Allelic reprogramming of the histone modification h3k4me3 in early mammalian development

ABSTRACT Histone modifications are fundamental epigenetic regulators that control many crucial cellular processes1. However, whether these marks can be passed on from mammalian gametes to

the next generation is a long-standing question that remains unanswered. Here, by developing a highly sensitive approach, STAR ChIP–seq, we provide a panoramic view of the landscape of

H3K4me3, a histone hallmark for transcription initiation2, from developing gametes to post-implantation embryos. We find that upon fertilization, extensive reprogramming occurs on the

paternal genome, as H3K4me3 peaks are depleted in zygotes but are readily observed after major zygotic genome activation at the late two-cell stage. On the maternal genome, we unexpectedly

find a non-canonical form of H3K4me3 (ncH3K4me3) in full-grown and mature oocytes, which exists as broad peaks at promoters and a large number of distal loci. Such broad H3K4me3 peaks are in

contrast to the typical sharp H3K4me3 peaks restricted to CpG-rich regions of promoters. Notably, ncH3K4me3 in oocytes overlaps almost exclusively with partially methylated DNA domains. It

is then inherited in pre-implantation embryos, before being erased in the late two-cell embryos, when canonical H3K4me3 starts to be established. The removal of ncH3K4me3 requires zygotic

transcription but is independent of DNA replication-mediated passive dilution. Finally, downregulation of H3K4me3 in full-grown oocytes by overexpression of the H3K4me3 demethylase KDM5B is

associated with defects in genome silencing. Taken together, these data unveil inheritance and highly dynamic reprogramming of the epigenome in early mammalian development. Access through

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BEING VIEWED BY OTHERS HISTONE H3K36ME2 AND H3K36ME3 FORM A CHROMATIN PLATFORM ESSENTIAL FOR DNMT3A-DEPENDENT DNA METHYLATION IN MOUSE OOCYTES Article Open access 03 August 2022

REPROGRAMMING OF THE HISTONE H3.3 LANDSCAPE IN THE EARLY MOUSE EMBRYO Article 09 November 2020 H2AK119UB1 GUIDES MATERNAL INHERITANCE AND ZYGOTIC DEPOSITION OF H3K27ME3 IN MOUSE EMBRYOS

Article 05 April 2021 ACCESSION CODES PRIMARY ACCESSIONS GENE EXPRESSION OMNIBUS * GSE71434 DATA DEPOSITS All data have been deposited to GEO with the accession number GSE71434. REFERENCES *

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CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS We appreciate help from members of the Xie laboratory for comments during preparation of the manuscript. We

thank J. Zhao and G. Qin for helping with pronuclei isolation. We are grateful to the animal core facility, the sequencing core facility, and biocomputing facility at Tsinghua University.

This work is supported by the funding provided by the National Basic Research Program of China 2016YFC0900301 (W.X.), 2015CB856201 (W.X.), 2012CB966701 (J.N.), the National Natural Science

Foundation of China 31422031 (W.X.), 91519326 (W.X.), 31171381 (J.N.), the Beijing Natural Science Foundation grant 5152014 (J.N.), Tsinghua University Initiative Scientific Research Program

20161080043 (W.X.), the funding from the THU-PKU Center for Life Sciences (W.X., J.N.), and the Youth Thousand Scholar Program of China (W.X.). AUTHOR INFORMATION Author notes * Bingjie

Zhang, Hui Zheng, Bo Huang and Wenzhi Li: These authors contributed equally to this work. AUTHORS AND AFFILIATIONS * Center for Stem Cell Biology and Regenerative Medicine, MOE Key

Laboratory of Bioinformatics, THU-PKU Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, 100084, China Bingjie Zhang, Hui Zheng, Bo Huang, Yunlong Xiang, Yu

Zhang, Qianhua Xu, Yuanyuan Li, Qiujun Wang, Jing Ma, Qiangzong Yin & Wei Xie * PKU-THU Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University,

Beijing, 100871, China Bo Huang * Center for Stem Cell Biology and Regenerative Medicine, School of Medicine, Tsinghua University, Beijing, 100084, China Wenzhi Li, Jia Ming, Bo Chen, 

Kehkooi Kee & Jie Na * Singapore Institute for Clinical Sciences, Agency for Science, Technology and Research (A*STAR), Singapore, 117609, Singapore Xu Peng & Feng Xu * State Key

Laboratory of Biomembrane and Membrane Engineering, School of Life Sciences, Tsinghua University, Beijing, 100084, China Xiaotong Wu & Anming Meng * Clinical and Translational Research

Center of Shanghai First Maternity and Infant Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, 200092, China Wenqiang Liu, Xiaochen Kou, Yanhong Zhao, Wenteng

He, Chong Li & Shaorong Gao * Institute of Molecular and Cell Biology, A*STAR, Singapore, 138673, Singapore Feng Xu Authors * Bingjie Zhang View author publications You can also search

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author inPubMed Google Scholar CONTRIBUTIONS B.Z. developed STAR ChIP–seq and conducted ChIP–seq experiments; B.H., Y.X. and J. Ma collected mouse oocytes and early embryos. B.H. performed

RNA-seq; H.Z. performed data analysis; W.L., J. Ming and B.Z. conducted KDM5B experiment. W.L., X.K., Y. Zhao, W.H., C.L. and B.H. collected mouse pronuclei. X.P., B.H. and F.X. advised the

development of STAR ChIP–seq. Y. Zhang and Q.Y. performed oocyte DNA methylome profiling. Y.L. and Q.W. conducted high-throughput sequencing; X.W. collected zebrafish oocytes; K.K., A.M.,

S.G., J.N. and W.X. supervised the project or related experiments. H.Z. and W.X. wrote the manuscript. CORRESPONDING AUTHORS Correspondence to Jie Na or Wei Xie. ETHICS DECLARATIONS

COMPETING INTERESTS B.Z. and W.X. are co-inventors on a filed patent application for STAR ChIP–seq. ADDITIONAL INFORMATION REVIEWER INFORMATION _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 VALIDATION OF STAR CHIP–SEQ IN MES CELLS. A, A brief

schematic of STAR ChIP–seq. Based on a previously described ChIP method28, micrococcal nuclease (MNase) is used to fragment native chromatin. Nucleosomes with certain histone modifications

are immunoprecipitated using specific antibodies. Proteinase K is added for protein digestion and is heat-inactivated. Shrimp alkaline phosphatase (rSAP) is directly added to the reaction

for 3′ DNA end repair, which is required for the poly-C addition in the subsequent TELP library preparation. After heat-inactivation of rSAP, the resulting DNA is subjected to the TELP

library preparation as previously described3 without purification. Briefly, poly-C tailing is conducted on denatured single-strand DNA using dCTP and terminal deoxynucleotidyl transferase

(TDT). Biotin-labelled anchor primer (indicated by ‘B’) containing poly-G is used for second strand DNA extension. The products are captured by magnetic streptavidin beads. After an adaptor

ligation to the opposite end of poly-C, the double-stranded DNA is amplified by primers (P1 and P2) containing Illumina adaptor sequences. The resulting library is ready to be sequenced. B,

A snapshot of the UCSC browser view shows enrichment of STAR ChIP–seq for histone modifications H3K4me3, H3K27ac and H3K27me3 using various numbers of mES cells. Data sets from mouse ENCODE

are also shown for comparison35. C, The Pearson correlation coefficients showing the comparison between STAR ChIP–seq of H3K4me3 using various numbers of mES cells and conventional ChIP–seq

of H3K4me3 from either this study using 1 × 106 mES cells or data from ENCODE35. D, A bar chart showing the percentages of conventional mESC H3K4me3 total/promoter peaks recaptured by STAR

ChIP using various numbers of mES cells. E, Box plots showing the H3K4me3 enrichment determined by conventional ChIP–seq at peaks that are either recaptured or missed by STAR ChIP–seq.

EXTENDED DATA FIGURE 2 VALIDATION OF H3K4ME3 STAR CHIP–SEQ AND RNA-SEQ IN MOUSE GAMETES AND EARLY EMBRYOS. A, Representative bright-field microscopic images showing the pre-implantation

embryos used for STAR ChIP–seq with time points of sample collection indicated. B, The Spearman correlation between the replicates (_n_ = 2) of RNA-seq samples and between RNA-seq in this

study and Deng _et al_.36 at stages when available. C, The Pearson correlation between the replicates (_n_ = 2) of H3K4me3 for mouse pre-implantation embryos at each stage. D, Scatter plots

showing the H3K4me3 enrichment levels (2-kb window) between replicates across the genome for mouse pre-implantation embryos at each stage. E, Scatter plot showing the H3K4me3 enrichment

levels (2-kb window) in MII oocytes between data sets generated using an in-house antibody or a commercial antibody (Millipore 04-745). F, A snapshot of the UCSC browser view shows H3K4me3

STAR ChIP–seq results using two different H3K4me3 antibodies (in-house or commercial/Millipore 04-745) in mES cells and MII oocytes. Annotated genes and repetitive elements are also shown.

G, Venn diagram showing the overlap between mES cell H3K4me3 (conventional ChIP–seq) promoter peaks and ICM H3K4me3 promoter peaks. H, Snapshots of the UCSC browser views showing the H3K4me3

and expression signals near _Foxa1_ (left), _Atp1b1_ (middle) and _Tbx3_ (right). EXTENDED DATA FIGURE 3 CHARACTERIZATION OF PROMOTER AND DISTAL H3K4ME3 IN MOUSE GAMETES AND EARLY EMBRYOS.

A, Scatter plots showing the relationship between the expression levels of all genes and their promoter H3K4me3 enrichment (_Z_-score-normalized) for each development stage. Mouse ES cell

data from ENCODE are used as control. Spearman correlation coefficients are also shown. B, Moving average plots (window = 100 genes, moving step = 1 gene) showing the relationship between

the expression levels of non-maternal genes and their promoter H3K4me3 enrichment (_Z_-score-normalized) for each stage after major ZGA. C, The genomic distributions of H3K4me3 peaks are

shown for early embryos and mES cells. TSS, transcription start site (±2.5 kb); TES, transcription end site (±2.5 kb). D, A bar chart showing the percentages of all mappable reads that can

be aligned to RepBase for mouse embryos of various stages. The error bars denote the standard deviations of two biological replicates of RNA-seq for each stage. E, Enrichment of repeats (log

ratio of observed/random) in H3K4me3 promoter and distal peaks compared to that in random peaks at each stage. F, The enrichment (log ratio of observed/random) of top repeat subfamily

members in distal H3K4me3 peaks in mouse embryos of various stages and mES cells. G, The snapshots of distal H3K4me3 signals near various types of repeats. EXTENDED DATA FIGURE 4

REPROGRAMMING OF H3K4ME3 ON THE PATERNAL GENOME. A, A snapshot of UCSC genome browser shows the enrichment of H3K4me3 in sperm from a published data set10 and this study. B, Snapshots

showing the allelic H3K4me3 signals and RNA-seq signals near the imprinted loci _Snrpn_ and _Impact_. Please note the maternal RNAs in zygotes, early two-cell and late two-cell embryos are

presumably inherited from oocytes. The paternal zygotic transcripts appear from the late two-cell stage onward for both genes. C, Bar chart showing the numbers of H3K4me3 peaks identified in

sperm (in regions covered by SNPs) and the paternal alleles of early embryos. D, Hierarchical clustering of sperm, early embryos and somatic cortex based on the paternal allele H3K4me3

enrichment. For sperm H3K4me3 data, only regions covered by SNPs were included for analysis. E, A bar chart showing the percentages of raw reads assigned to the maternal or paternal allele

for each stage. F, Scatter plots showing the H3K4me3 enrichment levels between SNP-assigned parental alleles from zygotes and pronuclei for both the paternal (left) and maternal genomes

(right). EXTENDED DATA FIGURE 5 NON-CANONICAL H3K4ME3 (NCH3K4ME3) IN OOCYTES AND EARLY EMBRYOS A, A snapshot of the UCSC browser view shows the enrichment of H3K4me3 in MII oocytes, isolated

maternal pronuclei, PN5 zygotes (maternal allele), and histone H3 (maternal allele) in PN5 zygotes. B, Scatter plots showing the comparisons between the enrichment of histone H3 (maternal)

in PN5 zygotes and H3K4me3 from either MII oocytes (left) or the maternal allele of PN5 zygotes (right). C, Hierarchical clustering of early embryo stages (maternal) based on global H3K4me3

enrichment. D, A bar chart showing the expression levels (log2[FPKM + 1]) of several marker genes for granulosa cells and oocytes, as well as a house-keeping gene (_Actb_), in oocyte samples

collected at different stages. ICMs are included as a non-oocyte control. E, Heat maps showing the expression (FPKM) of all genes and the H3K4me3 enrichment (normalized RPKM) at their

promoters (TSS ± 2.5 kb) for oocytes and early embryos. F, Hierarchical clustering of stages of oocytes and early embryos based on global H3K4me3 enrichment. G, A snapshot of UCSC browser

shows the H3K4me3 enrichment in oocyte of various stages as well as DNA methylation in MII oocyte. A PMD with ncH3K4me3 enrichment is shaded. EXTENDED DATA FIGURE 6 NON-CANONICAL DISTAL

H3K4ME3 IN OOCYTES CORRELATES WITH DNA HYPOMETHYLATION. A, Box plots showing the CpG density of H3K4me3 promoter and distal peaks in early embryos, mES cells, and the cortex. B, Pie charts

showing the percentage of the genome covered by PMDs (left) and the percentage of distal H3K4me3 marked regions covered by PMDs (right) in MII oocytes. C, A global view of H3K4me3 promoter

and distal peak density of chr12 in MII oocytes (top). Gene densities and PMD densities are also shown (bottom). Three gene desert regions are shaded. D, DNA methylation levels around active

genes are shown for those in embryonic day 16.5 (E16.5) female PGCs37, day 14 oocytes, and MII oocytes14. EXTENDED DATA FIGURE 7 THE TRANSITION FROM NCH3K4ME3 TO CANONICAL H3K4ME3 REQUIRES

ZYGOTIC TRANSCRIPTION BUT NOT DNA REPLICATION. A, Box plots of gene expression levels (log2[FPKM + 1]) show the effects of α-amanitin and aphidicolin on genes activated during ZGA (left).

Genes expressed at all stages (middle) and genes repressed at all stages (right) are also shown. B, Hierarchical clustering of two alleles of early embryos including those treated with

α-amanitin or aphidicolin based on the global H3K4me3 enrichment. C, A snapshot of the UCSC browser view shows the global view of H3K4me3 enrichment for maternal and paternal alleles in

various types of embryos including those treated with α-amanitin or aphidicolin. D, Bar charts showing normalized H3K4me3 and BrUTP fluorescence intensity of individual surrounded nucleolus

(SN)-stage oocytes. Each pair of bars for H3K4me3 and BrUTP represents an individual oocyte. Data from three independent experiments were pooled. GV, germinal vesicle. EXTENDED DATA FIGURE 8

NCH3K4ME3 IS NOT FOUND IN ZEBRAFISH. A, A snapshot of the UCSC genome browser shows the global view of H3K4me3 enrichment in oocytes (this study), the dome stage embryos38, 48 hours post

fertilization (hpf) embryos39 and DNA methylation level40 for various zebrafish developmental stages. B, The genomic distributions are shown for the H3K4me3 peaks in zebrafish oocytes,

embryos at the dome stage38 and 48 hpf embryos39. C, Box plots showing the CpG densities of H3K4me3 promoter peaks in zebrafish oocytes, embryos at the dome stage38 and embryos at 48 hpf39.

A random set of peaks that match the lengths of H3K4me3 peaks on the same chromosomes were used as a control in the analysis. D, Violin plots showing the distributions of DNA methylation

levels from gametes to somatic tissues in zebrafish (left panel) or mice (right panel). E, Box plot showing the lengths of distal PMDs in zebrafish and mouse oocytes, sperms, and somatic

tissues. F, Bar chart shows the percentages of the genome covered by distal PMDs in oocytes, sperms and somatic tissues in zebrafish or mouse. RELATED AUDIO REPORTER KERRI SMITH FINDS OUT

HOW THE CONCERT OF OUR GENES TAKES US FROM A SINGLE CELL TO… US. SUPPLEMENTARY INFORMATION SUPPLEMENTARY DATA This file contains Supplementary Table 1. (XLSX 39 kb) SUPPLEMENTARY DATA This

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al._ Allelic reprogramming of the histone modification H3K4me3 in early mammalian development. _Nature_ 537, 553–557 (2016). https://doi.org/10.1038/nature19361 Download citation * Received:

03 August 2015 * Accepted: 15 August 2016 * Published: 14 September 2016 * Issue Date: 22 September 2016 * DOI: https://doi.org/10.1038/nature19361 SHARE THIS ARTICLE Anyone you share the

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