Stability of the epigenetic landscape underpins maintenance of the cell-type-specific transcriptional profile. As one of the main repressive epigenetic systems, DNA methylation has been shown to be important for long-term gene silencing; its loss leads to ectopic and aberrant transcription in differentiated cells and cancer1. The developing mouse germ line endures global changes in DNA methylation in the absence of widespread transcriptional activation. Here, using an ultra-low-input native chromatin immunoprecipitation approach, we show that following DNA demethylation the gonadal primordial germ cells undergo remodelling of repressive histone modifications, resulting in a sex-specific signature in mice. We further demonstrate that Polycomb has a central role in transcriptional control in the newly hypomethylated germline genome as the genetic loss of Ezh2 leads to aberrant transcriptional activation, retrotransposon derepression and dramatic loss of developing female germ cells. This sex-specific effect of Ezh2 deletion is explained by the distinct landscape of repressive modifications observed in male and female germ cells. Overall, our study provides insight into the dynamic interplay between repressive chromatin modifications in the context of a developmental reprogramming system.
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We thank J. Elliot, T. Adejumo and B. Patel for help with FACS; C. Whilding for help with microscopy and immunofluorescence data analysis; L. Game for help with next-generation sequencing; Z. Agate-Bacon and G. Zimmerman for mouse husbandry; T. Carell (LMU Munich) for providing isotopically labelled deoxynucleoside standards; the members of the Hajkova laboratory for discussions and revisions of the manuscript; and A. Hocher and T. Warnecke for additional computational analysis. Work in the Hajkova laboratory is supported by MRC funding (MC_US_A652_5PY70) and an ERC grant (ERC-CoG-648879–dynamic modifications) to P.H. T.-C.H. was a recipient of an Imperial College London/Taiwan Top University Strategic Alliance PhD Scholarship.
The authors declare no competing interests.
Peer review information Nature thanks the anonymous reviewers for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 Summary of ULI-nChIP-seq and genomic distribution of H3K27me3 and H3K9me3 enrichment.
a, Experimental scheme of PGC isolation using ∆PE Oct4-GFP mice (GOF-18/∆PE-GFP)12. ~1000 PGCs were used for each ULI-nChIP-seq. b, Characteristics of the H3K27me3 and H3K9me3 ULI-nChIP-seq datasets. Fraction of paired-end reads based on the mappability. Uniquely aligned (dark green). Uniquely aligned duplicates (light green). Multiple aligned (blue). Unaligned (grey). R: biological replicates. c, d, Genome-wide correlation of biological replicates for H3K27me3 and H3K9me3 ULI-nChIP-seq (Pearson correlation coefficient, 10 kb bins)25 . e, Genomic distribution of H3K27me3 and H3K9me3 peaks. f, Distance of H3K27me3 and H3K9me3 peaks relative to transcription start site (TSS). g, Violin plot showing the peak intensity distribution in E10.5 and E13.5 male and female PGCs. Peaks identified by MACS2 peak calling pipeline with broad peak setting (Methods). Box plots plotted using Tukey’s method. The upper and lower hinges: the first and the third quartiles. The central line: the median. The upper end of the whisker represents the lowest value among either the third quartile plus 1.5 × IQR or the maximum value from the data set. The lower end of the whisker represents the largest value among either the first quartile minus 1.5 × (IQR) or the minimum value from the data set. h, Bar chart showing the fraction of mapped paired-end reads of H3K27me3 and H3K9me3 associated with TEs in the genome.
Extended Data Fig. 2 Dynamics of DNA methylation and H3K27me3 at promoters during gonadal reprogramming.
a, Whole-genome bisulphite sequencing (WGBS) data from E10.5 and E12.5 female and male PGCs9. Density plot depicting DNA methylation levels at all promoters. b, Density plot depicting H3K27me3 enrichment at all promoters. c, Violin plot of H3K27me3 levels at promoters that lost DNA methylation (DNA methylation > 0.2 at E10.5). d, f, Dynamics of DNA methylation and H3K27me3 at all promoters (d) and group A (f). DNA methylation and H3K27me3 enrichment are shown by colour gradient. Distribution of each dot’s value is shown using rug plot along x and y axis. e, Box plot shows H3K27me3 enrichment of low CpG density (CpG < 4.1%) promoters which gained H3K27me3 in the female PGCs following global loss of DNA methylation. P values were calculated using 2 tailed Mann-Whitney U test with Bonferroni correction. ***: P < 0.001. g, Comparison of sequence characteristics and H3K27me3 enrichment of the group A from E13.5 male and female germ cells. P values were calculated by 2 tailed Mann-Whitney U test. Box plots were plotted using Tukey’s method. The upper and lower hinges: the first and the third quartiles. The central line: the median. The upper end of the whisker represents the lowest value among either the third quartile plus 1.5 × IQR or the maximum value from the data set. The lower end of the whisker represents the largest value among either the first quartile minus 1.5 × (IQR) or the minimum value from the data set.
Extended Data Fig. 3 Dynamics of DNA methylation, H3K9me3 and H3K27me3 at promoters during gonadal reprogramming.
a, Heat map depicting the H3K27me3 and H3K9me3 enrichment (ULI-nChIP-seq), and DNA methylation rate (WGBS) at promoters. The promoters were grouped based on the pattern of dynamic change between DNA methylation and H3K27me3 (See also Fig. 2). Group A: loss of DNA methylation, gain H3K27me3 at E13.5. Group B: median loss of DNA methylation, high H3K27me3. Group C: low DNA methylation, High H3K27me3. Group D: loss of DNA methylation, low H3K27me3 at E13.5. Promoters that did not meet the criteria were grouped into non-classified. The expression levels of promoter-associated genes from RNA-seq are presented by TPM (Transcripts Per Kilobase Million) or z-score. The total number of promoters in each group are shown on the left. b, Box plot showing the quantitative measurement in each category, female and male PGCs, respectively. Box plots were plotted using Tukey’s method. The upper and lower hinges: the first and the third quartiles. The central line: the median. The upper end of the whisker represents the lowest value among either the third quartile plus 1.5 × IQR or the maximum value from the data set. The lower end of the whisker represents the largest value among either the first quartile minus 1.5 × (IQR) or the minimum value from the data set. P values were calculated using 2 tailed Mann-Whitney U test. c, Venn diagram showing the number of overlapped promoters between male and female PGCs. d, Gene ontology of expressed genes in group A (Supplementary Table 4).
a, Representative immunofluorescence (IF) staining shows EZH2 expression at different embryonic stages. EZH2 is highly expressed in PGCs, compared with surrounding somatic cells. Biological replicates n = 3. OCT4: PGC marker. DAPI indicates DNA. Scale bar: 10 μm. b, Functional domains of EZH2 protein and targeting strategy of Ezh2 allele. Open boxes: exons. Black arrowhead: loxP sites. c, Breeding scheme to generate germline Ezh2 knockout. Ezh2 Δ/Δ, Tg (Blimp1-Cre) refers to CKO in the figures. f: allele flanked by loxP sites (floxed). Δ: Deleted allele generated using Cre-mediated recombination. Tg (Blimp1-Cre): transgenic mice express Cre recombinase under the control of Blimp1 (Prdm1) promoter. d, Deleted alleles were confirmed by PCR genotyping using the primers shown by black arrows in b. #1: Ezh2f/∆, Tg (Blimp1-Cre)+/-. #2: Ezh2f/+. This experiment was repeated at least 3 times independently with similar results.
Extended Data Fig. 5 Global H3K9me3, H2A119ub and DNA methylation are not altered in PGCs following the loss of EZH2.
a–c, Representative IF staining for H3K9me3, H2A119ub and TET1 using cryosectioned genital ridges. MVH and OCT4: PGC marker. DAPI indicates DNA. Biological replicates n = 2. Each dot represents one cell. d, IF staining for 5-methylcytosine (5mC). 5mC is enriched in pericentromeric regions in the nucleus of somatic cells but depleted in both Ctrl and Ezh2 CKO germ cells. 3 independent experiment were repeated with similar results. DNA was stained by Propidium Iodide (PI). Scale Bar: 10 μm. e, Global 5mC and 5hmC levels (mean ± s.d.) were measured by LC-MS/MS. Each dot represents an individual biological replicate. adj. P values were calculated using one-way ANOVA and Tukey’s post-hoc multiple comparison test (2 tailed). f, Representative IF images of E13.5 Ctrl and Ezh2 CKO female and male gonads. The bar chart shows the total number of germ cells per female gonad. Biological replicates n = 2. Each dot represents an individual biological replicate. MVH: PGC marker. DAPI indicates DNA. Scale bar: 100 μm.
a, Catalytic core and accessory subunits of mammalian PRC2. b, RNA expression levels (TPM) of PRC2 components in PGCs at different embryonic stages. c, RNA expression levels (TPM) of Ezh1 in the Ctrl and Ezh2 CKO germ cells. d, Sample distance matrix of RNA-seq samples by non-supervised clustering (Methods). e, PCA Plot shows the distance of transcriptomes from different PGC developmental stages. Dash line circle indicates samples of the same developmental stage
Extended Data Fig. 7 Sex-specific transcription factor repertoire determines transcriptional activation upon loss of EZH2.
a, Gene ontology (GO) terms associated with E13.5 male differentially expressed (DE) genes (Ctrl vs Ezh2 CKO). b, Integrative Genomics Viewer (IGV) plot shows the H3K27me3 enrichment and RNA-seq read counts of Stra8. Mouse genome: mm9. c, Heat map depicting gene expression and the chromatin dynamics at promoters of meiotic DE genes. Box plot shows the H3K27me3 enrichment and RNA expression (TPM) of meiotic DE genes in male and female PGCs. Z scores were calculated for male and female separately. Box plots were plotted using Tukey’s method. The upper and lower hinges: the first and the third quartiles. The central line: the median. The upper end of the whisker represents the lowest value among either the third quartile plus 1.5 × IQR or the maximum value from the data set. The lower end of the whisker represents the largest value among either the first quartile minus 1.5 × (IQR) or the minimum value from the data set. P values were calculated using 2 tailed Mann-Whitney U test. ***: P < 0.01 d, Bar chart showing the odds ratio of 104 meiosis prophase genes19 in each groups of promoters (Fig. 2). P values were calculated by Fisher exact test. *: P < 0.05. **: P < 0.01. ***: P < 0.001. e, Promoters of upregulated genes in female Ezh2 CKO are significantly enriched for transcription factor motifs that relate to retinoid acid signalling pathway. Motif analysis was performed using Bioconductor package PWMEnrich. f, Heat map showing the relative gene expression of identified transcription factors in Ctrl and Ezh2 CKO PGC samples. g, Heat map showing gene expression of 45 Germline Reprogramming Responsive (GRR) genes9 in Ctrl and Ezh2 CKO samples. Differentially expressed genes are shown on the top (adj. P < 0.05) (Two-tailed Wald test using DESeq2; See Methods).
a, Representative images of the female germ cells immunosiained with meiosis specific synaptonemal complex protein SCP3 and germ cell marker MVH in Ctrl and Ezh2 CKO embryonic ovaries. n = 2 independent animals analysed. b, Representative images of leptotene/zygotene and pachytene germ cells from female gonads at E15.5 and E16.5. Meiotic prophase I progression was classified based on SCP1 and SYCP3 staining. Leptotene/zygotene: DNA aggregates/ knobs were identified with numerous thin threads of SYCP3 staining. Pachytene: thick threads stained with strong SCP1 and SCP3 signal. Percentage of staged germ cells in Ctrl and Ezh2 CKO gonads was shown. Each dot represents a biological replicate. n = 2 independent animals analysed. At least 200 germ cells were calculated for each biological replicate. c, d, Representative IF images of γH2AX positive cells in cryosectioned gonads. During normal meiosis progression, γH2AX signal shows DNA double strand breaks (DSBs) occurring during homologous recombination. Accumulation of γH2AX signal was identified in E15.5 Ctrl and Ezh2 CKO germ cells and was greatly reduced at E18.5. A number RAD51 foci can be identified at E16.5 but greatly decreased at E18.5. Filament-like, RAD51-positive structure was identified in about 10% of the Ezh2 CKO germ cells but not in Ctrl germ cells. MVH positive germ cells are indicated by yellow arrowhead. DAPI indicates DNA. Scale bar: 10 μm. n = 2 independent animals analysed. These experiments were repeated 2 times independently with similar results.
a, Volcano plot of TEs in E13.5 male PGCs (Ezh2 CKO vs Ctrl). Significantly upregulated TEs are labelled in red. Fold change > 1, FDR < 0.1. b, Multidimensional scaling (MDS) plot showing distance of Ctrl and Ezh2 CKO PGC RNA-seq samples based on TE expression. Only uniquely mapped reads were considered. c, Gene Set Enrichment Analysis (GSEA) of RNA-seq (E13.5♀ Ezh2 CKO vs Ctrl). Number of genes enriched in each gene set is shown by the circle size. d, GSEA plot showing genes upregulated in female Ezh2 CKO PGCs are enriched in p53 pathway and interferon alpha response. FDR q value < 0.25 was considered significant. NES: normalized enrichment score. e, Distance of H3K9me3 de novo peaks to transcription start sites (TSS) and transposable elements (TEs). f, Representative IGV plot showing H3K9me3 enrichment on IAP Ez elements. g, TE subfamilies enriched predominantly for H3K9me3 or H3K27me3. Each row represents one TE subfamily. Multiple mapped and uniquely mapped reads were taken into account. h, i, Heat map showing DNA methylation, H3K9me3 and H3K27me3 enrichment at individual copies of IAPLTR2_Mm and L1Md_Gf. Each row represents one uniquely mapped, single TE copy belonging to the respective TE subfamily.
a, Representative images of γH2AX staining in male gonads. OCT4 or MVH positive germ cells are indicated by the yellow arrowhead. γH2AX positive cells are indicated by the white arrowhead. Less than 1% of MVH positive were γH2AX positive in E18.5 male Ezh2 CKO gonads. n = 2 independent animals analysed. b, Gross appearance of Ctrl and Ezh2 CKO male gonads at E18.5. c, Numbers of Ctrl and Ezh2 CKO male germ cells per section. Data are represented as mean ± s.d. Biological replicate n = 2. d, e, Representative IF images of DAZL and MILI expression in MVH positive male germ cells in Ctrl and Ezh2 CKO embryos. DAPI indicates DNA. Scale bar: 10 μm. n = 2 independent animals analysed.
Extended Data Fig. 11 EZH2 is dispensable for transcriptional regulation in PGC-like cells (PGCLCs).
a, Experimental scheme of PGCLC induction and 4-OHT induced EZH2 depletion in PGCLCs. Equivalent mouse germ line stages are shown above the experimental timeline. Ezh2f/f, CreERT2 embryonic stem cells (ESCs) were firstly induced into epiblast-like cells (EpiLCs) (Methods). Ethanol (Ctrl) or 4-Hydroxytamoxifen (4-OHT) were then added to induce Cre-mediated deletion of Ezh2 during PGCLC induction. At day 4 of treatment, PGCLCs were sorted by FACS and subjected to RNA-seq. b, Representative IF images of whole mount day 4 Ctrl and Ezh2 KO PGCLC aggregates. Biological replicates = 3. Experiment were repeated independently with similar results. Scale bar: 50 μm. c, Histone modifications (H3K27me3, H3K9me3 and H2AK119ub) in Ctrl and Ezh2 KO PGCLCs. AP2γ positive cells: PGCLCs. Biological replicates = 3. Experiment were repeated independently with similar results. DNA was stained by DAPI (blue). Scale bar: 10 μm. d, Expression of germline associated genes and pluripotency associated genes from this study and published datasets62,63. TPM: Transcripts Per Kilobase Million. Each data point represents one biological replicate (mean ± s.d.). n = 3 biological independent experiments. e, IGV plot shows RNA-seq read coverage of Ezh2 transcripts. Reads from deleted exons were depleted in the KO samples. Mouse genome: mm9. f, Sample distance matrix of RNA-seq samples by non-supervised clustering. g, h, Volcano plot showing differentially expressed (DE) genes (g) and TEs (Multiple mapped plus uniquely mapped reads) (h) in Ctrl and Ezh2 KO PGCLCs. Significantly DE genes and TEs are labelled in red (P values calculated by two-tailed Wald test using DESeq2; See Methods).
a, Venn diagram depicting overlap of genes significantly upregulated in the Ezh2 CKO female PGCs and the Rnf2 CKO PGCs27. b, Upper Venn diagram showing the overlap between the EZH2 and SETDB1 regulated TEs in E13.5 mouse germ cells. Lower Venn diagram shows overlap between EZH2 regulated TEs in E13.5 germ cells with EED regulated TEs in ESCs23,33. c, Model depicting the relationship between DNA demethylation and changes in histone repressive modifications in gonadal PGCs undergoing epigenetic reprogramming.
This file contains uncropped immunoblots in Fig 1g and the gating strategy for isolating OCT4-GFP positive primordial germ cells.
Summary for ULI-nChIP libraries and RNA-seq libraries.
This table contains a summary of the CG content of promoters and their associated genes.
This file contains (1) TPM and lists of differentially expressed genes (Ezh2 CKO versus Ezh2 Ctrl PGCs) and (2) a summary of expression levels of DNA methyltransferases and relevant chromatin modifiers.
This table contains gene ontology terms related to Fig. 2 and RNA-seq data.
This table contains differentially expressed retrotransposons (Ezh2 CKO vs Ezh2 Ctrl PGCs).
This file contains detailed information for antibodies, primer sequences, mouse strains and essential kits.
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Huang, TC., Wang, YF., Vazquez-Ferrer, E. et al. Sex-specific chromatin remodelling safeguards transcription in germ cells. Nature 600, 737–742 (2021). https://doi.org/10.1038/s41586-021-04208-5
Nature Metabolism (2022)