Abstract
In mammals, 5-methylcytosine (5mC) and Polycomb repressive complex 2 (PRC2)-deposited histone 3 lysine 27 trimethylation (H3K27me3) are generally mutually exclusive at CpG-rich regions. As mouse embryonic stem cells exit the naive pluripotent state, there is massive gain of 5mC concomitantly with restriction of broad H3K27me3 to 5mC-free, CpG-rich regions. To formally assess how 5mC shapes the H3K27me3 landscape, we profiled the epigenome of naive and differentiated cells in the presence and absence of the DNA methylation machinery. Surprisingly, we found that 5mC accumulation is not required to restrict most H3K27me3 domains. Instead, this 5mC-independent H3K27me3 restriction is mediated by aberrant expression of the PRC2 antagonist Ezhip (encoding EZH inhibitory protein). At the subset of regions where 5mC appears to genuinely supplant H3K27me3, we identified 163 candidate genes that appeared to require 5mC deposition and/or H3K27me3 depletion for their activation in differentiated cells. Using site-directed epigenome editing to directly modulate 5mC levels, we demonstrated that 5mC deposition is sufficient to antagonize H3K27me3 deposition and confer gene activation at individual candidates. Altogether, we systematically measured the antagonistic interplay between 5mC and H3K27me3 in a system that recapitulates early embryonic dynamics. Our results suggest that H3K27me3 restraint depends on 5mC, both directly and indirectly. Our study also implies a noncanonical role of 5mC in gene activation, which may be important not only for normal development but also for cancer progression, as oncogenic cells frequently exhibit dynamic replacement of 5mC for H3K27me3 and vice versa.
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Data availability
Datasets generated in this study were uploaded to the NCBI Gene Expression Omnibus under accession number GSE242201. Additional data can be found in the Supplementary Information. Source data are provided with this paper.
Code availability
Scripts used to generate the figures presented are available under an GNU General Public License v3.0 on GitHub (https://github.com/julienrichardalbert/5mC-H3K27me3/releases/tag/v0.2).
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Acknowledgements
We thank T. Horii and I. Hatada (Gunma University, Japan) for providing the constitutive dCas9–SunTag/TET1CD epigenome-editing construct, B. Guichard (Institut Jacques Monod (IJM), France) and M. Möckel (IMB Mainz, Germany) for purifying the pA-Tn5 protein, C. McQuillen and H. Fanlo Ucar for technical assistance, D. Bourc’his for her insights and support, J. Barau, M. Leeb, A. Bogutz, S. Janssen and M. Pitasi for useful discussions, M. Lorincz and D. Holoch for critical reading, J. Marchand for installing and maintaining computational resources, N. Valentin and the IJM FACS facility. Work in the M.V.C.G. group is supported by the European Research Council (ERC-StG-2019 DyNAmecs) and by a Laboratoire d’Excellence Who Am I? (11-LABX-0071) emerging teams grant. This research was also funded by the Fondation pour la Recherche Médicale (FRM) postdoc France fellowship (SPF202110014238) to J.R.A. and the FRM (SPF202004011789) and Fondation ARC (ARCPDF12020070002563) postdoctoral fellowships to A.M.-S. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
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J.R.A., conceptualization, methodology, formal analysis, data curation, visualization, writing—original draft and writing—review and editing; T.U., investigation, visualization, writing—original draft and writing—review and editing; A.M.-S., investigation; A.L.B., investigation; A.S., investigation; A.D., investigation; M. Scarpa, investigation; M. Schulz, investigation; M.V.C.G., conceptualization, methodology, investigation, writing—review and editing, supervision, project administration and funding acquisition.
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Nature Structural & Molecular Biology thanks Gabriella Ficz and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Dimitris Typas, in collaboration with the Nature Structural & Molecular Biology team.
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Extended data
Extended Data Fig. 1 In vitro and in vivo H3K27me3 dynamics during the exit from naïve pluripotency.
a. 2D scatterplot showing H3K27me3 enrichment over genome-wide 10kb bins during in vitro differentiation of WT ESCs to EpiLCs (D7). Datapoints are coloured by 5mC levels in WT EpiLCs. 10kb bins with ≥10 CpGs covered by ≥5 reads are shown (n=252,559), and the density of datapoints is included. b. 2D scatterplot showing H3K27me3 enrichment over genome-wide 10kb bins during in vivo differentiation of E3.5 inner cell mass (ICM) cells of the blastocyst into the E7.5 epiblast. Datapoints are coloured by 5mC levels in E7.5 epiblasts. 10kb bins with ≥10 CpGs covered by ≥5 reads are shown (n=254,190), and the density of datapoints is included. c. Scatterplot showing H3K27me3 levels over CpG islands (CGIs) in WT ESCs (D0) and EpiLCs (D7). Datapoints are coloured by average 5mC levels in WT EpiLCs. CGIs with ≥10 CpGs covered by ≥5 reads are shown (n=15,714/16,009). CGI promoters of specific genes are indicated, contours are drawn at iso-proportions of the density of datapoints. d. UCSC Genome Browser screenshots of Polycomb-group target genes. WGBS and H3K27me3 CUT&Tag data derived from WT ESCs and EpiLCs (day 7) is shown. Refseq genes, CpG density, CGIs and individual CpGs are included. H3K27me3 peak calls are shown as rectangles. Promoter regions are highlighted in yellow. Coordinates: Hoxd12 chr2:74,672,861-74,678,432, Pax7 chr4:139,712,945-139,857,645, Pax2 chr19:44,712,108-44,849,206. e. Heatmap showing expression levels (RPKM) of Polycomb-group (PcG) target genes Hoxd12, Pax7 and Pax2 in vitro and in vivo (inner cell mass cells of embryonic day (E) 3.5 and E4.0 blastocysts, and E5.5 and E6.5 epiblasts. For each in vitro and in vivo dataset, the scale bar for each gene is set independently, either to the maximum expression level or 1, whichever is higher. f. UCSC Genome Browser screenshots of EpiLC-specific genes. Data as in d. Coordinates: Zhx2 chr15:57,676,294-57,758,534, Gpc6 chr14:116,904,924-116,947,162. g. Heatmap showing expression levels (RPKM) of EpiLC-specific genes Zhx2 and Gpc6, as in e. h. UCSC Genome Browser screenshots of EpiLC-silenced genes. Data as in d. in vivo data derived from the inner cell mass (ICM) of E3.5 blastocysts and E7.5 epiblasts is shown. Coordinates: Klf4 chr4:55,521,819-55,537,790, Prdm14 chr1:13,104,889-13,135,730, Tbx3 chr5:119,635,577-119,698,095. i. Heatmap showing expression levels (RPKM) of EpiLC-silenced genes Klf4, Prdm14 and Tbx3, as in e.
Extended Data Fig. 2 The transcriptomic landscape of TKO EpiLCs and genomic composition of SWRs.
a. Heatmaps showing expression levels (RPKM) of naïve (Klf4, Tbx3, Zfp42/Rex1, Fgf4 and Prdm14) and formative/primed (Pou3f1, Sox3, Etv5, Cdh2 and Fgf5) pluripotency markers in vitro and in vivo (inner cell mass cells of E3.5 and E4.0 blastocysts, and E5.5 and E6.5 epiblasts). For each in vitro and in vivo dataset, the scale bar for each gene is set independently to the maximum expression of that gene in WT and TKO samples. b. Principal Component Analysis (PCA) plot showing transcriptomic similarities between naïve ESCs with the inner cell mass cells of the blastocyst, and between EpiLCs with epiblasts. Both groups are arbitrarily circled by a dotted line. D0: ESCs; D2, D4, D7: EpiLCs; Oo.: oocyte; 1C: zygote; E2C: early 2-cell embryo; 2C: 2-cell embryo; L2C: late 2-cell embryo; 4C: 4-cell embryo; 8C: 8-cell embryo; Mo.: morula-stage embryo; ICM: inner cell mass cells of the blastocyst; TE: trophectoderm cells of the blastocyst; E5.5-, 6.5-, 7.5-: embryonic days; Epi: epiblast; ExE: extraembryonic ectoderm; Endo.: endoderm; Meso.: mesoderm; Ecto.: ectoderm; Prim. Str.: primitive streak. c. Pie charts showing the genomic distribution of H3K27me3 domain classes SWitch Regions (SWRs), ESC-Specific Regions (ESRs) and COnstitutive Regions (CORs). A fourth pie chart showing the total area of each genomic compartment is included. d. Motif analysis showing enriched motifs in the putative PRC binding sites within SWRs using ESRs as control sequences and two motif analysis algorithms.
Extended Data Fig. 3 H3K36me3 deposition is not strongly impacted in absence of DNA methylation.
a. 2D scatterplot showing H3K27me3 and H3K36me3 enrichment over genome-wide 10kb bins in WT ESCs. Data points are coloured by 5mC levels in WT ESCs. 10kb bins with ≥10 CpGs covered by ≥5 reads are shown (n=252,812), and the density of datapoints is included. b. 2D scatterplot showing H3K27me3 and H3K36me3 enrichment over genome-wide 10kb bins in WT EpiLCs. Data points are coloured by 5mC levels in WT EpiLCs. 10kb bins with ≥10 CpGs covered by ≥5 reads are shown (n=252,559), and the density of datapoints is included. c. 2D scatterplot showing H3K36me3 enrichment over genome-wide 10kb bins in WT and TKO ESCs. Data points are coloured by 5mC levels in WT ESCs as in a, and density of datapoints is included. d. 2D scatterplot showing H3K36me3 enrichment over genome-wide 10kb bins in WT and TKO EpiLCs. Data points are coloured by 5mC levels in WT EpiLCs as in b, and density of data points is included. e. 2D scatterplot showing H3K27me3 enrichment over genome-wide 10kb bins in WT and TKO EpiLCs, as in Fig. 1c. Data points are coloured by the relative change in H3K36me3 enrichment in TKO versus WT EpiLCs, and density of datapoints is included. f. Violin plot showing the distribution of H3K36me3 level change in TKO EpiLCs compared to WT EpiLCs. 10kb bins are categorized based on H3K27me3 dynamics in WT and TKO EpiLCs; No H3K27me3: insufficient H3K27me3 levels (CPM<1) in either WT or TKO EpiLCs, not DE: H3K27me3 levels do not significantly change between WT and TKO EpiLCs, Down TKO: relative loss of H3K27me3 in TKO EpiLCs, Up TKO: relative enrichment of H3K27me3 in TKO EpiLCs compared to WT EpiLCs.
Extended Data Fig. 4 TKO+Ezhip KO (QKO) ESCs differentiate to EpiLCs.
a. Heatmap showing Ezhip expression levels (RPKM) in WT and Suz12 KO and Eed KO ESCs. The value of the highest expression level in all four conditions is indicated. b. Representative brightfield images of EpiLCs. Scale bar = 1mm. Pictures were taken of D5 EpiLCs. c. Bar plots showing the expression level of naïve (Rex1/Zfp42, Nanog), general (Oct4), formative, (Otx2, Fgf5) and primed (Brachyury/T) pluripotency markers in D7 EpiLCs. Bars represent the mean, error bars the standard deviation, and individual data points are shown for each independent experiment (n=4). No statistical tests were performed. d. Heatmap showing relative enrichment of PRC2 subunit proteins on the chromatin of WT naïve and primed epiblast stem cells (EpiSCs). Data from Ugur et al.54. e. Heatmap depicting PRC2 subunit gene expression levels (RPKM) during EpiLC differentiation in WT and TKO cells.
Extended Data Fig. 5 Examples of SWR regions with maintenance of gene repression or activation.
a. Schema showing a hypothetical H3K27me3-to-5mC SWitch Region (SWR) that overlaps a genic promoter. In this case, dense methylation of the promoter region results in the maintained repression of expression. b. UCSC Genome Browser screenshot of an example SWR overlapping the Piwil1 germline gene promoter. The Refseq gene annotation, CpG density, individual CpGs, CpG islands and scale bar are included. Promoter region is highlighted in yellow. Note the anticorrelated enrichment of 5mC and H3K27me3 in WT cells, and the aberrant maintenance of H3K27me3 in TKO EpiLCs. Coordinates: chr5:128,733,977-128,756,805. c. Heatmap of Piwil1 expression levels (RPKM) in vitro and in vivo differentiation. ESCs: D0; EpiLCs: D2, D4, D7; E3.5-, E4.5-: embryonic days 3.5 and 4.5 inner cell mass cells; E5.5-, E6.5: embryonic days 5.5 and 6.5 epiblasts. d. UCSC Genome Browser screenshots of SWRs that overlap genes activated in WT EpiLCs (see schema, Fig. 3b). Coordinates: Pdyn chr2:129,673,296-129,713,102, Krt80 chr15:101,345,735-101,394,892, Pga5 chr19:10,666,677-10,680,350.
Extended Data Fig. 6 Implementation of an epigenome editing system for site-specific 5mC deposition at candidate SWRs.
a. Schema showing inducible constructs integrated into the Rosa26 locus in ESCs. One allele contains a construct composed of the TetOn array promoter for Doxycycline-dependent activation; DD: FKBP12-derived destabilizing domain, stabilized by the addition of the Shield-1 ligand; dCas9: catalytically dead Cas9, where two point mutations abrogate nucleolytic activity; 10xGCN4: an array of ten GCN4 epitopes (dCas9-SunTag). The homologous allele encodes a TetOn promoter, a destabilizing DD domain, a single-chain antibody (scFv) that recognizes the GCN4 epitope, green fluorescent protein (GFP) and the catalytic domain of DNMT3A (3ACD). Control cells contain an identical construct that encodes a catalytically inactive form of DNMT3A (d3ACD). A separate plasmid containing single guide RNAs (sgRNAs) was randomly integrated by piggyBac transposition. Resistance genes driven by an independent promoter are indicated. b. Western blot validating the inducible dCas9-SunTag/scFv-GFP-3ACD cell line. The two proteins (dCas9-SunTag and scFv-GFP-3ACD) encoded by the two inducible constructs are not detected in the absence of Dox, detected at intermediate levels after Dox induction, and robust expression after the addition of both Dox and Shield-1. c. UCSC Genome Browser screenshots of candidate SWR regions adjacent to the genes: Zdbf2, Celsr2, Atp4a, Arsi, Pga5, Pdyn, and Krt80. The target site for 5mC editing using 3ACD and TET1 is shown; gRNAs are indicated in red (forward strand) or blue (reverse strand). H3K27me3 CUT&Tag in TKO EpiLCs is included. The location of H3K27me3 CUT&RUN quantitative PCR and BS-pyro amplicons are also shown. CpG density, CpG islands (CGIs) and Refseq gene annotations have been added for reference. Coordinates: Zdbf2 chr1:63,246,946-63,275,173; Celsr2 chr3:108,401,129-108,424,638; Atp4a: chr7:30,703,888-30,733,844; Arsi chr18:60,899,912-60,924,326; Pga5 chr19:10,660,312-10,683,383; Pdyn chr2:129,683,316-129,707,017; Krt80 chr15:101,366,622-101,390,127.
Extended Data Fig. 7 Targeted 5mC deposition and concomitant H3K27me3 loss at the Zdbf2 SWR and site-directed 5mC editing at other candidate SWRs.
a. BS-pyro of the Zdbf2 SWR showing 5mC deposition in induced cells (+Dox +Shield-1, green) expressing dCas9 and the 3ACD construct compared to uninduced control cells (-Dox -Shield-1, gray). Cells were collected 7 days after the addition of Dox and Shield-1. 9 replicates are shown, and each replicate represents the average 5mC level across 4 CpGs. b. CUT&RUN-qPCR showing lower average levels of H3K27me3 in induced cells (green) over the Zdbf2 SWR. Data represent mean of n=8 and n=9 replicates for induced and uninduced samples, respectively. c. CUT&RUN-qPCR showing similar levels of H3K27me3 enrichment at the positive control Pax5 promoter locus relative to a negative (background) control, β-actin. Data represents mean of replicates, with each replicate indicated as a dot (d3ACD + Dox n=5, 3ACD +Dox n=8, 3ACD -Dox n=9, 3ACD +Dox n=8). d. BS-pyro results at the Zdbf2, Celsr2, Pdyn, Pga5 and Krt80 candidate SWRs. Each data point represents the average 5mC level over the pyrosequencing amplicon for each replicate, and bars represent the mean of independent experiments (n=2, except Zdbf2 g1 n=10). a, c-d Data are represented as mean ± standard error of independent experiments. Replicates and their mean are shown. P-values were calculated by two-tailed unpaired t-test assuming equal variance.
Extended Data Fig. 8 Targeted 5mC deposition in TKO + Dnmt1 ESCs at the Zdbf2 and Celsr2 SWRs.
a. Western Blot showing DNMT1 protein levels in TKO + Dnmt1 ESCs. Clone B5 was selected for subsequent experiments. b. LUminometric Methylation Assay (LUMA) showing global 5mC levels in ESCs grown in serum conditions. Data points represent technical replicates (n=2). N.D.: not detected. c. Schema of constitutive epigenome editing constructs randomly integrated into ESCs grown in serum conditions. d. BS-pyro of the Zdbf2 and Celsr2 SWRs. Data points represent the average 5mC level over the amplicon for each replicate. e. Bar plot showing expression levels of Zdbf2 and Celsr2 by RT-qPCR. Levels were normalized to the average Ct of two housekeeping genes (Rplp0 and Rrm2) (ΔCt method). d-e Data are represented as mean ± standard error of independent experiments, Replicates and their mean are shown (n=3, except for d3ACD Zdbf2 gRNA n=2). P-values were calculated by two-tailed unpaired t-test assuming equal variance.
Extended Data Fig. 9 Targeted 5mC demethylation of candidate SWRs using the constitutively expressed dCas9-SunTag/scFv-GFP-TET system.
a. BS-pyro results from EpiLCs (day 4) at candidate SWRs Pdyn (gRNA 1-2), Pga5 (gRNA 1-2), Krt80 (gRNA 3-4) and Arsi (gRNA 1-2). All cells constitutively express the dCas9-SunTag/scFv-GFP-TET1CD construct. Cells expressing target gRNAs are in purple, and control cells expressing scrambled gRNAs are in grey. TKO EpiLCs were used as a negative control (orange, n=1, except for Arsi, n=2). Each data point represents the average 5mC level over the pyrosequencing amplicon (3-9 CpGs: 5 for Pdyn, 5 for Pga5, 3 for Krt80, and 9 for Arsi) for each independent experiment (n=4, except TET1 Arsi gRNA, n=3). TKO samples were not considered for statistical analysis. b. CUT&RUN-qPCR results showing similar enrichment of H3K27me3 at the positive control Pax5 locus across cell lines relative to β-actin (negative control locus). Data represent mean of n=6 independent experiments. No significant differences between target and scrambled gRNA samples were detected. c. CUT&RUN qPCR showing H3K27me3 levels normalized to the positive control Pax5 CGI promoter (ΔCt method) for the Zdbf2 (left) and Celsr2 (right) SWRs. Data represent n=6 independent experiments for each line. a-c. Data are represented as mean ± standard error of independent experiments, replicates and their mean are shown. P-values were calculated by two-tailed unpaired t-test assuming equal variance. d. 2D scatterplot showing H3K27me3 levels over genome-wide 10kb bins (n=273,121) in epigenome-edited cells targeting the Zdbf2 and Celsr2 SWRs. The 10kb bins overlapping SWRs are indicated. e. 2D scatterplot showing H3K27me3 levels over genome-wide 10kb bins (n=273,121) in epigenome-edited cells targeting the Arsi and Atp4a SWRs. The 10kb bin overlapping the Atp4a SWR is indicated.
Extended Data Fig. 10 dTET1 epigenome editing to intermediate effects in EpiLCs.
a. BS-pyro of the Zdbf2 and Celsr2 SWRs treated with: TET1 with no target gRNA (scrambled), a catalytically inactive TET1 (dTET1) and target gRNA, TET1 and target gRNA, and in TKO EpiLCs. Each data point represents the average 5mC level over the pyrosequencing amplicon (4-5 CpGs) for each independent experiment (n=3, except TKO n=2). b. MA plot showing H3K27me3 enrichment level changes in EpiLCs targeting TET1 (left) or dTET1 (right) to the Zdbf2 SWR compared to the Celsr2 SWR. c. UCSC Genome browser screenshots of the Zdbf2 and Celsr2 SWR regions showing H3K27me3 level enrichment by CUT&RUN in WT ESCs and epigenome-edited EpiLCs. d. Bar plot showing expression levels of Zdbf2 and Celsr2 by RT-qPCR. Levels were normalized to the average Ct of two housekeeping genes (Rplp0 and Rrm2) (ΔCt method). n=3 independent experiments were conducted (except Zdbf2 TKO, n=2). a,d: Data are represented as mean ± standard error of independent experiments, Replicates and their mean are shown. P-values were calculated by two-tailed unpaired t-test assuming equal variance. TKO EpiLCs datasets were not included in the statistical tests.
Supplementary information
Supplementary Information
Supplementary Figs. 1–9.
Supplementary Tables
Supplementary Table 1: Datasets generated and mined. Supplementary Table 2: Oligonucleotide sequences. Supplementary Table 3: Statistical source data underlying supplementary figures.
Source data
Source Data Figs. 2d and Extended Data Figs. 6b and 8a
Unprocessed western blots.
Source Data Figs. 4b–d and 5b–d and Extended Data Figs. 4c, 7a–d, 8b,d,e, 9a–c and 10a,d.
Statistical source data.
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Richard Albert, J., Urli, T., Monteagudo-Sánchez, A. et al. DNA methylation shapes the Polycomb landscape during the exit from naive pluripotency. Nat Struct Mol Biol 32, 346–357 (2025). https://doi.org/10.1038/s41594-024-01405-4
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DOI: https://doi.org/10.1038/s41594-024-01405-4
