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Temporal and regional X-linked gene reactivation in the mouse germline reveals site-specific retention of epigenetic silencing

Nature Structural & Molecular Biology volume 32, pages 926–939 (2025)Cite this article

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Abstract

Random X-chromosome inactivation is a hallmark of female mammalian somatic cells. This epigenetic mechanism, mediated by the long noncoding RNA Xist, occurs in the early embryo and is stably maintained throughout life, although inactivation is lost during primordial germ cell (PGC) development. Using a combination of single-cell allele-specific RNA sequencing and low-input chromatin profiling on developing mouse PGCs, we provide a detailed map of X-linked gene reactivation. Despite the absence of Xist expression, PGCs still harbor a fully silent X chromosome at embryonic day 9.5 (E9.5). Subsequently, X-linked genes undergo gradual and distinct regional reactivation. At E12.5, a substantial part of the inactive X chromosome resists reactivation, retaining an epigenetic memory of its silencing. Our findings define the orchestration of reactivation of the inactive X chromosome, a key event in female PGC reprogramming with direct implications for reproduction.

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Fig. 1: scRNA-seq of polymorphic PGCs during embryonic development.
Fig. 2: Progression of X-chromosome reactivation in developing PGCs.
Fig. 3: Kinetics of reactivation of X-linked genes over the entire X chromosome in developing PGCs during reversal of random XCI.
Fig. 4: Differential timing of X-linked gene reactivation is associated with timing of silencing and chromosomal location.
Fig. 5: Enrichment of repeats and DNA methylation correlates with the latest reactivated loci of the Xi.
Fig. 6: Repressive histone mark contributes to resistance to early gene reactivation.

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Data availability

All scRNA-seq, WGBS and CUT&RUN datasets were deposited to the GEO under accession numbers GSE243943 and GSE243464. The WGBS datasets of E10.5 PGCs and E12.5 PGCs were respectively extracted from the DDBJ (DRA000607)54 and the GEO (GSE76973)55. Other data are available from the corresponding author upon request. Source data are provided with this paper.

Code availability

No code was developed for this study. Details and specificities (tools, version and options) to replicate the bioinformatic pipelines are provided in the Methods.

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Acknowledgements

We acknowledge the M.A.S. and D.B. laboratories for insightful discussions. We thank K. Harnish, Cambridge Stem Cell Institute Genomics Facility and the MGX-Montpellier GenomiX platform for advice and deep sequencing of the libraries. We thank the pathogen-free barrier animal facility of Gurdon Institute and the PCEA and ZEFI of IGMM, UMR5535, in Montpellier. We are grateful to J. Barau, D. Helmlinger and L. Bonneville for their advice and reagents related to the low-input CUT&RUN. We thank Y. Moyano-Rodríguez, T. Forné and E. J. Kremer for proofreading the manuscript. This work was supported by a Centre National de la Recherche Scientifique et Institut National de la Santé et de la Recherche Médicale (CNRS-INSERM) ATIP-Avenir grant, the Amorçage Jeune Equipe from Fondation Recherche Médicale (FRM AJE202005011598) and Agence National de la Recherche under the ‘Investissements d’avenir’ program (ANR-16-IDEX-0006) to M.B., by the Wellcome Trust (096738 and 092096) and Cancer Research UK program (C6946/A14492) to M.A.S., by a PhD fellowship from La Ligue Nationale Contre le Cancer to C.R. and by FRM (SPE20150331826) and Marie Sklodowska-Curie Individual (H2020-MSCA-IF-2015, no. 706144) fellowships to M.B. We acknowledge financial support to MGX from the France Génomique National infrastructure, funded as part of the ‘Investissement d’Avenir’ program managed by Agence Nationale pour la Recherche (contract ANR-10-INBS-09).

Author information

Author notes

  1. These authors contributed equally: Clara Roidor, Laurène Syx.

Authors and Affiliations

  1. IGMM, University of Montpellier, CNRS, Montpellier, France

    Clara Roidor, Emmanuelle Beyne, Karim Chebli & Maud Borensztein

  2. INSERM U900, Mines ParisTech, Institut Curie, PSL Research University, Paris, France

    Laurène Syx & Nicolas Servant

  3. CRBM, University of Montpellier, CNRS, Montpellier, France

    Peggy Raynaud

  4. INSERM U934, CNRS UMR3215, Institut Curie, PSL Research University, Paris, France

    Dina Zielinski, Aurélie Teissandier, Marius Walter & Deborah Bourc’his

  5. Wellcome/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK

    Caroline Lee, M. Azim Surani & Maud Borensztein

  6. Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK

    Caroline Lee & M. Azim Surani

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Contributions

M.B. conceptualized the study with M.A.S. and performed the scRNA-seq experiments. C.R. performed the chromatin analysis, handled the mouse colonies and collected the embryos with the help and supervision of K.C. L.S., E.B., P.R. and D.Z. performed the bioinformatics analysis, supervised by N.S. and M.B. A.T. performed the repeats and WGBS bioinformatics analysis. C.L. contributed to the animal husbandry and sample collection. M.W. and D.B. conducted the WGBS experiments. M.B., M.A.S. and D.B. secured the funding. M.B. wrote the original draft. C.R. and L.S. reviewed and edited the manuscript, with input from coauthors.

Corresponding author

Correspondence to Maud Borensztein.

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Extended data

Extended Data Fig. 1 Differential gene expression upon female PGC development and key markers of soma and PGC.

(A) PCA of the 30 most differentially expressed genes (DEGs) that contributed to lineage segregation. The different stages are denoted by different colours. Orange arrows represent genes contributing to PGC lineage, and blue arrows represent genes contributing to soma lineages. (B-D) Volcano plots represent differentially expressed genes (DEGs) between the two developmental stages of female PGCs. Differential expression was measured by TMM method (trimmed mean of M values, see Method section), followed by Benjamini–Hochberg correction. Genes with an adjusted P value < 0.05 were considered significant. A few transcriptional changes have been observed in migratory PGCs. Changes arise once PGCs colonize the gonads. Some examples of DEGs are highlighted. Red dots represent upregulated genes, and blue dots represent downregulated genes. X-linked genes are shown in orange.

Source data

Extended Data Fig. 2 Clustering of PGCs by sex.

(A) Pseudotime representation of the scRNA-seq data based on the first principal component for XX females (pink), XO females (red), and XY males (blue). (B) Level of Xist expression and degree of reactivation in each single cell. Each dot represents a single cell. Most female soma cells exhibit high Xist expression and a low number of biallelically expressed genes. Genes with RPRT expression < 4 were considered to be unexpressed.

Source data

Extended Data Fig. 3 Contribution to the kinetics of X-chromosome reactivation.

(A) Representation of the Gene Ontology analysis of Biological process performed on the best correlated genes with X-linked gene reactivation (adj.p-value ≤ 0.05) (Supplementary Data 3). Correlation and anti-correlation between gene expression levels (autosomes and X chromosomes) and the percentage of X-linked gene reactivation (allelic ratio >0.15 and <0.85 for X-linked genes) were measured using two-sided Pearson’s correlation and the Benjamini–Hochberg correction. (B) Comparison of reactivation timing for 7 X-linked genes between our study (129 x Mus musculus castaneus PGCs) and published scRT-PCR digested by restriction enzyme (Mus musculus domesticus x Mus musculus mollossinus PGCs) 21. In the extracted data from Sugimoto and Abe, an arbitrary threshold of ≥ 50% of biallelic cells has been applied to consider the gene reactivated. (C) Distance to escapee genes. Distribution of genomic distances to escapees (Mb) for each X-linked gene reactivation class. The transcription start site (TSS) of each gene was used to measure the distance from the closest escaping gene. No significant differences were found between reactivation classes by the KW test (p-value = 0.13), despite very late-reactivated genes being statistically further to escapees than early-reactivated genes by MW test (p-value = 0.02). Boxplots represent the medians with lower and upper quartiles. (D) Reactivation classes in female PGCs compared to the in vitro PGC-like cell system 32. (E) Xist RNA entry sites are regions of the X chromosome showing early accumulation of Xist RNA upon initiation of X-chromosome inactivation and are thought to be the closest to the Xist locus in 3D spatial proximity. Allelic expression of X-linked genes classified on the basis of their relative position to Xist RNA entry sites (as identified during XCI induction in ESC 6): inside (TSS located inside a Xist RNA site, 17 informative genes), next to (TSS located less than 100 kb away from an entry site, 17 informative genes) and outside (over 100 kb from an entry site, 163 informative genes). Box plots represent medians (centre lines) with lower and upper quartiles (box limits). Whiskers represent 1.5× the interquartile range. Outliers are represented by dots.

Source data

Extended Data Fig. 4 Validation of H3K27me3 low-input CUT&RUN in enriched regions from both chromosomes.

Integrative Genomics Viewer tracks for total H3K27me3 normalized reads (dark blue) and allele-specific reads (light blue: CAST; red: B6) at the HoxC locus (A) and Sox9 locus (B) in Count Per Million mapped reads (CPM, bin 10). Orange blocks are statistically-called H3K27me3 broad domains by MACS2, compared to IgG (top track). Each replicate is shown at E11.5 and E12.5. Location is given in mm10 genome, with gene isoforms extracted from Integrative Genome viewer and UCSC.

Extended Data Fig. 5 CUT&RUN tracks at escapee and early-reactivated X-linked locus.

Integrative Genomics Viewer tracks for total H3K27me3 normalized reads (dark blue) and allele-specific reads (light blue: Xi/CAST; red: Xa/B6) at the Kdm5c region (very-late reactivated gene) (A) and Smc1a region (early-reactivated gene) (B) in Count Per Million mapped reads (CPM, bin 10). Orange blocks are statistically-called H3K27me3 broad domains by MACS2, compared to IgG. Each replicate is shown at E11.5 and E12.5. Location is given in mm10 genome, with gene isoforms extracted from Integrative Genome viewer and UCSC.

Extended Data Fig. 6 CUT&RUN tracks at candidate X-linked gene locus.

(A) Integrative Genomics Viewer tracks for total H3K27me3 normalized reads (dark blue) and allele-specific reads (light blue: Xi/CAST; red: Xa/B6) at the Gjb1 (silent gene), Zmym3 (very-late reactivated gene) and Nono (early-reactivated gene) locus. Each replicate is shown at E11.5 and E12.5. (B-D) Integrative Genomics Viewer tracks for total H3K27me3 normalized reads (dark blue) and allele-specific reads (light blue: Xi/CAST; red: Xa/B6) at (B) the Med14 early reactivated gene, (C) the Kif4 early-reactivated gene, and (D) the Cacna1f silent gene in Count Per Million mapped reads (CPM, bin 10). Orange blocks are statistically-called H3K27me3 broad domains by MACS2, compared to IgG. Location is given in mm10 genome, with gene isoforms extracted from Integrative Genome viewer and UCSC.

Extended Data Fig. 7 H3K27me3 occupancy at active and inactive X chromosomes.

(A) Heatmaps of H3K27me3 occupancy in E11.5 and E12.5 female PGCs. Profile plots and their corresponding heatmaps centred on the transcription start sites (TSS) +/- 3 kb of all X-linked genes for H3K27me3 repressive histone marks. The purple-to-yellow gradient indicates low to high sum score of H3K27me3 in the corresponding regions clustered by k-mean =5 on total H3K27me3 at E11.5. Genome B6: Xa and Genome Cast: Xi. (B) Repartition of reactivation classes and silent genes in the different clusters, calculated by k-mean, based on their H3K27me3 occupancy at E11.5. Silent gene class has been characterised as genes silenced from both X chromosomes in all female PGC scRNA-seq. Numbers in pie charts are TSS.

Supplementary information

Supplementary Figures

Supplementary Figs. 1–4.

Reporting Summary

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Supplementary Data 1

Information on the scRNA-seq datasets.

Supplementary Data 2

Allelic ratio of informative X-linked genes and escapees from Fig. 3 heatmaps and their reactivation classes.

Supplementary Data 3

Correlation between gene expression and percentage of X-linked gene reactivation (XX soma and PGC cells).

Supplementary Data 4

DEGs at E12.5 PGCs. Differential expression was measured by the trimmed mean of M values (Methods), followed by Benjamini–Hochberg correction. Genes with an adjusted P value < 0.05 were considered significant.

Supplementary Data 5

Primers for allele-specific DNA methylation assays.

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Roidor, C., Syx, L., Beyne, E. et al. Temporal and regional X-linked gene reactivation in the mouse germline reveals site-specific retention of epigenetic silencing. Nat Struct Mol Biol 32, 926–939 (2025). https://doi.org/10.1038/s41594-024-01469-2

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