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. 2020 Mar 4;6(10):eaay4768.
doi: 10.1126/sciadv.aay4768. eCollection 2020 Mar.

Polycomb Regulation Is Coupled to Cell Cycle Transition in Pluripotent Stem Cells

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Free PMC article

Polycomb Regulation Is Coupled to Cell Cycle Transition in Pluripotent Stem Cells

Helena G Asenjo et al. Sci Adv. .
Free PMC article

Abstract

When self-renewing pluripotent cells receive a differentiation signal, ongoing cell duplication needs to be coordinated with entry into a differentiation program. Accordingly, transcriptional activation of lineage specifier genes and cell differentiation is confined to the G1 phase of the cell cycle by unknown mechanisms. We found that Polycomb repressive complex 2 (PRC2) subunits are differentially recruited to lineage specifier gene promoters across cell cycle in mouse embryonic stem cells (mESCs). Jarid2 and the catalytic subunit Ezh2 are markedly accumulated at target promoters during S and G2 phases, while the transcriptionally activating subunits EPOP and EloB are enriched during G1 phase. Fluctuations in the recruitment of PRC2 subunits promote changes in RNA synthesis and RNA polymerase II binding that are compromised in Jarid2 -/- mESCs. Overall, we show that differential recruitment of PRC2 subunits across cell cycle enables the establishment of a chromatin state that facilitates the induction of cell differentiation in G1 phase.

Figures

Fig. 1
Fig. 1. Recruitment of the PRC2 core subunit Ezh2 to bivalent genes increases during S and G2-M phases of the cell cycle.
(A) Flow cytometry dot plot analysis of FUCCI-mESCs indicating sorting gates used to obtain cell populations enriched in G1 (Gate 1), S (Gate 2), and G2-M (Gate 3) cell cycle phases (left). Sorted cells were stained with propidium iodide and analyzed by flow cytometry (right). (B) Venn diagrams of bivalent genes previously published in (–38). (C) Heatmaps of normalized Ezh2 ChIP-seq reads around the TSS of HC bivalent promoters at different cell cycle phases. Heatmap of hypermethylated promoters is shown as a negative control. (D) Average binding profile of Ezh2 around the TSS of HC bivalent promoters in G1 (red), S (green), and G2-M (blue). (E) Quantification of Ezh2-binding signal at the promoter regions (−0.5 to +1.5 kb relative to TSS) of HC bivalent genes in indicated cell cycle phases. (F) Average binding profile of Ezh2 around the TSS of HC bivalent (black) and hypermethylated (gray) promoters in G1 phase. (G) Hierarchical clustering analysis of binding of Ezh2 to the promoter region (−0.5 to +1.5 kb relative to TSS) of HC bivalent genes at indicated phases of the cell cycle. Binding relative to the average is presented. (H) Genome browser view of Ezh2 ChIP-seq data across cell cycle at the Hoxd gene cluster. Suz12 binding was analyzed using published data (19). (I and J) Histogram showing enrichment of Ezh2 (I) or H3K27me3 (J) at PRC2 target promoter regions (Dach1, Sox7, Ascl1, Pax3, Msx1, and Nkx2-2) in G1 (black), S (gray), and G2-M (red) assayed by ChIP-qPCR. Active (Oct4, Nanog, and Hprt1) and hypermethylated (Myf5 and λ-5) gene promoters were used as negative controls. Means ± SEM of three (I) or four (J) experiments is shown. (E, I, and J) Asterisk (*) marks statistically significant differences.
Fig. 2
Fig. 2. Binding of Jarid2 to target promoters increases during S and G2-M phases of the cell cycle.
(A) Heatmaps of normalized ChIP-seq reads showing the binding of Jarid2 around the TSS of HC bivalent promoters at different cell cycle phases. Heatmap of hypermethylated promoters is shown as a negative control. (B) Average binding profile of Jarid2 around the TSS of HC bivalent promoters in G1 (red), S (green), and G2-M (blue). (C) Hierarchical clustering analysis of binding of Jarid2 to the promoter region (−0.5 to +1.5 kb relative to TSS) of HC bivalent genes at indicated phases of the cell cycle. Binding relative to the average is presented. (D) Heatmaps comparing the binding of Jarid2 and Ezh2 around the TSS of HC bivalent promoters in G2-M. (E) Linear regression analysis showing the correlation between the binding signals of Jarid2 and Ezh2 at HC bivalent promoters (−0.5 to +1.5 kb relative to TSS) at indicated cell cycle phases. (F) Genome browser view of Ezh2 and Jarid2 binding across cell cycle at the Adra2c bivalent gene. Suz12 binding was analyzed using published data (19).
Fig. 3
Fig. 3. Binding of EPOP and EloB to bivalent promoters is enhanced in G1 phase.
(A) Heatmaps showing the binding of EPOP around the TSS of HC bivalent promoters in G1 and G2-M. Heatmap of hypermethylated promoters is shown as a negative control. (B) Average binding profile of EPOP around the TSS of HC bivalent promoters in G1 (red) and G2-M (blue). (C) Boxplot of EPOP-binding signal at the promoter regions (−0.5 to +1.5 kb relative to TSS) of HC bivalent genes in indicated cell cycle phases. (D) Histogram showing enrichment of EPOP to PRC2 target promoter regions (Dach1, Sox7, Ascl1, Pax3, Msx1, and Nkx2-2) in G1 (black), S (gray), and G2-M (red) assayed by ChIP-qPCR. Active (Oct4, Nanog, and Hprt1) and hypermethylated (Myf5 and λ-5) gene promoters were used as negative controls. Means ± SEM of three experiments is shown. (E) Heatmaps showing the binding of EloB around the TSS of HC bivalent promoters in G1, S, and G2-M. Heatmap of hypermethylated promoters is shown as a negative control. (F) Average binding profile of EloB around the TSS of HC bivalent promoters in G1 (red), S (green), and G2-M (blue). (G) Quantification of EloB-binding signal at the promoter regions (−0.5 to +1.5 kb relative to TSS) of HC bivalent genes in indicated cell cycle phases. (H) Whole-cell lysate Western blots comparing Ezh2, Jarid2 and EPOP protein levels in G1, S, and G2-M. Lamin B was used as a loading control. (C, D, and G) Asterisk (*) marks statistically significant differences.
Fig. 4
Fig. 4. RNA synthesis is down-regulated and Ser5-RNAPII is accumulated at PRC2 target promoters during S and G2-M phase.
(A) Average RNA production from HC bivalent (left) and active (right) promoters in G1 (red), S (green), and G2-M (blue). (B) Boxplot comparing 4sU-seq reads mapped to the proximal promoter region (TSS to +3Kb) of HC bivalent genes in indicated cell cycle phases. (C) Genome browser view of RNA synthesis at indicated cell cycle phases at the bivalent gene Nes. Ezh2 binding was analyzed using published data (46). (D) Heatmaps showing the binding of Ser5-RNAPII around the TSS of HC bivalent promoters in G1, S, and G2-M. Heatmap of hypermethylated promoters is shown as a negative control. (E) Average binding profile of Ser5-RNAPII around the TSS of HC bivalent gene promoters in G1 (red), S (green), and G2-M (blue). (F) Quantification of Ser5-RNAPII–binding signal at the promoter regions (−0.5 to +1.5 kb relative to TSS) of HC bivalent genes in indicated cell cycle phases. (G) Hierarchical clustering analysis of binding of Ser5-RNAPII to the promoter region (−0.5 to +1.5 kb relative to TSS) of HC bivalent genes at indicated phases of the cell cycle. Binding relative to the average is presented. (H) Genome browser view of the binding of Ser5-RNAPII across cell cycle at the Hoxd gene cluster. Ezh2 binding was analyzed using published data (46). (I) Analysis by ChIP-qPCR of Ser5-RNAPII binding at PRC2 target promoter regions (Dach1, Sox7, Ascl1, Pax3, Msx1, and Nkx2-2) in G1 (black), S (gray), and G2-M (red) assayed by ChIP-qPCR. Active (Oct4 and Nanog) and hypermethylated (Myf5 and λ-5) gene promoters were used as controls. Means ± SEM of three experiments is shown. (J) Venn diagram showing the overlap between HC bivalent genes overtly repressed in G2-M (FC > 1.5) and genes displaying accumulation of Ser5-RNAPII at their promoter region in G2-M (cluster I in Fig. 3G). (K) Linear regression analysis of the binding signals of Ezh2 and Ser5-RNAPII at HC bivalent promoters (−0.5 to +1.5 kb relative to TSS) at indicated cell cycle phases. (L) Histogram displaying the percentage of genes showing changes (G1 > G2-M or G1 < G2-M) of binding for Ezh2 (FC > 2), Jarid2 (FC > 1.3), Ser5-RNAPII (FC > 2), EPOP (FC > 1.5), EloB (FC > 1.5), and RNA production (FC > 1.5) during cell cycle transition. Genes that showed no difference between analyzed phases were excluded to calculate the percentage. (B, F, and I) Asterisk (*) marks statistically significant differences. FC, fold change.
Fig. 5
Fig. 5. Cell cycle–dependent regulation of PRC2 is more accused at the promoter of developmental transcription factors.
(A) Venn diagram showing overlap between HC bivalent genes that are targets of Ser5-RNAPII, Ezh2, Jarid2, EPOP, and EloB in asynchronous populations using published (23, 36) and our (Jarid2) datasets. (B) Gene Ontology (GO) analysis of common target and remaining genes. Bars represent the number of genes that fall into indicated GO categories. P value is shown next to each category. (C) Average binding profile of Ezh2, Jarid2, EPOP, Ser5-RNAPII, and RNA synthesis around the TSS of HC bivalent genes in G1 (red), S (green), and G2-M (blue) comparing common target and remaining HC bivalent genes (as defined in Fig. 5A).
Fig. 6
Fig. 6. Jarid2 −/− mESCs display altered cell cycle–dependent regulation of bivalent genes.
(A) Analysis by ChIP-qPCR comparing the enrichment of H3K27me3 at PRC2 target promoter regions (Dach1, Sox7, Ascl1, Pax3, Msx1, and Nkx2-2) in Jarid2 fl/fl (black bars) and Jarid2 −/− (gray bars) in G2-M phase. Active (Oct4 and Nanog) gene promoters were used as negative controls. Means ± SEM of four experiments is shown. (B) Average RNA synthesis (top) and binding of Ser5-RNAPII (bottom) around the TSS of common target bivalent genes in G1 (left) and G2-M (right) in Jarid2 fl/fl (black lines) and Jarid2 −/− (gray lines) mESCs. (C) Quantification of RNA synthesis (top) and Ser5-RNAPII binding (bottom) in Jarid2 fl/fl and Jarid2 −/− cells in G1 and G2-M at common target genes. (D) Analysis by ChIP-qPCR comparing the binding of Ser5-RNAPII to PRC2 target promoter regions (Dach1, Sox7, Ascl1, Pax3, Msx1, and Nkx2-2) in Jarid2 fl/fl (black bars) and Jarid2 −/− (gray bars) in G2-M phase. Active (Oct4, Nanog, and Hprt) and hypermethylated (Myf5 and λ-5) gene promoters were used as controls. Means ± SEM of three experiments are shown. (E) Schematic diagram of observations described here. (A, C, and D) Asterisk (*) marks statistically significant differences.

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