PCGF3/5-PRC1 initiates Polycomb recruitment in X chromosome inactivation

Abstract

Recruitment of the Polycomb repressive complexes PRC1 and PRC2 by Xist RNA is an important paradigm for chromatin regulation by long noncoding RNAs. Here, we show that the noncanonical Polycomb group RING finger 3/5 (PCGF3/5)-PRC1 complex initiates recruitment of both PRC1 and PRC2 in response to Xist RNA expression. PCGF3/5-PRC1-mediated ubiquitylation of histone H2A signals recruitment of other noncanonical PRC1 complexes and of PRC2, the latter leading to deposition of histone H3 lysine 27 methylation chromosome-wide. Pcgf3/5 gene knockout results in female-specific embryo lethality and abrogates Xist-mediated gene repression, highlighting a key role for Polycomb in Xist-dependent chromosome silencing. Our findings overturn existing models for Polycomb recruitment by Xist RNA and establish precedence for H2AK119u1 in initiating Polycomb domain formation in a physiological context.

Fig. 1. H2AK119u1 mediates PRC2 recruitment by Xist RNA.

A. Schematic of the experiment. Tamoxifen (+OHT). Doxycycline (+dox). B. Examples of H3K27me3 and EZH2 immunofluorescence following 24h Xist induction. C. Percentage of cells with domains for Xist, H3K27me3 and EZH2 in tamoxifen treated and untreated cells after Xist induction for 24h. A minimum of 800 cells were counted in 4 biological repeats. Error bars indicate standard deviation. D. Xist RNA FISH illustrating deletion of Ring1A/B has no effect on Xist RNA domain formation.

Fig. 2. Xist dependent localisation and dynamics of non-canonical PRC1 complexes.

A. Images illustrating focal enrichment of eGFP-PCGF fusion proteins following induction of Xist RNA for 24h in 3E-H mESCs sublines (+ dox). Focal enrichment is not seen in uninduced cells (- dox). Images are maximum intensity projections of six consecutive z-stacks. Scale bar is 10μm. B. FRAP of eGFP-PCGF fusion proteins within Xist RNA domains. C. FRAP of eGFP-PCGF fusion proteins within randomly selected regions of the nucleoplasm. In (B) and (C) data from single cells was fitted with a bi-exponential equation. Curves represent average of the fit from a number (n) of cells.

Fig. 3. RYBP/YAF2 interaction with H2AK119u1 contributes to Xist dependent enrichment of non-canonical PRC1 complexes.

Images illustrating distribution of eGFP-PCGF proteins in Rybp^-/-Yaf2^-/- mESC lines complemented with wild-type mCherry-RYBP, or mCherry-RYBP TF-AA mutant after Xist induction (+dox) for 24 h. Images are maximum intensity projections of six consecutive z-stacks. Scale bar is 10 μm.

Fig. 4. PCGF3/5-PRC1 initiates Polycomb recruitment to facilitate chromosome silencing by Xist RNA.

A. Schematic representation of PCGF knockout experiments. Tamoxifen (+OHT). Doxycycline (+dox). B. Images illustrating accumulation of H2AK119u1 over Xist RNA domains in the presence and absence of PCGF proteins. C. Scoring data representing a minimum of 300 cells in three replicates is summarised in the bar chart. Error bars show standard deviation. Significant differences (Student t-test) are indicated (**p<0.001). D. Immunofluorescence analysis of H2AK119u1 and H3K27me3 domains in Pcgf3^-/- :Pcgf5^-/- and Pcgf3^fl/fl:Pcgf5^fl/fl female embryos at E7.5. Images show representative single focal plane scans through epiblast. Scale bar is 20 μm. E. Genes differentially expressed between Xist induced (+dox) and uninduced (control) in Pcgf3^fl/flPcgf5^-/- (left panel) and Pcgf3^-/-Pcgf5^-/- (right panel) mESCs on Xist transgene bearing chromosome 16. Genes with FDR< 0.05 are depicted in violet; FDR >0.05 and <0.1 are in green. F. Heatmap showing expression change between Xist induced (+dox) and uninduced (control) mESCs for all genes significantly misregulated on chromosome 16 in Pcgf3^fl/flPcgf5^-/- (FDR<0.1) together with expression changes for the same genes in Pcgf3^-/-Pcgf5^-/- mESCs.