Epigenetic reprogramming of the zygote involves dynamic incorporation of histone variant H3.3. However, the genome-wide distribution and dynamics of H3.3 during early development remain unknown. Here, we delineate the H3.3 landscapes in mouse oocytes and early embryos. We unexpectedly identify a non-canonical H3.3 pattern in mature oocytes and zygotes, in which local enrichment of H3.3 at active chromatin is suppressed and H3.3 is relatively evenly distributed across the genome. Interestingly, although the non-canonical H3.3 pattern forms gradually during oogenesis, it quickly switches to a canonical pattern at the two-cell stage in a transcription-independent and replication-dependent manner. We find that incorporation of H3.1/H3.2 mediated by chromatin assembly factor CAF-1 is a key process for the de novo establishment of the canonical pattern. Our data suggest that the presence of the non-canonical pattern and its timely transition toward a canonical pattern support the developmental program of early embryos.
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We thank H. Sugishita and A. Inoue (RIKEN) for sharing recombinant pAG-MN protein and a CUT&RUN protocol, T. Wakayama (Yamanashi University) for sharing B6;129 F1 ESCs and K. Hayashi (Kyushu University) for sharing a confocal microscope. We also thank the members of our laboratory and common research facilities of the Medical Institute of Bioregulation, Kyushu University, for technical assistance. This work was supported by grants from a MEXT Grant-in-Aid for Scientific Research on Innovative Areas (JP19H05756 and JP19H05758; T.I. and A.O.), the Kato Memorial Bioscience Foundation (T.I.), a JSPS Grant-in-Aid for Scientific Research (B) (JP16H04687; K.I.) and a JSPS Grant-in-Aid for Specially Promoted Research (JP18H05214; H.S.)
The authors declare no competing interests.
Peer review information Peer reviewer reports are available. Beth Moorefield was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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a, Western blotting showing the specificity of antibodies. Anti-H3.1/H3.2, anti-H3.3 and anti-pan-H3 antibodies were used to label recombinant H3.1 or H3.3. Results from two biological replicates are shown. Uncropped blot images are shown in the Source Data. b, Genome browser snapshots of the H3.3 ULI-NChIP-seq data. The H3.3 UNI-NChIP-seq data generated from 300 mESCs (this study) with prior H3.3 ChIP-seq data (GSE59189)4 shown for comparison. Enrichment is shown by log2 ratios between H3.3 ULI-NChIP and input. The midline corresponds to log2 ratio = 0. Higher and lower enrichment over input is indicated in magenta and blue, respectively. c, Hierarchical clustering and correlation analysis of H3.3 ULI-NChIP-seq data. Correlation between the data was analyzed by considering H3.3 enrichment in genome-wide 10-kb bins. Heatmaps show Spearman’s correlation coefficients between the samples. d, Plots showing average H3.3 enrichment at genic regions of the indicated samples. e, Plots showing CpG methylation level around genic regions. Genes were categorized by indicated CpG density at promoters as well as their expression levels. DNA methylation data was obtained from GSE63417 (zygote) and GSE56697 (2-cell and 4-cell). f, Plots showing average H3.3 enrichment at distal DNase I-hypersensitive sites. DNase I-hypersensitive sites distal to genic regions (top) were identified using previously published data (GSE76642)23. Bottom, H3.3 enrichment at the distal DNase I-hypersensitive sites. g, Genome browser snapshots of the H3.3 ULI-NChIP-seq data. Arrows indicate the positions of indicated repetitive elements. Source data
Extended Data Fig. 2 Examination of possible factors affecting H3.3 distribution and H3.3 ULI-NChIP-seq data with A/B compartments or histone modifications.
a, Stacked bar charts showing the complexity and quality of the H3.3 ULI-NChIP-seq mapped reads. The proportion of PCR duplicates and MAPQ score were used to assess the complexity and quality of the mapped reads, respectively. b, Genome browser snapshots showing normalized H3.3 enrichment in adult neurons, MII oocytes, zygotes, and 2-cell embryos. H3.3 ChIP-seq data for hippocampal neurons was obtained from GSE69806 (ref. 30). c, Immunostaining for H3.3S31P in FGOs, MII oocytes, zygotes, and 2-cell embryos. Representative images from two independent experiments are shown. n = 16 FGOs, 19 MII oocytes, 12 zygotes, and 15 2-cell embryos. Scale bar = 5 μm. d, Immunostaining for H3.3 in FGOs, MII oocytes, zygotes, and 2-cell embryos. Representative images from two independent experiments are shown. n = 10 FGOs, 15 MII oocytes, 11 zygotes, and 6 2-cell embryos. Scale bar = 5 μm. e, Genome browser snapshots showing normalized H3.3 enrichment and A/B compartments. Assignment of A/B compartment was performed using published Hi-C data (GSE82185)32. f, Bar graph showing the overlap between histone modification and genomic regions that show gain or loss of H3.3 during the zygote-to-2-cell progression. The numbers below indicate the number of bins (10 kb) analyzed.
a, Hierarchical clustering and correlation analysis of H3.3 ULI-NChIP-seq data. Heatmaps show Spearman’s correlation coefficients between the samples. Two biological replicates were analyzed for each developmental stage. b, Heatmaps showing genome-wide H3.3 and H3K4me3 enrichment. Published H3K4me3 dataset (GSE71434)39 was used. The analyses were performed for 10-kb bins genome-wide. The data is sorted by the H3.3 enrichment level in growing oocytes. Number of bins = 510,178. c, Heatmaps showing H3.3 (left) and H3K4me3 enrichment (right) around genic regions. The H3.3 patterns were subjected to k-means clustering. Published H3K4me3 dataset (GSE71434)39 was used.
a, Scatter plots showing normalized H3.3 enrichment in the maternal or paternal allele in zygotes (left) or 2-cell embryos (right). Bin size = 500 kb. b, Scatter plots showing the correlation between H3.3 enrichment in the maternal (left) or paternal (right) allele and H3K4me3 in 2-cell embryos. c, Heatmaps showing normalized allelic H3.3, H3K4me3 and H3K27me3 enrichment data. Published histone modification data (GSE73952 and GSE97778)33,34 are used. The analyses were performed for 10-kb bins showing H3.3 enrichment either in zygotes (left) or 2-cell embryos (right). The data were sorted by H3.3 enrichment level on the maternal allele. Enrichment is shown as log2 ratios between ChIP and input.
a, b, The effect of aphidicolin (a) and α-amanitin (b) on DNA replication and transcription, respectively. The incorporation of EdU and EU was analyzed by microscopy. Scale bars = 50 μm. n = 12 (control for aphidicolin), 14 (aphidicolin-treated), 9 (control for α-amanitin) and 10 (α-amanitin-treated) embryos from two independent experiments. c, Heatmaps showing normalized H3.3 enrichment around genic regions. The H3.3 patterns were subjected to k-means clustering. Enrichment is shown as log2 ratios between H3.3 and input. d, Violin plots showing the size of H3.3-enriched regions in the indicated samples.
a, Experimental design to compare histone levels between the zygote and the 2-cell stage. Triton pre-extraction was performed before fixation to remove free histones. Zygotes and 2-cell embryos were then pooled and processed under the same conditions. Images were also acquired with identical parameters. Scale bar, 50 μm. b, Immunostaining for pan-H3 in PN5 zygotes and 2-cell embryos (left). Scale bar =10 μm. Right, plots showing the pan-H3 signal intensity in zygotes and 2-cell embryos. Signal intensities were normalized to those of DAPI signals. P-values (Bonferroni-adjusted two-tailed Mann-Whitney U tests) are indicated. Error bars indicate mean ± s.d.. n = 12 (female PN), 12 (male PN) and 30 (nuclei in late 2-cell embryos) from two independent experiments. c, Plots showing the H3.1/H3.2 signal intensity in control (DMSO-treated) and aphidicolin-treated 2-cell embryos. Signal intensities were normalized to those of DAPI. P-values (two-tailed Mann-Whitney U tests) are indicated. Error bars indicate mean ± s.d.. n = 48 (control) and 46 (Aphidicolin) from four independent experiments. d, Plots showing the H3.3 signal intensity in control and aphidicolin-treated 2-cell embryos. The signal intensities were normalized by DAPI signal intensity. P-value (two-tailed Mann-Whitney U tests) is indicated. Error bars indicate mean ± s.d.. n = 38 (control) and 34 (Aphidicolin) from three independent experiments. Data for graphs in b–d are available as source data. Source data
a, Images showing the expression of EGFP or EGFP-p150DN after mRNA injection. Images were acquired 3 h after mRNA injection. p150DN accumulates at female and male pronuclei in zygotes. Scale bar = 50 μm. b, Plots showing the average H3.3 enrichment around TSSs in EGFP-injected and p150DN-injected 2-cell embryos. c, Stacked bar charts showing the developmental progression of non-injected embryos. Cultured embryos were observed at indicated time points (post hCG) and their developmental stages were determined. The stages are shown by indicated colors. n = 64 embryos analyzed in three independent experiments. d, Heatmaps showing levels of differentially expressed transcripts among three experimental groups. Clustering of differentially expressed transcripts between control (EGFP-injected) 8-cell, blastocyst and p150DN-injected 8-cell was performed. The differential expression is indicated by z-score. Source data
a, Western blotting showing the efficiency of p150 knockdown. Representative images from three independent biological replicate samples are shown. β-actin, loading control. Uncropped blot images are shown as Source Data. b, Genome browser snapshots showing normalized H3.3 enrichment in control and p150-depleted mESCs. Enrichment is shown by log2 ratios between H3.3 and input. c, Genome browser snapshots of the H3.3 ULI-NChIP-seq and H3.3 CUT&RUN data in mESCs. d, Scatter plots showing H3.3 enrichment obtained from H3.3 ULI-NChIP-seq and CUT&RUN in mESCs. The number of 10-kb bins = 252,405. e, Heatmaps showing genome-wide H3.3 enrichment. H3.3 ULI-NChIP-seq and CUT&RUN data obtained by using mESCs were analyzed. The number of 10-kb bins, 252,405. f, Plots showing read enrichment around genic regions. CUT&RUN data using control (normal) IgG was generated for control and p150-depleted mESCs, and read enrichment was analyzed at genic regions. Data from two biological replicates were merged. Note that reads are enriched exactly at the TSSs in p150-depleted mESCs, possibly due to digestion of accessible chromatin by protein A/G-MNase. g, Bar graph indicating relative number of aligned reads. The number of reads from spike-in DNA was used for normalization. h, Plots showing H3.3 enrichment around genic regions. H3.3 CUT&RUN data generated from control and p150-depleted ESCs were analyzed. H3.3 enrichment after spike-in normalization (scaled) is also shown. i, Expression of the indicated transcripts during early mouse development. The published RNA-seq data46 was used. j, Quantitative real-time PCR confirmation performed in this study. Gapdh was used for normalization. Log2 fold change of each transcript between control and p150-depleted mESCs are shown. Each dot represents the data from two biological replicates. Error bars indicate mean ± s.d.. Data for graphs are available as source data. Source data
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Ishiuchi, T., Abe, S., Inoue, K. et al. Reprogramming of the histone H3.3 landscape in the early mouse embryo. Nat Struct Mol Biol (2020). https://doi.org/10.1038/s41594-020-00521-1