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. 2023 Oct;56(10):e13457.
doi: 10.1111/cpr.13457. Epub 2023 Mar 23.

PR-DUB safeguards Polycomb repression through H2AK119ub1 restriction

Rui Li  1 , Dandan Huang  2 , Yingying Zhao  1 , Ye Yuan  1 , Xiaoyu Sun  1 , Zhongye Dai  1 , Dawei Huo  1 , Xiaozhi Liu  3 , Kristian Helin  4   5 , Mulin Jun Li  6   7 , Xudong Wu  1   8
Affiliations

PR-DUB safeguards Polycomb repression through H2AK119ub1 restriction

Rui Li et al. Cell Prolif. 2023 Oct.

Abstract

Polycomb group (PcG) proteins are critical chromatin regulators for cell fate control. The mono-ubiquitylation on histone H2AK119 (H2AK119ub1) is one of the well-recognized mechanisms for Polycomb repressive complex 1 (PRC1)-mediated transcription repression. Unexpectedly, the specific H2AK119 deubiquitylation complex composed by additional sex comb-like proteins and BAP1 has also been genetically characterized as Polycomb repressive deubiquitnase (PR-DUB) for unclear reasons. However, it remains a mystery whether and how PR-DUB deficiency affects chromatin states and cell fates through impaired PcG silencing. Here through a careful epigenomic analysis, we demonstrate that a bulk of H2AK119ub1 is diffusely distributed away from promoter regions and their enrichment is positively correlated with PRC1 occupancy. Upon deletion of Asxl2 in mouse embryonic stem cells (ESCs), a pervasive gain of H2AK119ub1 is coincident with increased PRC1 sampling at chromatin. Accordingly, PRC1 is significantly lost from a subset of highly occupied promoters, leading to impaired silencing of associated genes before and after lineage differentiation of Asxl2-null ESCs. Therefore, our study highlights the importance of genome-wide H2AK119ub1 restriction by PR-DUB in safeguarding robust PRC1 deposition and its roles in developmental regulation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIGURE 1
FIGURE 1
Non‐promoter pervasive H2AK119ub1 is associated with RING1B sampling. (A) Pie plot showing genomic distribution of H2AK119ub1‐occupied regions in mESCs. (B) Boxplots comparing H2AK119ub1 CUT&Tag signals in mESCs across different genomic regions. (C) Heatmaps showing signals for H2AK119ub1 (CUT&Tag), H3K27me3, RING1B and SUZ12 occupancy (ChIP‐seq) at promoters and non‐promoters in mESCs. All rows are centred on H2AK119ub1 peaks and further divided into promoter and non‐promoter clusters. (D) Up: Metaplots showing different H2AK119ub1 levels at non‐promoter regions in mESCs (C1–C4 clusters indicate CUT&Tag signal from low to high). Down: Metaplots showing RING1B ChIP‐seq signal across different levels of H2AK119ub1 clusters. (E) Boxplots comparing RING1B ChIP‐seq signal across C1–C4 clusters (**p value <0.01; ***p value <0.001). (F) Bar plots illustrating the binding frequency of RING1B across different levels of H2AK119ub1 clusters (the binding frequency indicates the proportion of H2AK119ub1 peaks overlapped with RING1B peaks in specific clusters).
FIGURE 2
FIGURE 2
ASXL2 loss results in pervasive gain of H2AK119ub1 at non‐promoter regions. (A) Genome typing of Asxl2 deletion (two clones) by CRISPR/Cas9 techniques. (B,C) WB assays to compare the levels of designated proteins or histone modifiations in WT and Asxl2 KO mESCs. (D) Venn diagrams showing the overlap of H2AK119ub1 peaks (q‐value <0.1) between WT and Asxl2 KO mESCs at promoter and non‐promoter regions. (E) Boxplots comparing H2AK119ub1 signals of WT and Asxl2 KO among WT‐unique, shared and Asxl2 KO‐unique groups at promoter and non‐promoter regions. (F) Histogram plots comparing the log2 fold change of H2AK119ub1 signals in Polycomb/non‐Polycomb promoter and non‐promoter groups between Asxl2 KO and WT mESCs. The H2AK119ub1 signals are the average read densities of each 2‐kb bin. (G) Genome browser view of H2AK119ub1 profile in WT and Asxl2 KO mESCs. (H) H2AK119ub1 ChIP‐qPCR analysis of designated non‐promoter regions near to two PcG target genes T, Nkx2‐5 (as illustrated in G) in WT and Asxl2 KO mESCs. Data are represented as the mean ± SD of replicates (n = 3) (*p < 0.05 and two‐tailed unpaired t test).
FIGURE 3
FIGURE 3
RING1B redistribution is associated with H2AK119ub1 gain at non‐promoter regions in Asxl2‐null mESCs. (A) Boxplots comparing differences of RING1B, RYBP and SUZ12 ChIP‐seq signals at defined genomic regions in WT and Asxl2 KO mESCs. (B) Heatmaps illustrating RING1B, SUZ12 and RYBP ChIP‐seq signals at promoter regions in WT and mESCs. (C) Venn diagrams showing the overlap of RING1B peaks (q‐value<0.1) between WT and Asxl2 KO mESCs at promoter (left) and non‐promoter (right) regions. (D) Heatmap showing the RING1B signal changes simultaneously as H2AK119ub1 signals decrease or increase in Asxl2 KO versus WT mESCs. The signals are the average read densities of each 2‐kb bin. (E) Stacked bar plot showing the proportion of simultaneous decrease and increase groups as outlined in (D) at promoter and non‐promoter regions. (F) Snapshots of both H2AK119ub1‐ and RING1B‐gained loci at promoter and non‐promoter regions.
FIGURE 4
FIGURE 4
RING1B loss from promoters is associated with impaired gene silencing in Asxl2‐null mESCs. (A) Heatmaps illustrating RING1B ChIP‐seq signal at the promoters of with the RING1B binding unchanged (3049 peaks, log2 fold change between −0.5 and 0.5) or lost (3316 peaks, log2 fold change < −1.5) in Asxl2 KO mESCs versus WT mESCs. All rows are centred on TSS. (B) Boxplots comparing log2 fold change of gene expression in Asxl2 KO mESCs versus WT mESCs between RING1B‐unchanged and RING1B‐lost clusters. (C) Snapshots comparing RING1B signals at the promoters of designated genes in WT and Asxl2 KO mESCs. (D) RT‐qPCR analysis of mRNA levels of designated genes in WT and Asxl2 KO mESC. Data are represented as the mean ± SD of replicates (n = 3) (*p < 0.05 and two‐tailed unpaired t test). (E) Model for PR‐DUB in safeguarding PcG functions. Normally robust deposition of PRC1 is restricted to target promoters though its sampling may produce low H2AK119ub1 levels at the genome wide. Upon PR‐DUB deficiency, the pervasive gain of H2AK119ub1 at non‐promoters is associated with titration of PRC1 away from target promoters and thereby leads to compromised maintenance of gene silencing.
FIGURE 5
FIGURE 5
ASXL2 loss results in aberrant lineage differentiation. (A) Schematic diagram showing differentiation strategy of ESC to MES. (B) Heatmap illustrating the proportion of genes with up‐ and down‐regulation according to log2 fold change of gene expression between WT‐MES and WT‐ESC, Cluster 1 (WT log2 fold change > 3), Cluster 2 (WT log2 fold change < −3), Cluster 3 (WT −0.5 < log2 fold change<0.5). (C) GO enrichment analysis of genes in Cluster 1 and Cluster 3 S1, top 10 enriched items are shown according to –log10 adjusted‐p. (D) RT‐qPCR analysis of the mRNA levels of non‐MES lineage genes Pax6, Hox10 and Neurog1 in designated groups of cells. (E) ChIP‐qPCR analysis of RING1B binding at the promoters of Pax6, Hox10 and Neurog1 in designated groups of cells. Data are represented as the mean ± SD of replicates (n = 3) (*p < 0.05 and two‐tailed unpaired t test for D and E).
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