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Epigenomic analysis of gastrulation identifies a unique chromatin state for primed pluripotency

Abstract

Around implantation, the epiblast (Epi) transits from naïve to primed pluripotency, before giving rise to the three germ layers. How chromatin is reconfigured during this developmental window remains poorly understood. We performed a genome-wide investigation of chromatin landscapes during this period. We find that enhancers in ectoderm are already pre-accessible in embryonic day 6.5 (E6.5) Epi when cells enter a primed pluripotent state. Unexpectedly, strong trimethylation of histone H3 at lysine 4 (H3K4me3) emerges at developmental gene promoters in E6.5 Epi and positively correlates with H3K27me3, thus establishing bivalency. These genes also show enhanced spatial interactions. Both the strong bivalency and spatial clustering are virtually absent in preimplantation embryos and are markedly reduced in fate-committed lineages. Finally, we show that KMT2B is essential for establishing bivalent H3K4me3 at E6.5 but becomes partially dispensable later. Its deficiency leads to impaired activation of developmental genes and subsequent embryonic lethality. Thus, our data characterize lineage-specific chromatin reconfiguration and a unique chromatin state for primed pluripotency.

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Fig. 1: Global view of chromatin states during gastrulation in mouse embryos.
Fig. 2: Epigenetic regulation of lineage-restricted putative enhancers.
Fig. 3: Super bivalency identified in primed pluripotent Epi in vivo.
Fig. 4: Loss of super bivalent H3K4me3 is associated with aberrant activation of developmental genes.
Fig. 5: TET proteins promote super bivalent H3K4me3 in E6.5 Epi.
Fig. 6: Super bivalent genes show strong spatial clustering.
Fig. 7: Dynamic chromatin regulation of developmental genes from naïve pluripotency to committed lineages in vivo.

Data availability

All data have been deposited to GEO with the accession number GSE125318. Source data for Extended Data Fig. 6 are available online.

Code availability

Software and code used to analyze these data are listed in the Nature Research Reporting Summary.

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Acknowledgements

We thank members of the Xie laboratory for comments during the preparation of the manuscript. We appreciate the support from the animal core facility, the sequencing core facility and biocomputing facility at Tsinghua University. This work was funded by the Beijing Municipal Science & Technology Commission (Z181100001318006 to W.X.), National Natural Science Foundation of China (31422031 and 31725018 to W.X.), Beijing Advanced Innovation Center for Structural Biology (100300001 to W.X.), the National Basic Research Program of China (2015CB856201 to W.X.) and the THU-PKU Center for Life Sciences (W.X.). W.X. is a recipient of an HHMI International Research Scholar.

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Authors and Affiliations

Authors

Contributions

Y.X., Y.Z. and W.X. conceived and designed the project. Y.X. dissected early lineages and performed STAR ChIP–seq and RNA-seq library construction. Y.Z. and Y.X. analyzed NGS data. Y.Z. performed STEM-seq experiments. Q.X. collected early embryos, performed in vitro transcription of sgRNAs and electroporation experiments. C.Z. designed and established the Kmt2b zygotic knockout system. B.L. conducted miniATAC-seq. B.Z. conducted STAR ChIP–seq. K.Z. performed sisHi-C library construction of Kmt2b−/− embryos and Z.D. generated sisHi-C libraries of EpiLC. S.G. prepared EpiLC supervised by S.K. Y.W., X.W. and L. Li helped with the preparation of the manuscript. L. Liu performed the genotyping of Kmt2b KO mice. Y.L. and Q.W. performed NGS sequencing. W.X. supervised the project and related experiments. Y.Z, Y.X. and W.X. prepared the manuscript and figures with help from all the authors.

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Correspondence to Wei Xie.

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Extended data

Extended Data Fig. 1 Global view of histone modifications and chromatin accessibility in mouse early lineages.

a, Snapshots showing two replicates of H3K4me3, H3K27ac, H3K27me3, and ATAC-seq in E6.5Epi, E6.5VE, Ect, PS, Mes, and End at E7.5. mESC data from ENCODE47 are also shown for comparison. The genome browser view scales were adjusted based on the global data range. b, The Pearson correlation between two replicates for H3K4me3, H3K27me3, H3K27ac, and ATAC-seq in post-implantation lineages. The correlation between our dataset and a published dataset73 for H3K27me3 in E6.5 epiblast is also shown.

Extended Data Fig. 2 Global view of histone modifications, chromatin accessibility, and dynamics of putative enhancers during mouse early lineage specification.

a, Hierarchical clustering of two replicates for histone modifications and ATAC-seq data at promoters among post-implantation tissues. b, The heatmap showing the Spearman correlation between gene expression and enrichment of histone modifications and open chromatin at promoters in the post-implantation embryos. c, Heatmaps showing gene expression and related histone modifications/open chromatin enrichment at promoters for lineage-specific genes. d, Heatmaps showing tissue-specific enhancers from mesoderm to heart (left), and endoderm to liver (right). GO terms enriched for each tissue-specific enhancers are also listed. Somatic enhancers were obtained from a previous study47. e, Boxplots showing the dynamics of DNA methylation for tissue-specific enhancers34,47 during development. The median of each dataset is shown by the center line. The bottom, top edges and whiskers represent the twenty-fifth and seventy-fifth percentiles, and 1.5 times the interquartile range (IQR), respectively.

Extended Data Fig. 3 Bivalency establishment in early embryo.

a, Snapshots showing H3K4me3 and H3K27me3 enrichment at developmental genes Hoxd cluster and Gata2 in various cell types (n=2). Gene expression is also shown (log2 transformed FPKM). mESC and somatic tissue data from ENCODE47 are shown. Published data in 8-cell embryos and ICM28,29 are also shown for comparison. The genome browser view scales were adjusted based on the global data range. b, Average plots showing H3K4me3 and H3K27me3 enrichment at the Hox genes, bivalent genes inactive at all stages examined (8-cell, ICM, E5.5Epi, E6.5Epi, E6.5VE, and mESC), and housekeeping genes. The H3K4me3 enrichment is normalized against H3K4me3 signals at housekeeping (HK) gene promoters for each lineage. Arrows indicate E6.5 Epi. c, Average plots showing H3K4me3 and H3K27me3 enrichment at inactive bivalent genes (inactive at E6.5Epi, Ect, PS, Mes, and End), and housekeeping genes. The H3K4me3 enrichment is normalized against H3K4me3 signals at housekeeping (HK) gene promoters for each lineage. Arrows indicate E6.5 Epi.

Extended Data Fig. 4 Bivalency states in E6.5Epi and E6.5VE.

a, The UCSC genome browser views showing the enrichment of H3K4me3 (n=2), H3K27me3 (n=2) and H3 (n=1) at developmental genes Hoxa cluster, Pax6, as well as a housekeeping gene Rpn1 in mouse E6.5Epi and E6.5VE. b, Average plots showing H3K4me3 (n=2), H3K27me3 (n=2) and H3 (n=1) enrichment at the Hox gene cluster, inactive bivalent genes (inactive in E6.5Epi and E6.5VE), and housekeeping genes in E6.5Epi and E6.5VE. c, The scatter plot showing the enrichment of H3K4me3 (ULI-NChIP–seq), either done in this study (left) or in a previous study50 (right), and H3K27me3 (STAR ChIP–seq) of all bivalent genes (n=3,992) for E6.5 epiblast. The average H3K4me3 enrichment of housekeeping gene (HK.ave) is shown for each tissue. The number of super bivalent genes (top right) and Pearson correlation of H3K4me3 and H3K27me3 for each tissue (bottom right) are shown. d, The Venn diagram shows the overlap of super bivalent genes identified by ULI-NChIP-seq (Uli) and STAR ChIP-seq in E6.5 epiblast. The P-values was calculated by Fisher’s test.

Extended Data Fig. 5 Super bivalency marks primed pluripotent state in early lineages and somatic tissues.

a, Snapshots comparing the enrichment of H3K4me3, H3K27me3, and H3K27ac in E6.5 epiblast and somatic tissues from ENCODE47 at developmental genes Neurod1, Pcp4l1, and Foxa1. The heatmaps showing related gene expression levels. The genome browser view scales were adjusted based on the global data range. b, Left, the boxplot showing the expression levels of super bivalent genes and housekeeping genes in E6.5Epi34 and cortex47. The median of each dataset is shown by the center line. The bottom, top edges and whiskers represent the twenty-fifth and seventy-fifth percentiles, and 1.5 times the interquartile range (IQR), respectively. Two-sided P-values calculated by t-test are also shown. Super bivalent genes are identified in E6.5Epi (inactive). C-active, E6.5Epi super bivalent genes that become active in cortex (FPKM > 5); C-inactive, E6.5Epi super bivalent genes that remain inactive in cortex (FPKM < 2). Right, average plots showing the enrichment of H3K4me3 around the active and inactive super bivalent genes and housekeeping genes in E6.5Epi and cortex. c, A similar analysis as b in E6.5Epi and heart. H-active, E6.5Epi super bivalent genes that become active in heart (FPKM > 5); H-inactive, E6.5Epi super bivalent genes that remain inactive in heart (FPKM < 2).

Extended Data Fig. 6 Loss of super bivalent H3K4me3 is associated with aberrant developmental gene activation.

a, Gene expression of Kmt2b in pre- and post-implantation embryos is shown as bar graphs using previously published datasets28,34. b, Left, a schematic showing Kmt2b knockout strategy using Cas9/CRISPR as previously described61. Inner and outer primers for genotyping are shown. Right, the genotyping results of identified wild-type and Kmt2b-/- embryos using extra-embryonic tissues at E6.5. One representative image from three independent experiments is shown. Uncropped gel is also shown in Source data. M, DNA ladder; +/+, wild-type; +/-, heterozygote; -/-, homozygote. c, Average plots showing H3K4me3 enrichment at super bivalent/non-super bivalent/housekeeping genes (defined in E6.5 epiblast) for wild-type (n=2) and Kmt2b-/- E6.5Epi (n=3) and E8.5 head (n=2). The H3K4me3 enrichment is normalized against H3K4me3 signals at housekeeping (HK) gene promoters for each lineage. d, The morphology of wild-type and Kmt2b KO embryos from E6.5 to E9.5. Three independent experiments were performed. e, Hierarchical clustering based on gene expression in wild-type and Kmt2b KO embryos from E6.5 to E9.5. Arrow indicates E9.5 Kmt2b-/- embryo.

Source data

Extended Data Fig. 7 WT vs. Tet1/2 DKO embryos in E6.5 epiblasts.

a, Bar chart showing the expression levels of Kmt2b in wild-type (n=3) and Tet1/2 DKO (n=2) E6.5 epiblasts. The error bar represents the S.D with the barplot showing the the mean value. b, The morphology of wild type and Tet1/2 DKO embryos at E8.75. Two independent experiments were performed. c, Hierarchical clustering based on gene expression in wild-type and Tet1/2 DKO embryos from E6.5 to E8.5.

Extended Data Fig. 8 Spatial interactions of super bivalent genes in E6.5 epiblast.

a, The snapshots showing the ‘Virtual 4C’ (converted from Hi-C datasets) among bivalent genes in 8-cell, ICM, E6.5Epi, E6.5VE, mESC, and fibroblast. The enrichment of H3K4me3 and H3K27me3 in E6.5Epi (n=2) and E6.5VE (n=2) are shown below. Magnified views of interactions between bivalent genes are also shown. b, Heatmaps showing the inter-chromosomal interactions between chromosomes 2 and 11. Boxes show the zoomed-in views of interactions between Dlx, Hoxd cluster (chr2) and Hoxb clusters (chr11). A UCSC genome browser of H3K27me3 enrichment in wild-type E6.5Epi is shown on the top to indicate the positions of Polycomb targets.

Extended Data Fig. 9 Super bivalent genes show strong spatial clustering.

a, A Venn diagram shows the overlap between ELRI genes10 (n=108), top 100 Polycomb nucleation genes55, and super bivalent genes identified in E6.5Epi (see Methods). The P-values showing the overlap among pairwise comparison were calculated by Fisher test. b, Boxplots showing the normalized interaction frequencies among different gene groups in 8-cell, ICM, E6.5Epi, E6.5VE, mESC, EpiSC, EpiLC and fibroblast for ELRI bivalent genes(top) and non-ELRI bivalent genes (bottom). Super, super bivalent genes; Non-super, non-super bivalent genes; HK, housekeeping genes; Inactive, non-bivalent inactive genes (Methods). The median of each dataset is shown by the center line. The bottom, top edges and whiskers represent the twenty-fifth and seventy-fifth percentiles, and 1.5 times the interquartile range (IQR), respectively. Two-sided P-values calculated by t-test are also shown.

Extended Data Fig. 10 Strong spatial clustering of developmental genes in E6.5 epiblast.

a, Boxplots showing the normalized interaction frequencies among genes in each gene group in wild-type (n = 5) and Kmt2b-/- (n=3) E6.5 Epi. P-values calculated by two-sided t-test are also shown. b, Left, a schematic diagram shows the relative spatial position of a selected gene defined by its interactions with developmental genes divided by its interactions with housekeeping genes. Right, boxplots showing the log ratios of such bivalent/housekeeping gene interactions for different gene groups (inactive, active developmental genes, housekeeping genes) in E6.5 epiblast.

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Original uncropped gel of Extended Data Figure 6b. Boxes indicate the cropped regions

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Xiang, Y., Zhang, Y., Xu, Q. et al. Epigenomic analysis of gastrulation identifies a unique chromatin state for primed pluripotency. Nat Genet 52, 95–105 (2020). https://doi.org/10.1038/s41588-019-0545-1

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