Abstract
Histone modifications have critical roles in regulating the expression of developmental genes during embryo development in mammals1,2. However, genome-wide analyses of histone modifications in pre-implantation embryos have been impeded by the scarcity of the required materials. Here, by using a small-scale chromatin immunoprecipitation followed by sequencing (ChIP–seq) method3, we map the genome-wide profiles of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3), which are associated with gene activation and repression4,5, respectively, in mouse pre-implantation embryos. We find that the re-establishment of H3K4me3, especially on promoter regions, occurs much more rapidly than that of H3K27me3 following fertilization, which is consistent with the major wave of zygotic genome activation at the two-cell stage. Furthermore, H3K4me3 and H3K27me3 possess distinct features of sequence preference and dynamics in pre-implantation embryos. Although H3K4me3 modifications occur consistently at transcription start sites, the breadth of the H3K4me3 domain is a highly dynamic feature. Notably, the broad H3K4me3 domain (wider than 5 kb) is associated with higher transcription activity and cell identity not only in pre-implantation development but also in the process of deriving embryonic stem cells from the inner cell mass and trophoblast stem cells from the trophectoderm. Compared to embryonic stem cells, we found that the bivalency (that is, co-occurrence of H3K4me3 and H3K27me3) in early embryos is relatively infrequent and unstable. Taken together, our results provide a genome-wide map of H3K4me3 and H3K27me3 modifications in pre-implantation embryos, facilitating further exploration of the mechanism for epigenetic regulation in early embryos.
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Acknowledgements
The authors would like to thank S. Liu from the University of British Columbia for advice with the ULI-NChIP–seq experiments. This work was supported by the National Natural Science Foundation of China (31325019, 31430056, 91319306, 31401266, 31501196, 31401247, 31501197, 31322031, 31371288 and 31571365), the Ministry of Science and Technology of China (2015CB964503, 2016YFA0100400 and 2015CB964800), the Science and Technology Commission of Shanghai Municipality (14CG16 and 15XD1503500), and the Major Program of Development Fund for Zhangjiang National Innovation Demonstration Zone (ZJ2014-ZD-002).
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Contributions
Y.G. and S.G. conceived and designed the experiments. X.L. performed the ChIP experiments. C.W. performed computational analysis. W.L. performed the micromanipulation of embryos. J.L., C.L., X.K., J.C., Y.Z., H.G. and H.W. assisted with the sample preparation. X.L., C.W., Y.Z. and Y.G. designed and performed the data analysis. X.L., C.W., Y.G. and S.G. wrote the manuscript.
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The authors declare no competing financial interests.
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Nature thanks R. Schultz and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
Extended Data Figure 1 Ultra-low-input ChIP–seq in ES cells and pre-implantation embryos.
a, Sample correlation for the ChIP–seq experiments using 200, 1,000 and bulk mES cells. Each point represents the normalized reads count of a RefSeq promoter for corresponding samples. Pearson’s correlation coefficients of the two experiments were calculated using all the RefSeq promoters. b, Genome browser view of the H3K4me3 and H3K27me3 RPM (reads per million reads) at the Fgf4 and Fbln2 gene regions for the mES cells in duplicate. The ChIP–seq RPM (reads per million mapped reads) was smoothened by the mean of five pixels using the UCSC genome browser. c, Correlation of the H3K4me3 samples for embryo developmental stages and cell lines. Each point represents the normalized reads count of a RefSeq promoter for the replicates. Pearson’s correlation coefficients of the two replicates were calculated using all the RefSeq promoters. d, Correlation of the H3K27me3 samples for embryo developmental stages and cell lines. Each point represents the normalized reads count of a RefSeq promoter for the replicates. Pearson’s correlation coefficients of the two replicates were calculated using all the RefSeq promoters. e, f, Genome browser view of the H3K4me3 RPM at the Fgf4 (e) and the H3K27me3 RPM at the Fbln2 and Gata6 (f) gene regions for embryos and cell lines. The ChIP–seq RPM was smoothened by the mean of five pixels using the UCSC genome browser.
Extended Data Figure 2 Features of the chromatin states in pre-implantation embryos.
a. Number of high-enrichment and low-enrichment H3K4me3-only regions during embryogenesis. The high-enrichment H3K4me3-only domains were defined as segments retained using a stringent ChromHMM threshold (fourfold over input file) and the low-enrichment H3K4me3-only domains were the additional segments when the stringent threshold was not used. b, Emission parameters of the ChromHMM segmentations. Colour indicates the enrichment of chromatin modifications in each specific segmentation. The whole genome was classified into: 1, non-marked regions; 2, H3K4me3-only regions; 3, H3K27me3-only regions; and 4, bivalent regions. c, Genome distribution of the different classes of the ChromHMM segmentations across the mouse developmental stages and cell lines. The analysed regions are labelled to the left of the heat map, and segmentations are labelled at the top of the heat map. Colour represents relative percentage scaled by row. d, RNA expression level and relative protein level of the H3K4me3 and H3K27me3 writers. RNA-seq data and mass spectrometric data on early mouse embryos (unpublished) were used in this analysis. The expression level was evaluated using a log-scaled FPKM, and the protein level was evaluated using the relative ratio over all the stages. In this figure, H3K4me3 writers included Rbbp5, Dpy30, Ash2l, Wdr5 and Setd1a. H3K27me3 writers included Jarid2, Ezh2, Eed, Suz12 and Rnf2.
Extended Data Figure 3 Dynamics of the histone-modification landscape in embryogenesis and during differentiation.
a, b, Left panel, alluvial plots showing the global dynamics of the bivalent (a) and non-marked (b) regions during early embryo development. Each line represents a 200-bp bin defined based on the ChromHMM categories. For each panel, the total regions were those classified as this state in at least one analysed stage. Right panel, the percentage of the analysed states (that is, bivalent or non-marked) in each stage compared to the total regions. The global dynamics (solid line) and chromosome X dynamics (dashed line) are plotted separately. c, Averaged distance to the nearest TSS of the stable and dynamic H3K4me3-only marked regions. The stable H3K4me3-only regions were defined as those regions that retained the H3K4me3 mark in the next stage, while the dynamic H3K4me3-only regions were defined as those regions that lost the modification in the next stage. d, Averaged domain length of the stable and dynamic H3K4me3-only marked regions. e, Averaged signal density of the stable and dynamic H3K4me3-only marked regions. The signal density was calculated as H3K4me3 ChIP–seq RPKM (reads per million reads per kb). f–i, Left panel, alluvial plots showing the global dynamics of H3K4me3-only (f), bivalent (g), H3K27me3-only (h) and non-marked (i) regions during the first cell lineage differentiation and cell line derivation. Each line represents a 200-bp bin defined according to the ChromHMM categories. For each state, the total regions were those classified as this state in at least one analysed stage. The morula stage was plotted in the middle, and the two differentiated lineages were plotted on either side. Right panel, the percentage of the analysed states in each stage compared to the total regions. The global dynamics (solid line) and chromosome X dynamics (dashed line) are plotted separately.
Extended Data Figure 4 Establishment and dynamics of H3K4me3 and H3K27me3 on promoters.
a, b, Heat map of the H3K4me3-marked (a) and H3K27me3-marked (b) transcript dynamic during early mouse embryo development and differentiation. The H3K4me3-marked transcripts were defined with an overlap of at least 200 bp with the ChromHMM H3K4me3 segmentations, including the H3K4me3-only and bivalent segmentations. H3K27me3-marked transcripts were defined as having an overlap of at least 200 bp with the ChromHMM H3K27me3 segmentations, including the H3K27me3-only and bivalent segmentations. The transcript clustering was based on the binary score; see the Methods section for the detailed clustering method. c, d, Correlation between the H3K4me3 (c) or H3K27me3 (d) signal intensity and local CpG ratio in all pre-implantation developmental stages and cell lines. ChIP–seq signal intensity was evaluated using a weighted ChIP–seq signal of ±2 kb around the TSS. Pearson correlation coefficients were calculated and are shown in the top right of each panel. e, f, Averaged H3K4me3 (e) and H3K27me3 (f) signal intensity on the HCPs, ICPs and LCPs throughout all pre-implantation developmental stages and cell lines. The definition of the three class promoters is described in the Methods.
Extended Data Figure 5 Distinct features of the establishment of H3K4me3 and H3K27me3 in embryogenesis and differentiation.
a, Profiles of the H3K27me3 signal density on the HCPs, ICPs and LCPs from MII to morula stage. The H3K27me3 signal density was calculated as H3K27me3 RPM with 50-bp resolution. b, Averaged local max CpG ratio of the new H3K27me3-marked transcripts. The newly established H3K27me3-marked transcripts were defined as transcripts belongs to non-marked state in the previous stage and marked by H3K27me3 in the present stage. c, Averaged H3K27me3 signals of the HCPs, ICPs and LCPs in the two-cell stage, classified by the methylation level. The published whole-genome bisulphite sequencing data was used in this analysis20. The methylated genes were defined as genes with an averaged promoter methylation level ≥0.6, and the unmethylated genes were defined as genes with an averaged promoter methylation level ≤0.25. d, e, Profiles of the H3K4me3 (d) and H3K27me3 (e) signal density on the HCPs, ICPs and LCPs in ICM, TE, ES cells and TSCs. The H3K4me3 and H3K27me3 signal density was calculated as H3K27me3 RPM with 50-bp resolution. f, g, Scatter plot of the signal intensities of H3K4me3 (f) and H3K27me3 (g) with the promoter regions and DNA methylation levels. Our reduced representation bisulphite sequencing (RRBS) data published in another project44 was used in this analysis. The red and blue fitting curves represent the ChIP–seq intensity and methylation level in the corresponding regions, respectively. The horizontal axis, from left to right, represents the transcripts ranked from high to low according to their promoter H3K4me3 or H3K27me3 intensities, and the y axis indicates the averaged methylation level of corresponding promoter Pearson correlations of the H3K4me3 intensities and DNA methylation levels were calculated and included in the panel f and Pearson correlations of the H3K27me3 intensities and DNA methylation levels in panel g. h, Correlation of H3K4me3 and H3K27me3 signal intensity throughout early embryo developmental stages and cell lines.
Extended Data Figure 6 Identification and regulation of H3K4me3 broad domains on promoters.
a, Bar plot showing the distribution of the H3K4me3 domain length at the promoter regions during mouse development. The two dashed lines represent the cut-offs between the plotted narrow, medium and broad domains. The percentages of transcripts classified into these three groups and control are listed in the top right of the panel. The representative genes Ndrg2, Ctcf and Ldb1, whose promoters were covered by narrow, medium and broad domains, respectively, are displayed. b, Distribution of the H3K4me3 domain length at the promoter regions in all pre-implantation developmental stages and cell lines. The dashed line represents the cut-off between the medium and broad domains. c, Heat map showing the H3K4me3 RPM on all the RefSeq transcripts of the four states of H3K4me3 domains for the morula, ICM and ES cell samples. Each row represents a promoter region with ±10 kb around a TSS. d, Genome browser view of H3K4me3 broad domains establishment at Nanog and Pou5f1 genes from the two-cell stage to the ICM stage. ChIP–seq RPM was smoothed by the mean of 5 pixels using the UCSC genome browser and visualized. e, Depletion of H3K4me3 demethylase inhibited the formation of blastocysts. The siRNA mixture of each predicated H3K4me3 demethylase was injected into MII oocytes separately or mixed together. The injected oocytes were then performed ICSI. The data are represented as the mean ± s.d. (n = 3). f, Knockdown of Kdm5b (si-Kdm5b) inhibited the formation of expanding and hatching of blastocysts. Injection of Kdm5b mRNA (oe-Kdm5b) as well as control RNA was also performed for comparison. The data are represented as the mean ± s.d. (n = 3). g, Heat map showing the breadth of H3K4me3 domains on promoters in control, oe-Kdm5b and si-Kdm5b samples. The H3K4me3 domains were classified into four groups: broad domains (dark red), medium domains (red), narrow domains (pink) and control domains (grey). The dashed lines represent the classification based on control samples. h, Box plot showing the averaged H3K4me3 domain breadth in different samples on promoter covered by broad (left), medium (middle) and control (right) H3K4me3 domains in control samples. i, Genome browser view of control, oe-Kdm5b and si-Kdm5b samples at Zfp85 gene locus. ChIP–seq RPM was smoothed by the mean of 5 pixels using the UCSC genome browser and visualized.
Extended Data Figure 7 H3K4me3 breadth is highly associated with transcription activity and cell identity.
a, Box plot showing the averaged expression level of the broad, medium, narrow and control H3K4me3 domains in all pre-implantation developmental stages and cell lines. The number of the transcript is listed with the group name. b, Heat map showing the gain and loss of the broad domains during the transition from the morula stage to the ICM and TE. The transcripts were clustered using a k-means function into seven groups. c, Averaged expression level of the transcript in the morula, ICM and TE samples for the clusters in b. The expression level was evaluated using the averaged log2 FPKM. d, Tendencies of the four types of H3K4me3 domains in the next stage during stable cell line derivation. Each panel represents a specific type of H3K4me3 domain. Each bar represents the types and percentages of the transcripts in the next stage. e, Broad domains inherited from the TE to TSCs. A gene ontology analysis was performed on the overlapped genes from the TE to TSCs, and representative genes were labelled. The P value was calculated using multiple Student’s t-tests. f, g, Bar plot of the H3K4me3 domain length at the Id2 (f) and Elf5 (g) promoter regions (left panel) and the corresponding expression level (right panel) in embryo and cell lines. The dashed line represents the cut-off for broad domains.
Extended Data Figure 8 Dynamics of the bivalent promoters in embryogenesis and differentiation.
a, Heat map showing the dynamics of the bivalent genes during early mouse embryo development. Each row represents one RefSeq transcript. Red indicates a transcript that is only marked by H3K4me3, blue represents a transcript that is marked by H3K27me3, and dark green indicates a transcript in a bivalent state. b, Number of bivalent transcripts in MII oocytes, pre-implantation embryos and cell lines. c, Tendencies of the H3K4me3-only promoters, H3K27me3-only promoters, bivalent promoters and non-marked promoters in the next stage during early mouse embryo development. Each bar represents the type and percentage of the transcript in the next stage. d, Bar plot showing the possibility of the H3K27me3-marked condition for transcripts with a broad, medium or narrow H3K4me3 domains. Red represents transcripts without the H3K27me3 mark, and dark green represents transcripts with the H3K27me3 mark, indicating a bivalent state.
Extended Data Figure 9 Inheritance of the ICM-bivalent promoters in ES cell derivation.
a, Tendencies of the H3K4me3-only promoters, H3K27me3-only promoters, bivalent promoters and non-marked promoters in the next stage during cell line derivation. Each bar represents the type and percentage of the transcript in the next stage. The ICM-bivalent genes, which are covered only by H3K4me3 signals, or only by H3K27me3 signals or maintain bivalent states in the ES cell samples, were defined as ES-cell-activated, ES-cell-repressed and ES-cell-maintained, respectively. b, c, Genome browser view of the H3K4me3 and H3K27me3 density at activated bivalent gene Jade1 (b) and repressed bivalent gene Cxcl10 (c) in ES cell and ICM samples. The ChIP–seq RPM was smoothed by the mean of 5 pixels using the UCSC genome browser. d, H3K4me3 and H3K27me3 signal intensities of the three types of ICM-bivalent transcripts, ES-cell-activated, ES-cell-repressed and ES-cell-maintained, defined in a. e, Expression level during early mouse embryo development of the three types of ICM bivalent transcripts, ES-cell-activated, ES-cell-repressed and ES-cell-maintained, defined in a.
Supplementary information
Supplementary Data
This file contains Supplementary Table 1, a summary of chromatin state of TSS in MII oocytes, pre-implantation embryos, ESCs and TSCs. (XLSX 1751 kb)
Supplementary Data
This file contains Supplementary Table 2, functional annotation of transcripts that maintain H3K27me3 from MII Oocyte to embryo development. (XLSX 17 kb)
Supplementary Data
This file contains Supplementary Table 3, a summary of genes covered by broad H3K4me3 in MII oocytes, pre-implantation embryos, ESCs and TSCs. (XLSX 1834 kb)
Supplementary Data
This file contains Supplementary Table 4, a list of siRNA used in the experiment. (PDF 101 kb)
Supplementary Data
This file contains Supplementary Table 5, a summary of quality information of ChIP data and correlation of replicates in each stage. (XLSX 23 kb)
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Liu, X., Wang, C., Liu, W. et al. Distinct features of H3K4me3 and H3K27me3 chromatin domains in pre-implantation embryos. Nature 537, 558–562 (2016). https://doi.org/10.1038/nature19362
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DOI: https://doi.org/10.1038/nature19362
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