In female mammals, one of the two X chromosomes becomes inactivated during development by X-chromosome inactivation (XCI). Although Polycomb repressive complex (PRC) 1 and PRC2 have both been implicated in gene silencing, their exact roles in XCI during in vivo development have remained elusive. To this end, we have studied mouse embryos lacking either PRC1 or PRC2. Here we demonstrate that the loss of either PRC has a substantial impact on maintenance of gene silencing on the inactive X chromosome (Xi) in extra-embryonic tissues, with overlapping yet different genes affected, indicating potentially independent roles of the two complexes. Importantly, a lack of PRC1 does not affect PRC2/H3K27me3 accumulation and a lack of PRC2 does not impact PRC1/H2AK119ub1 accumulation on the Xi. Thus PRC1 and PRC2 contribute independently to the maintenance of XCI in early post-implantation extra-embryonic lineages, revealing that both Polycomb complexes can be directly involved and differently deployed in XCI.
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The allele-specific RNA-seq dataset is publicly available through Gene Expression Omnibus (GEO). The accession number is GSE168140. Raw data of Figs. 3c and 5c are provided in Supplementary Tables 2 and 3, respectively. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.
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We thank members of the Heard and Koseki laboratories for helpful discussion; Tim Pollex for critical reading of the manuscript; the mouse facilities in Curie Institute and RIKEN for breeding the mice; D. Reinberg and R. Margueron for antibodies and advice; R. Margueron for providing Ezh2fl/fl, Rosa26::CreERT2 mice. We acknowledge the NGS core facility of the Genome Information Research Center at the Research Institute for Microbial Deseases of Osaka University for support in RNA-seq. This work was funded by the LabEx DEEP (ANR-11-LABX-0044), and an ERC Advanced Investigator award ERC-ADG-2014 671027 to E.H.; Core Research for Evolutional Science and Technology and Precursory Research for Embryonic Science and Technology from the Japan Science and Technology Agency–Agency for Medical Research and Development, and Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science to Koseki; JPMJPR11SE to O.M., from PRESTO, JST. 17H06426 to K.N. and 18H05532 to C.O., from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT).
The authors declare no competing interests.
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a, Features of all fifteen E7.5 ∆Ring1A/B female embryos. Morphologies of embryos: N: normal, ABN: abnormal E7.5 morphology or (*) small E6.5-like morphology. H2AK119ub1 accumulation status on the Xi for embryonic and/or extraembryonic lineages. H3K27me3 accumulation status on the Xi for all lineages: embryonic and extraembryonic. 15 out of 27 female embryos were ∆Ring1A/B among 68 embryos analysed. b, Quantification of H2AK119ub1 and H3K27me3 Xi focus expression in nuclei from sections in Fig. 1b (upper part). Both embryo (e) and ectoplacental cone (epc) nuclei from ∆Ring1A/B E7.5 (OR1-1) embryos are analysed as positive (strong* or weak* expression as an Xi focus) or negative. Percentage and number of cells analysed are given for both histone marks. c, Quantification of H3K27me3 Xi accumulation in nuclei from sections in Fig. 1b (lower part). Results are given for control and ∆Ring1A/B E7.5 female embryo (OR3-2). All nuclei are analysed as positive (strong* or weak* expression as an Xi focus) or negative. Percentage and number of cells analysed are given. d, Another example of ∆Ring1A/B E7.5 female embryo (OR1–9) showing lack of H2AK119ub1 and presence of H3K27me3 on the Xi on sections. Upper part: General view of the embryo with DAPI staining is shown on the left. i: higher magnification of embryonic region (e) analyzed by immuno-RNA FISH for H2AK119ub1 and Xist. Quantification of H2AK119ub1 Xi accumulation in nuclei from the represented section. Lower part: Consecutive section. General view of the whole embryo with DAPI staining is shown on the left. i: higher magnification of embryonic region (e) immunostained for H3K27me3. Note; because the DAPI image of this cryosection was accidently lost, a consecutive section was DAPI stained and displayed here. Quantification of H3K27me3 Xi foci expression in nuclei from the represented section. All nuclei are analysed as positive (strong* or weak* expression as an Xi focus) or negative. Percentage and number of cells analysed are given for both histone marks. Scale bars: 100 µm for the whole embryo and 10 µm for the enlarged images indicated with white rectangles.
Extended Data Fig 2 ∆Ring1A/B E8.5 embryos are deprived of H2AK119ub1, but not H3K27me3 on the Xi in extraembryonic tissues.
a, Female specific lethality upon deletion of Ring1A/B at E8.5. E8.5 control and ∆Ring1A/B male and female embryos were analysed. Percentage and number are given. A significantly lower than expected number of ∆Ring1A/B female embryos at E8.5 (13.5% instead of 25%, P = 0.033) and an increased number of empty deciduae (15.4%), suggesting female ∆Ring1A/B embryo lethality at this stage. *empty decidua that is, dead embryo. Under the assumption that all genotypes are observed in 25% of the cases, one-sided binomial distribution was used to calculate p-values less than the expected number of embryos. b, H2AK119ub1/H3K27me3 accumulation status on the Xi analysed on sections for three E8.5 ∆Ring1A/B female embryos. Percentage of H3K27me3-positive cells on the Xi is given. c, ∆Ring1A/B E8.5 embryos are deprived of H2AK119ub1, but not H3K27me3 on the Xi in extraembryonic lineages, for example epc. Analyses on longitudinal sections; boxed regions (e and epc) are shown with higher magnification. Upper part: control embryo studied by immuno-RNA FISH for two histone marks, H2AK119ub1 (left), H3K27me3 (right) and Xist; consecutive sections. Lower part: ∆Ring1A/B female embryo (RAB8–57). Quantifications of H2AK119ub1 and H3K27me3 Xi accumulation in nuclei from these sections are shown below. All nuclei are analysed as positive (strong* or weak* expression as an Xi focus) or negative. Percentage and numbers of cells analyzed are given. e: embryo proper, epc: ectoplacental cone. Scale bars: 100 µm for the whole embryos and 10 µm for the enlarged images.
a, Quantification of Atrx and Huwe1 biallelic expression on both Xa and Xi in embryonic and extraembryonic lineages in three ∆Ring1A/B E7.5 embryos totally deprived of H2AK119ub1 accumulation on the Xi: ∆1, ∆2 and ∆3 (OR1–7, OR1–8 and OR8–15, respectively) and in one control (OR2–17). Percentage and numbers of cells analyzed for each lineage including TGCs. *Not significant due to low number of analyzed cells. b, Another example of ∆Ring1A/B female embryo (∆1, OR1–7) showing escape of Atrx in extraembryonic tissues. Three different regions are shown. 1 (ve and e), i: higher magnification showing a cell from the embryo proper in which Atrx is monoallelically expressed from the Xa. 2 (exe, ve and TGC), i: higher magnification of a TGC in which Atrx is biallelically expressed on both Xa and Xi; 3 (epc), i: higher magnification showing a cell from epc in which Atrx is biallelically expressed on both Xa and Xi. Arrowheads: Xist-coated Xi. Arrows: Xa. Scale bars: 100 µm for DAPI staining of the whole embryos and 10 µm for the enlarged images indicated with white rectangles. e; embryo proper, epc; ectoplacental cone, ve; visceral endoderm, exe; extraembryonic ectoderm, TGC; trophoblast giant cell. Three independent embryos (∆1: OR1–7, ∆2: OR1–8 and ∆3: OR8–15) were examined and all showed similar results. ∆2 (OR1–8) is shown in Fig. 2a. c, H2AK119ub1 level on the Xi in extraembryonic lineages correlates with the degree of derepression on the Xi. Quantification of Atrx and Huwe1 biallelic expression on both Xa and Xi in embryonic and extraembryonic lineages in two ∆Ring1A/B E7.5 embryos which partially lost H2AK119ub1 accumulation on the Xi in extraembryonic lineages: ∆4 and ∆5 (OR1–4 and OR1–9 respectively; see Extended Data Fig. 1a). Percentage and numbers of cells analyzed for each lineage including TGCs.
Extended Data Fig 4 Morphology of ∆Ring1A/B TGCs derived from E7.5 ectoplacental cone and quantification of H3K27me3 and H2AK119ub1 Xi accumulation.
a, Female-specific lethality of TGCs upon Ring1A/B (PRC1) deletion. Morphology of EPC outgrowths, containing TGCs were recorded from days 1 to 4 of culture. Each TGC culture was categorized into three categories, Growing (G), Arrested (A), and Dying (D), based on their morphologies. Representative pictures are shown on the left. Scale bar: 100 µm. Summary chart is shown on the right. ∆Ring1A/B female EPC outgrowth showed more severe phenotypes such as Arrested (A) and Dying (D) than male mutants and controls. b, Quantification of H3K27me3 Xi accumulation in control and ∆Ring1A/B TGCs illustrated in Fig. 2d. c, Quantification of H3K27me3 and H2AK119ub1 Xi accumulation in nuclei from sections in Fig. 4b. d, Quantification of H2AK119ub1 Xi accumulation in ∆Ezh2 TGCs as illustrated in Fig. 4d. b–d, All nuclei are analysed as positive (strong* or weak* expression as an Xi focus) or negative. Percentage and numbers of cells analyzed are given.
(Right) Schematic of B6-ChrXTMSM chromosome used in this study. Telomeric half of the B6 X chromosome is replaced with MSM/Ms X chromosome. (Left) Allele-specific expression ratio in 3 independent EPCs of Ring1A/B control, ∆Ring1A/B, Eed control and ∆Eed E7.5 embryos are represented as heat maps. Maternal expression in red and paternal expression in blue. Total 136 informative genes (122 for Ring1A/B and 132 for Eed) including 5 constitutive escapees (magenta), Xist (Blue), significant escapees upon PRC1 deletion (green), significant escapees upon PRC2 deletion (orange), not informative genes (gray) and silenced genes even in the absence of PRC1 or PRC2 (black) are shown. Values are allele-specific expression ratios in each EPC.
(Left) X-linked genes in embryonic and extraembryonic lineages on the Xi are silenced by XCI. CGIs on the Xi are heavily methylated in embryonic lineages, but maintained as hypo-methylated in extraembryonic lineages. (Middle) Ring1A/B knockout resulted in a depletion of all PRC1 subcomplexes on the Xi, but PRC2 is still retained on the Xi, at least in extraembryonic lineages. In this situation, PRC2 accumulation on the Xi is retained at E7.5 but lost at E8.5 in embryonic lineages (here E7.5 is shown). (Right) Ezh2 or Eed knockout resulted in a depletion of PRC2 on the Xi, but PRC1 is still retained on the Xi, in extraembryonic lineages. (Middle and Right) In these situations, many of X-linked genes undergo robust reactivation from the Xi only in extraembryonic lineages. In embryonic lineages, however, X-linked genes are still silenced in the absence of PRC1 or PRC2. DNA methylation of CGIs and/or some other factor(s) might compensate a lack of PRC1 or PRC2 to secure a tight silencing of X-linked genes on the Xi in embryonic lineages.
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Masui, O., Corbel, C., Nagao, K. et al. Polycomb repressive complexes 1 and 2 are each essential for maintenance of X inactivation in extra-embryonic lineages. Nat Cell Biol 25, 134–144 (2023). https://doi.org/10.1038/s41556-022-01047-y