Abstract
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.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
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.
References
Lyon, M. F. Gene action in the X-chromosome of the mouse (Mus musculus L.). Nature 190, 372–373 (1961).
Żylicz, J. J. & Heard, E. Molecular mechanisms of facultative heterochromatin formation: an X-chromosome perspective. Annu. Rev. Biochem. 89, 255–282 (2020).
Brockdorff, N. et al. The product of the mouse Xist gene is a 15 kb inactive X-specific transcript containing no conserved ORF and located in the nucleus. Cell 71, 515–526 (1992).
Brown, C. J. et al. The human XIST gene: analysis of a 17 kb inactive X-specific RNA that contains conserved repeats and is highly localized within the nucleus. Cell 71, 527–542 (1992).
Ku, M. et al. Genomewide analysis of PRC1 and PRC2 occupancy identifies two classes of bivalent domains. PLoS Genet. 4, e1000242 (2008).
Lock, L. F., Takagi, N. & Martin, G. R. Methylation of the Hprt gene on the inactive X occurs after chromosome inactivation. Cell 48, 39–46 (1987).
Gendrel, A.-V. et al. Smchd1-dependent and -independent pathways determine developmental dynamics of CpG island methylation on the inactive X chromosome. Dev. Cell 23, 265–279 (2012).
Chu, C. et al. Systematic discovery of Xist RNA binding proteins. Cell 161, 404–416 (2015).
McHugh, C. A. et al. The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3. Nature 521, 232–236 (2015).
Monfort, A. et al. Identification of Spen as a crucial factor for Xist function through forward genetic screening in haploid embryonic stem cells. Cell Rep. 12, 554–561 (2015).
Moindrot, B. et al. A pooled shRNA screen identifies Rbm15, Spen, and Wtap as factors required for Xist RNA-mediated silencing. Cell Rep. 12, 562–572 (2015).
Dossin, F. et al. SPEN integrates transcriptional and epigenetic control of X-inactivation. Nature 578, 455–460 (2020).
Brockdorff, N. Polycomb complexes in X chromosome inactivation. Philos. Trans. R. Soc. Lond. B 372, 20170021 (2017).
Silva, J. et al. Establishment of histone H3 methylation on the inactive X chromosome requires transient recruitment of Eed-Enx1 Polycomb group complexes. Dev. Cell 4, 481–495 (2003).
Plath, K. et al. Role of histone H3 lysine 27 methylation in X inactivation. Science 300, 131–135 (2003).
de Napoles, M. et al. Polycomb group proteins Ring1A/B link ubiquitylation of histone H2A to heritable gene silencing and X inactivation. Dev. Cell 7, 663–676 (2004).
Dixon-McDougall, T. & Brown, C. J. Independent domains for recruitment of PRC1 and PRC2 by human XIST. PLoS Genet. 17, e1009123 (2021).
Wang, H. et al. Role of histone H2A ubiquitination in Polycomb silencing. Nature 431, 873–878 (2004).
Gao, Z. et al. PCGF homologs, CBX proteins, and RYBP define functionally distinct PRC1 family complexes. Mol. Cell 45, 344–356 (2012).
Cao, R. et al. Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science 298, 1039–1043 (2002).
Wang, J. et al. Imprinted X inactivation maintained by a mouse Polycomb group gene. Nat. Genet. 28, 371–375 (2001).
Pintacuda, G. et al. hnRNPK Recruits PCGF3/5-PRC1 to the Xist RNA B-repeat to establish Polycomb-mediated chromosomal silencing. Mol. Cell 68, 955–969 (2017).
Almeida, M. et al. PCGF3/5–PRC1 initiates Polycomb recruitment in X chromosome inactivation. Science 356, 1081–1084 (2017).
Nesterova, T. et al. Systematic allelic analysis defines the interplay of key pathways in X chromosome inactivation. Nat. Commun. 10, 3129 (2019).
Endoh, M. et al. Polycomb group proteins Ring1A/B are functionally linked to the core transcriptional regulatory circuitry to maintain ES cell identity. Development 135, 1513–1524 (2008).
Okamoto, I., Otte, A. P., Allis, C. D., Reinberg, D. & Heard, E. Epigenetic dynamics of imprinted X inactivation during early mouse development. Science 303, 644–649 (2004).
Borensztein, M. et al. Xist-dependent imprinted X inactivation and the early developmental consequences of its failure. Nat. Struct. Mol. Biol. 24, 226–233 (2017).
Corbel, C., Diabangouaya, P., Gendrel, A. V., Chow, J. C. & Heard, E. Unusual chromatin status and organization of the inactive X chromosome in murine trophoblast giant cells. Development 140, 861–872 (2013).
Takagi, N. & Abe, K. Detrimental effects of two active X chromosomes on early mouse development. Development 109, 189–201 (1990).
Mugford, J. W., Yee, D. & Magnuson, T. Failure of extra-embryonic progenitor maintenance in the absence of dosage compensation. Development 139, 2130–2138 (2012).
Sakata, Y. et al. Defects in dosage compensation impact global gene regulation in the mouse trophoblast. Development 144, 2784–2797 (2017).
Corbel, C. & Heard, E. Transcriptional analysis by nascent RNA FISH of in vivo trophoblast giant cells or in vitro short-term cultures of ectoplacental cone explants. J. Vis. Exp. 114, e54386 (2016).
Takada, T. et al. Mouse inter-subspecific consomic strains for genetic dissection of quantitative complex traits. Genome Res. 18, 500–508 (2008).
Su, I. H. et al. Ezh2 controls B cell development through histone H3 methylation and Igh rearrangement. Nat. Immunol. 4, 124–131 (2003).
Sugishita, H. et al. Variant PCGF1-PRC1 links PRC2 recruitment with differentiation-associated transcriptional inactivation at target genes. Nat. Commun. 12, 5341 (2021).
Farcas, A. M. et al. KDM2B links the Polycomb repressive complex 1 (PRC1) to recognition of CpG islands. eLife 18, e00205 (2012).
Andergassen, D., Smith, Z. D., Kretzmer, H., Rinn, J. L. & Meissner, A. Diverse epigenetic mechanisms maintain parental imprints within the embryonic and extraembryonic lineages. Dev. Cell 56, 2995–3005 (2021).
da Rocha, S. T. et al. Jarid2 is implicated in the initial Xist-induced targeting of PRC2 to the inactive X chromosome. Mol. Cell 53, 301–316 (2014).
Cooper, S. et al. Jarid2 binds mono-ubiquitylated H2A lysine 119 to mediate crosstalk between Polycomb complexes PRC1 and PRC2. Nat. Commun. 7, 13661 (2016).
Dorbrinić, P., Szczurek, A. T. & Klose, R. J. PRC1 drives Polycomb-mediated gene repression by controlling transcription initiation and burst frequency. Nat. Struct. Mol. Biol. 28, 811–824 (2021).
Jadhav, U. et al. Replicational dilution of H3K27me3 in mammalian cells and the role of poised promoters. Mol. Cell 78, 141–151 (2020).
Li, H. Exploring single-sample SNP and INDEL calling with whole-genome de novo assembly. Bioinformatics 28, 1838–1844 (2012).
Li, H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv https://doi.org/10.48550/arXiv.1303.3997 (2013)
Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).
Takahashi, S. et al. Genome-wide stability of the DNA replication program in single mammalian cells. Nat. Genet. 51, 529–540 (2019).
Perry, J., Palmer, S., Gabriel, A. & Ashworth, A. A short pseudoautosomal region in laboratory mice. Genome Res. 11, 1826–1832 (2001).
Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. Embnet J. 17, 10–12 (2011).
Magoc, T. & Salzberg, S. L. FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27, 2957–2963 (2011).
Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).
Kent, W. J. et al. The Human Genome Browser at UCSC. Genome Res. 12, 996–1006 (2002).
Degner, J. F. et al. Effect of read-mapping biases on detecting allele-specific expression from RNA-sequencing data. Bioinformatics 25, 3207–3212 (2009).
Trapnell, C. et al. Transcript assembly and quantification by RNA-seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010).
Acknowledgements
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).
Author information
Authors and Affiliations
Contributions
O.M., C.C., H.K. and E.H. conceived the main idea of the whole analysis. O.M. and C.C. designed and conducted the experiments. K.N. and C.O. analysed allele-specific RNA-seq data. T.A.E. performed statistical analyses of all data. M.N. designed Eed conditional knockout mouse. F.K., P.D., M.K. and Y.K. bred all mice used in this study. O.M., C.C., F.K. and P.D. prepared frozen sections of embryos. C.C., O.M. and F.K. performed RNA FISH and immuno-RNA FISH on sections. O.M., C.C. and F.K. derived TGCs and performed RNA FISH and immuno-RNA FISH for TGCs. O.M. and C.C. analysed the data and wrote the paper. H.K. and E.H. edited the paper.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Nature Cell Biology thanks Christine Disteche and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data
Extended Data Fig 1 Replicates of E7.5 ∆Ring1A/B female embryos.
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.
Extended Data Fig 3 Replicates of E7.5 ∆Ring1A/B female embryos analyzed by RNA FISH.
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.
Extended Data Fig 5 Details of allele-specific RNA-seq.
(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.
Extended Data Fig. 6 A model for the role of Polycomb complexes in the maintenance of XCI.
(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.
Supplementary information
Supplementary Information
Supplementary Tables 1–4, Information and Discussion.
Supplementary Tables
The excel file includes Supplementary Tables 1–5.
Source data
Source Data Fig. 1
Statistical source data.
Source Data Fig. 2
Statistical source data.
Source Data Fig. 3
Raw numeric data.
Source Data Fig. 4
Raw numeric data.
Source Data Fig. 5
Raw numeric data.
Source Data Extended Data Fig. 1
Raw numeric data.
Source Data Extended Data Fig. 2
Raw numeric data.
Source Data Extended Data Fig. 4
Raw numeric data.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41556-022-01047-y