Abstract
Parental DNA methylation and histone modifications undergo distinct global reprogramming in mammalian pre-implantation embryos, but the landscape of epigenetic crosstalk and its effects on embryogenesis are largely unknown. Here we comprehensively analyse the association between DNA methylation and H3K9me3 reprogramming in mouse pre-implantation embryos and reveal that CpG-rich genomic loci with high H3K9me3 signal and DNA methylation level (CHM) are hotspots of DNA methylation maintenance during pre-implantation embryogenesis. We further profile the allele-specific epigenetic map with unprecedented resolution in gynogenetic and androgenetic embryos, respectively, and identify 1,279 allele-specific CHMs, including 19 known imprinting control regions (ICRs). Our study suggests that 22 ICR-like regions (ICRLRs) may regulate allele-specific transcription similarly to known ICRs, and five of them are confirmed to be important for mouse embryo development. Taken together, our study reveals the widespread existence of allele-specific CHMs and largely extends the scope of allele-specific regulation in mammalian pre-implantation embryos.
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Data availability
All the ChIP-seq, WGBS and RNA-seq datasets generated in this study are summarized in Supplementary Table 2 and have been deposited in the Genome Sequence Archive (https://bigd.big.ac.cn/gsa/) under accession no. CRA004123. All public ChIP-seq, DNase-seq, ATAC-seq, WGBS and RNA-seq datasets used in this study are summarized in Supplementary Table 1. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.
Code availability
The computational pipeline for CHM calling and scoring for allele-specific regulatory potentials (PCAR) is available at https://github.com/TongjiZhanglab/PCAR.
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Acknowledgements
We thank C. Zhao and W. Wang for assistance with data analysis. This work was supported by the National Key Research and Development Program of China (2017YFA0102600 (Y.Z.) and 2016YFA0100400 (S.G.)), the National Natural Science Foundation of China (32030022 (Y.Z.), 31970642 (Y.Z.), 31721003 (S.G.), 31820103009 (S.G.), 31871448 (W.L.) and 31801059 (C.W.)) and the Peak Disciplines (Type IV) of Institutions of Higher Learning in Shanghai (Y.Z. and S.G.).
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S.G. and Y.Z. conceived and designed the research. H.Y., Z.Y., C.W. and Y.Z. designed and performed computational analysis. W.L., D.B., Y.L. and Y.S. performed experiments. H.Y., W.L., Z.Y. and Y.Z. wrote the manuscript.
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Extended data
Extended Data Fig. 1 Features of stable CHMs in mouse pre-implantation embryos.
a, Line chart of the percentage of 1-kb bins escaping DNA demethylation at each given stage (that is, DNA methylation level ≥ 0.5 at the given stage and all previous stages). b, c, Boxplots comparing DNA methylation level (b) and H3K9me3 signals (c) at stable CHMs (n = 6,655), stage-specific CHMs (n = 16,461) and highly methylated non-CHM CpG islands (CGIs) (n = 1,632). Significance between stable CHMs and highly methylated non-CHM CGIs was evaluated by two-sided Wilcoxon rank sum test, *** indicates a p-value < 0.001 (p-value for DNA methylation level at 2-cell: 2.3×10−243; p-value for DNA methylation level at other stages and for H3K9me3 signals at all stages: < 2.2×10−308). In the boxplots, the centre lines mark the median, the box limits indicate the 25th and 75th percentiles, and the whiskers extend to 1.5 × the interquartile range from the 25th and 75th percentiles. d, Pie plot showing status of stable CHMs in gametes. e, f, Scatter plots showing DNA methylation level (e) and H3K9me3 signals (f) of stable CHMs in gametes. g, Boxplots comparing the expression levels between potential target genes of CHMs (n = 1,129) and other genes (n = 34,538). FPKM, fragments per kilobase of exon per million mapped fragments. Potential target genes were defined as the nearest genes within 300 kb of CHMs located in promoter and potential enhancers. CHMs located in PADs were removed. Significance between the groups was evaluated by two-sided Wilcoxon rank sum test, *** indicates a p-value < 0.001 and n.s. indicates not significant (p-value at 2-cell, 8-cell, Morula, ICM: 0.33, 4.9×10−19, 2.6×10−25 and 2.8×10−12). The meaning of boxplots is identical to that in b, c. h, Gene Ontology analysis of potential target genes of CHMs. The p-values were calculated based on a one-sided Fisher’s exact test.
Extended Data Fig. 2 CHMs are hotspots of DNA methylation maintenance in GG and AG pre-implantation embryos.
a, Line charts showing DNA methylation level from SNP-trackable data (C57BL: maternal, DBA: paternal) around hypermethylated CpG-rich regions in GG and AG embryos. b, Line charts showing H3K9me3 signals from SNP-trackable data around H3K9me3-marked CpG-rich regions in GG and AG embryos. c, d, Scatter plots showing the Pearson’s correlation of allelic DNA methylation level (c) and H3K9me3 signals (d) between GG / AG embryos and strain-hybrid embryos in SNP-trackable CpG-rich 1 kb bins, covering ≥ 100 SNP-trackable reads of corresponding WGBS data. e, f, i, j, Bar plot showing the percentage of highly methylated CpGs (DNA methylation level ≥ 0.5) at 2-cell stage maintaining high methylation status during GG (e) and AG (f) pre-implantation embryogenesis and in maternal (C57BL) (i) and paternal (DBA) (j) allele. g, h, k, l, Boxplots comparing DNA methylation level (left) and H3K9me3 signals (right) at stable CHMs (n = 8,384 / 6,433), stage-specific CHMs (n = 15,171 / 19,082) and highly methylated non-CHM CGIs (n = 731 / 703) in GG (g) / AG (h) embryos, or at SNP-trackable (that is, covering ≥ 20 SNP-trackable reads of corresponding WGBS data) stable CHMs (n = 2,072 / 1,499), stage-specific CHMs (n = 3,474 / 4,300) and highly methylated non-CHM CGIs (n = 94 / 96) in maternal (C57BL) (k) and paternal (DBA) (l) allele. Significance between stable CHMs and highly methylated non-CHM CGIs was evaluated by two-sided Wilcoxon rank sum test, ***: p-value < 0.001, **: p-value < 0.01, *: p-value < 0.05. p-values in each panel (from left to right): 4.5×10−44, 4.8×10−5, 3.3×10−69, < 2.2×10−308, < 2.2×10−308, < 2.2×10−308 (g); 4.3×10−126, 3.5×10−239, < 2.2×10−308, < 2.2×10−308, < 2.2×10−308, 1.0 × 10−255 (h); 0.012, 7.2×10−19, 5.6×10−12, 2.8×10−35 (k); 2.3×10−14, 6.8×10−22, 4.5×10−3, 1.4×10−9 (l). The meaning of boxplots is identical to that in Extended Data Fig. 1.
Extended Data Fig. 3 Status of allele-specific CHMs in gametes and E6.5 embryos.
a, Pie plots showing status of allele-specific CHMs in public strain-hybrid 2-cell (upper) and ICM (bottom) embryos. SNP-trackable allele-specific CHMs are required to cover ≥ 100 SNP-trackable reads of corresponding WGBS data. b, The UCSC Genome Browser view showing DNA methylation level and H3K9me3 signals in pre-implantation GG and AG embryos surrounding known gamete ICR Zrsr1/Commd (highlighted by blue), which is not identified as an allele-specific CHM. CHMs in GG and AG embryos are indicated by orange and green rectangles respectively. c, The UCSC Genome Browser view showing status of allele-specific CHMs showed consistent parent-of-origin DNA methylation and H3K9me3 asymmetry in gametes. The genomic location of the mCHM and pCHM are indicated by orange and green rectangle respectively. d, Scatter plots with distribution information showing DNA methylation level and H3K9me3 signals of mCHMs (upper) and pCHMs (bottom) in gametes according to their presence and absence in corresponding gamete. ICRLRs selected for validation were labelled and highlighted as orange and green dots respectively. e, The UCSC Genome Browser view showing status of allele-specific CHMs not identified as CHMs in corresponding gametes. The genomic location of mCHM and pCHM are indicated by orange and green rectangle respectively. f, The UCSC Genome Browser view showing status of allele-specific CHMs in E6.5 embryos. The genomic location of mCHM and pCHM are indicated by orange and green rectangle respectively.
Extended Data Fig. 4 DNA methylation asymmetry at allele-specific CHMs influenced by H3K9me3 depletion.
a, Representative immunofluorescence staining for H3K9me3 (green), DAPI (grey) and merged images in control and H3K9me3-depleted embryos at the zygote stage. Data is reproducible in three independent experiments. Scale bar, 20 μm. b, The UCSC Genome Browser view of a representative mCHM showing the decreased DNA methylation level in H3K9me3-depleted embryos at the morula stage. mCHM is indicated with orange rectangles. DNA methylation change after H3K9me3-depleted represents smoothed DNA methylation level change (H3K9me3-depleted - WT) in 200-bp windows.
Extended Data Fig. 5 Allele-specific expressed genes and transposable elements surrounding ICRs and ICRLRs.
a, b, Heatmap showing expression pattern of allele-specific expressed gene (a) and transposable element (b) surrounding ICRs and ICRLRs within 300 kb in pre-implantation embryos. Maternal-specific expressed genes (a) and transposable element (b) are indicated with orange and paternal-specific expressed genes (a) and transposable element (b) are indicated with green. ICRs are highlighted in red and transient gDMRs reported are highlighted in blue. Known imprinted genes are marked with asterisk. c, d, Pie plots showing allele-specific status of monoallelic expressed genes and transposable elements nearby ICRs (c) and ICRLRs (d) in hybrid pre-implantation embryos. SNP-trackable monoallelic expressed genes and transposable elements are required to cover ≥ 100 SNP-trackable reads of corresponding RNA-seq data. ‘Consistent’ indicates SNP-trackable monoallelic expressed genes and transposable elements showing similar allele preference in hybrid embryos. ‘Inconsistent’ indicates SNP-trackable monoallelic expressed genes and transposable elements showing no or contrast allele preference in hybrid embryos.
Extended Data Fig. 6 Functions of 5 identified ICRLRs in the development of early embryos.
a, UCSC Genome Browser view of ICRLRs selected for validation. The maternal and paternal ICRLRs are indicated by orange and green rectangles respectively. b, Box plots showing the expression levels of genes (n = 6) harbouring validated maternal ICRLRs in pre-implantation GG/AG embryos. The meaning of boxplots is identical to that in Extended Data Fig. 1. c, Representative images of blastocyst-stage embryos produced from control, mCHM_245-KO, mCHM_177-KO, mCHM_328-KO, mCHM_4-KO and pCHM_824-KO embryos. Data is reproducible in two independent experiments. Scale bar, 150 μm. d, Schematic diagram of generating ICRLR-KO GG and AG embryos. e, DNA sequencing showing effective paternal-specific (left) and maternal-specific (right) mCHM_177-KO. f, Bar plot showing the ratios of decidua at the E9.5 stage in the control, maternal-specific and paternal-specific mCHM_177-KO groups. Control embryos (two biologically independent experiments, sample size: 48, 46), maternal-specific (three biologically independent experiments, sample size: 56, 36, 45) and paternal-specific (three biologically independent experiments, sample size: 68, 40, 31) mCHM_177-KO are indicated in grey, orange and green respectively. Significance between groups was evaluated by two-sided Welch’s t-test, n.s. indicates not significant (p-value between maternal-specific and paternal-specific mCHM_177-KO: 0.36). g, DNA sequencing showing effective maternal-specific (upper) and paternal-specific (bottom) Brd2-KO. h, Bar plot for the ratios of blastocyst of control and BC051142-KO embryos. Control (two biologically independent experiments, sample size: 36, 30) and BC051142-KO embryos (two biologically independent experiments, sample size: 33, 36) are indicated in grep and black. Sample size refers to the number of 2-cell stage embryos. i, Genomic schematic diagram showing expression details of genes expressed in at least 1 stage in GG and AG pre-implantation embryos within 300 kb of mCHM_177. The gene Brd2 was indicated as green. Allele-specific score was calculated by PCAR.
Supplementary information
Supplementary Tables
Supplementary Table 1 Summary of the public ChIP-seq, DNase-seq, ATAC-seq, WGBS and RNA-seq datasets used in this study. Supplementary Table 2 Summary of the ChIP-seq, WGBS and RNA-seq datasets generated in this study. Supplementary Table 3 Summary of the allele-specific CHMs identified in pre-implantation embryos. The scores were calculated based on features of known ICRs. Allele-specific CHMs with equal or higher scores than any known ICR were regarded as ICRLRs. ICRLRs with functional validation in this study were marked with green. Supplementary Table 4 Sequences for siRNAs, PCR primers and sgRNAs; mutants information
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Yang, H., Bai, D., Li, Y. et al. Allele-specific H3K9me3 and DNA methylation co-marked CpG-rich regions serve as potential imprinting control regions in pre-implantation embryo. Nat Cell Biol 24, 783–792 (2022). https://doi.org/10.1038/s41556-022-00900-4
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DOI: https://doi.org/10.1038/s41556-022-00900-4
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