-3×FLAG-GFP was inserted biallelic at the 5′ end of the endogenous Rnf2 ORF (right). All degron mESC lines constitutively express osTir1. Western blot of PcG proteins and histone modifications in wild-type, Eed-null mESCs, and two independent clones of dRybp Eed-null mESCs (left). Western blot of PcG proteins and histone modifications in wild-type mESCs, Rybp-null mESCs, and two independent clones of dSuz12 Rybp-null mESCs (middle). Western blot of PcG proteins and histone modifications in wild-type mESCs, Ring1-null mESCs, and two independent clones of dRnf2 Ring1-null mESCs (right). IAA treatment (250 μM) for 24 hours leads to degradation of AID-fusion protein. * indicates unspecific signal. Lamin B1 (Lmn B1) serves as loading control. (B) Proliferation assays of dRybp Eed-null, dSuz12 Rybp-null, and dRnf2 Ring1-null mESCs grown under serum conditions for 72 hours without (untreated) or with 250 μM IAA. Live cells were quantified by flow cytometry every 24 hours. Displayed are the median and SD of four replicate measurements starting at 24 hours IAA (time point, 0 hours). (C) Representative images of alkaline phosphatase (AP) staining of dRybp Eed-null, dSuz12 Rybp-null, and dRnf2 Ring1-null mESC colonies cultured in the absence (untreated) or presence of 250 μM IAA for 5 days. (D) Box plots display SD of density quantifying the degree of mESC colony heterogeneity in response to 5 days of IAA treatment. Statistical significance calculated using unpaired t test (****P < 0.0001).

  • Fig. 3 cPRC1 and vPRC1 maintain H2AK119ub1 at “shared” Polycomb target genes.

    (A) Density scatterplots compare ChIP-seq signals of Rnf2 (top, ±5 kb around TSS) and H2AK119ub1 (H2Aub1) (bottom, ±10 kb around TSS) in untreated and IAA-treated dRybp Eed-null (48 hours, left), dSuz12 Rybp-null (48 hours, middle), and dRnf2 Ring1-null (24 hours, right) mESCs. r, Pearson’s correlation coefficient; “shared” TSS signal, violet; “variant-specific” TSS signal, green. (B) Genomic screenshots of Rnf2 (violet) and H2AK119ub1 (H2Aub1, orange) enrichments at two representative “shared” Polycomb target genes in untreated and IAA-treated dRybp Eed-null, dSuz12 Rybp-null, and dRnf2 Ring1-null mESCs. Superimposed are levels in wild-type mESCs (black line). (C) Meta plots of Rnf2 (top) and H2AK119ub1 (bottom) enrichments at “shared” Polycomb target genes in untreated and IAA-treated dRybp Eed-null, dSuz12 Rybp-null, and dRnf2 Ring1-null mESCs as indicated. (D) Box plots compare ChIP-seq signal of Rnf2 (left, ±5 kb around TSS) and H2AK119ub1 (H2Aub1) (right, ±10 kb around TSS) at “shared” and “variant-specific” Polycomb target genes in wild-type mESCs (white), untreated (green), and IAA-treated (red) dRybp Eed-null, dSuz12 Rybp-null, and dRnf2 Ring1-null mESCs. Significance (P value) was calculated using Wilcoxon rank test (****P < 2.2 × 10−16; ns, not significant).

  • Fig. 4 Combined cPRC1 and vPRC1 disruption triggers activation of lineage-specific genes.

    (A) Volcano plots show gene expression changes of IAA-treated relative to untreated dRybp Eed-null, dSuz12 Rybp-null, and dRnf2 Ring1-null mESCs at 24 hours (top) and 48 hours (bottom). Down (blue), number of repressed genes; up (red), number of up-regulated genes. Differential gene expression (Padj < 0.1) was calculated by taking into account two independent clones per genotype, each sequenced in triplicate. (B) Venn diagrams show overlap of differentially expressed genes in dRybp Eed-null (red circles), dSuz12 Rybp-null (green circles), and dRnf2 Ring1-null (blue circles) mESCs at 24 and 48 hours of IAA treatment. (C) Analysis of Gene Ontology (GO) categories of up-regulated genes in response to IAA treatment of dRybp Eed-null, dSuz12 Rybp-null, and dRnf2 Ring1-null mESCs at 24 and 48 hours. The color indicates the significance, and the size represents the fraction of genes in each category. (D) Box plots of gene expression changes at “shared” (violet) and “variant-specific” (green) Polycomb target genes in dRybp Eed-null, dSuz12 Rybp-null, and dRnf2 Ring1-null mESCs after 24 and 48 hours of IAA addition. (E) Dot charts display dynamic expression changes after 24 and 48 hours of IAA treatment at selected mESC pluripotency markers (gray) or lineage-specific PcG target genes.

  • Fig. 5 Polycomb-repressed genes are marked high PRC2, cPRC1, and vPRC1 occupancy.

    (A) Scatterplots show changes in Rnf2 (top, ±5 kb around TSS) and in H2AK119ub1 (bottom, ±10 kb around TSS) signals relative to gene expression changes in IAA-treated dRybp Eed-null, dSuz12 Rybp-null, and dRnf2 Ring1-null mESCs. “Shared” genes, violet; “variant-specific” genes, green. (B) Box plots show changes in Rnf2 (±5 kb around TSS) and H2AK119ub1 (±10 kb around TSS) signals of up-regulated (up), unchanged, and down-regulated (down) genes after 48 hours of IAA treatment of dRybp Eed-null, dSuz12 Rybp-null, and dRnf2 Ring1-null mESCs. Significance (P value) calculated using Wilcoxon test (****P < 2.2 × 10−16). (C) Box plots display wild-type levels of H3K27me3 (±10 kb around TSS) and Rnf2 (±5 kb around TSS) of genes up-regulated (red) or unchanged (gray) at 6, 24, and 48 hours of IAA treatment of dRybp Eed-null, dSuz12 Rybp-null, and dRnf2 Ring1-null mESCs. Significance (P value) calculated using Wilcoxon test (**P < 0.01; ***P < 0.001; ****P < 1 × 10−4; ns, not significant).

  • Fig. 6 Combined Pcgf1 and Suz12 depletion reduces H2AK119ub1, triggers activation of lineage-specific genes and results in loss of mESC self-renewal.

    (A) Schematic representation of dSuz12 Pcgf1-null mESCs. Pcgf1-null mutation in mESCs expressing osTIR1 and endogenous Suz12 fused to AID-3×FLAG-GFP. Western blot of dSuz12 mESCs and two independent clones of dSuz12 Pcgf1-null mESCs show IAA-dependent fusion protein degradation and confirm Pcgf1 LOF mutation. * indicates unspecific band. Lamin B1 (Lmn B1) serves as loading control. (B) Proliferation assays of two independent clones of dSuz12 Pcgf1-null mESCs grown under serum conditions for 72 hours without (untreated) or with 250 μM IAA. Live cells were quantified by flow cytometry every 24 hours. Displayed is the median and SD of four replicate measurements starting at 24 hours IAA (time point, 0 hours). (C) Representative image of AP staining of dSuz12 Pcgf1-null mESC colonies cultured in the absence (untreated) or presence or 250 μM IAA for 5 days (+IAA). (D) Density scatterplots compare ChIP-seq signals of Rnf2 (left, ±5 kb around TSS) and H2AK119ub1 (H2Aub1) (right, ±10 kb around TSS) in dSuz12 Pcgf1-null mESCs without and with IAA treatment for 48 hours. r, Pearson’s correlation coefficient; “shared” TSS signal, violet; “variant-specific” TSS signal, green. (E) Meta plots of Rnf2 (left) and H2AK119ub1 (right) enrichments at “shared” Polycomb target genes in untreated and IAA-treated dSuz12 Pcgf1-null mESCs for 48 hours. (F) Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis compares expression of panel of “shared” Polycomb target genes in two independent clones of dSuz12 Pcgf1-null mESCs without and with IAA treatment for 48 hours. Gene expression is normalized to GAPDH and is shown relative to the average expression in untreated cells for each individual cell line. Error bars show SD (n = 2). (G) Cartoon illustrates model of parallel, independent mechanisms of PRC2/cPRC1 and vPRC1 targeting to ensure robust repression of lineage-specific genes and safeguard against exit from mESC pluripotency and spontaneous differentiation. (H) Cartoon displays shared PcG target genes embedded in small (left) or large (right) Polycomb repressive chromatin domains (red). PRC1 is critical for repression of PcG target genes located within large Polycomb domains marked by relatively high levels of PRC2/cPRC1 and vPRC1, as well as repressive histone modifications. Silencing of TSSs with smaller Polycomb domains is PRC1 independent and may be buffered by additional repressive chromatin modifications or the lack of activating transcription factors (TFs).

Supplementary Materials

  • Supplementary Materials

    Parallel PRC2/cPRC1 and vPRC1 pathways silence lineage-specific genes and maintain self-renewal in mouse embryonic stem cells

    J. A. Zepeda-Martinez, C. Pribitzer, J. Wang, D. Bsteh, S. Golumbeanu, Q. Zhao, T. R. Burkard, B. Reichholf, S. K. Rhie, J. Jude, H. F. Moussa, J. Zuber, O. Bell

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    • Figs. S1 to S9
    • Tables S1 to S3
    • Uncropped Western blots of proteins from Fig. 2A and fig. S3B.
    • Uncropped Western blots of proteins from figs. S5B, S6A, and S9A.

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