Mapping H3K4me2 in mouse (C57BL6/N) gametes and pre-implantation embryos.

(A) The IGV view shows the global landscape of H3K4me2 signals in mouse sperm, GV oocytes, MII oocytes, and pre-implantation embryos. The H3K4me2 data in sperm was obtained from ENCODE (Lambrot et al.). ICM was isolated from the blastocyst. (B) Immunofluorescence of H3K4me2 in mouse (C57BL6/N) GV oocytes, MII oocytes, PN5 zygote, early two-cell embryo, late two-cell embryo, four-cell embryo, eight-cell embryo, morula, and blastocyst; the scale bar is also shown. (C) H3K4me2 enrichment near typical genes and their expression levels are shown. H3K4me2 signal, ATAC-seq, and gene expression are shown.

Comparison of H3K4me2 between sperms and oocytes.

(A-B) Heatmaps showing H3K4me2 and H3K4me3 expressed genes between sperm and GV oocyte in the promoter and distal region at different stages. CpG densities are also mapped and shown. (C) The enriched GO terms of H3K4me2 marked genes in GV and sperm-specific or shared genes are shown along with the associated p-values. (D) The results of the GREAT analysis for distal peaks in GV and sperm-specific or shared genes are shown along with the associated p-values. (E-F) The IGV views show H3K4me2 expressions in GV and sperm-specific or shared genes at the representative promoters (left, shaded) or distal accessible regions (right, shaded).

Resetting of H3K4me2 during mouse pre-implantation development.

(A) The number of H3K4me2 peaks in mouse embryo stem cells (mESCs), mouse gametes, and pre-implantation embryos was calculated. (B) The IGV view shows H3K4me2 signals in mESCs, mouse gametes, and pre-implantation embryos; Ncc1 gene sites are also shown. (C) Venn diagrams illustrate the overlap of H3K4me2 marked genes between sperm/GV oocytes, separately (left) or jointly (right), and four-cell embryos. The enriched GO terms for the shared and non-overlapping genes are also shown.

The inhibition of LSD2 resulted in abnormal embryonic development

(A) The dynamic changes in the Kdm1a (blue line) and Kdm1b (red line) levels in oocytes and early embryos (based on published RNA-seq data43). (B) a.The immunofluorescence intensity of LSD2 in the experimental group (5 mM TCP) and the control group are shown using a box diagram. b. Immunofluorescence images showed the immunofluorescence intensity of LSD2 of two-cell embryos in the experimental group (5 mM TCP) (right) and the control group (left) ; the scale bar is also shown. (C) The developmental morphology of mouse embryos in the experimental group (5 mM TCP) and the control group after 28 h and 40 h of culture are shown along with the scale bar. (D) The figure illustrates the embryonic development of mice at different stages after culture in vitro in the experimental group (5 mM TCP) and the control group. (E) a.The immunofluorescence intensity of H3K4me2 in the experimental group (5 mM TCP) and the control group are shown using a box diagram. b. Immunofluorescence images showed the immunofluorescence intensity of H3K4me2 of two-cell embryos in the experimental group (5 mM TCP) (right) and the control group (left) ; the scale bar is also shown.

Parental H3K4me2 was erased by maternal LSD2 during sperm-egg fusion

(A) Immunofluorescence images showed the immunofluorescence intensity of H3K4me2 in single-cell embryos in the experimental group (5 mM TCP) and the control group (C); the scale bar is also shown. (B) The immunofluorescence intensity of H3K4me2 in single-cell embryos in the experimental group (5 mM TCP) and the control group are represented as box and whisker plots. (C) The immunofluorescence intensity of H3K4me2 in male and female embryonic prokaryotes of the experimental group (5 mM TCP) and the control group are represented as box and whisker plots.

Inhibition of LSD2 affects gene expression

(A) A volcano map of genes with differential H3K4me2 expression after TCP inhibition of LSD2 expression. (B) The GO analysis showed enrichment of biological processes after an increase in the expression of H3K4me2. (C) The peak distribution area of H3K4me2 in the experimental group (5 mM TCP) and the control group (D) The IGV view shows the H3K4me2 signals in typical genes in the experimental group (5 mM TCP) and the control group. (E) A volcano map of the differential expression of H3K4me2 genes in the experimental group (5 mM TCP) and the control group. (F) The Venn diagram illustrates the intersection of differentially expressed genes and minor ZGA and major ZGA genes between the experimental group (5 mM TCP) and the control group (G) The distribution of differential ATAC peaks in the functional regions of the genome.

Pervasive H3K4me2 in CpG-rich regulatory regions in mouse embryos.

(A-B) H3K4me2 and H3K4me3 enrichment near TSS (TSS ±5 kb) and PMD (3× PMD) in mouse gametes, pre-implantation embryos, and mESC. (C) The IGV view shows promoter H3K4me2 signals and widespread distal H3K4me2 signals in mouse oocytes and early embryos. (D) The IGV view shows H3K4me2 signals and the distribution of DNA methylation in mouse oocytes and early embryos at the PMD (partially methylation district) region. (E) Heat maps show the enrichment of H3K4me2 and H3K4me3 at the promoters, including that in all stages shared and four-cell/eight-cell specific. ATAC-seq, promoter CpG density, and GO analysis results are presented. (F) Heat maps of the distal peaks of H3K4me2 expressed in specific stages among eight-cell, ICM, and mESC. ATAC-seq are shown.

Chromatin states at specific stages and the H3K4me2 mode chart during mouse maternal-to-zygotic transition.

(A) Heat maps of H3K4me2, H3K4me3, and ATAC-seq are shown, “All active” and “All inactive” promoters and the corresponding gene expression are presented. The CpG densities are also shown. (B) The bar graph shows that the H3K4me2 distal peaks overlapped with the CcREs four/eight-cell stage. Random peaks are shown as a control. (C) Heat maps of the chromatin states of the overlap between CcREs and the distal peaks of H3K4me2 and H3K27ac are presented. Clustering was conducted using ATAC-seq, H3K4me2 in the four-cell, eight-cell, and ICM stages. The results of the GREAT analysis for each cluster are shown (right). Data on histone modification of H3K27ac in mice was obtained from the GEO database, GSE72784. (D) Dynamic reprogramming of H3K4me2 during mouse parental-to-zygotic transition. A schematic model of “H3K4me2 resetting” during parental-to-zygotic transition is shown. After fertilization, the parental H3K4me2 is almost globally erased. De novo H3K4me2 promoters and distal regions in CpG-rich and DNA hypomethylated regions are re-established at the late two-cell stage, probably as a priming chromatin state. This leads to the resolution of primed promoters to “active” promoters with H3K4me3, or “poised”. A similar transition occurs for putative enhancers.

Validation of H3K4me2 CUT&RUN data in Human K562 cell line, oocytes and early embryos.

(A) The IGV views showing H3K4me2 distributions by CUT&RUN using various numbers of Human K562 cell line. The ENCODE references are added for comparison. GEO accession: GSM733651 . (B) The Pearson correlation coefficients showing the comparison between CUT&RUN of H3K4me2 using various numbers of Human K562 cells and conventional ChIP–seq data from ENCODE. (C) Scatter plots showing the correlations of biological replicates (n=2) of H3K4me2 signals (2kb-bin, whole genome) by CUT&RUN for each stage of mouse oocytes and early embryos. The Pearson correlation coefficients are also shown. (D) Scatter plots comparing the H3K4me2 signals with ATAC-seq (Assay for Transposase-Accessible Chromatin with high throughput sequencing) at promoters or distal accessible regions in mouse oocytes and early embryos. Spearman correlation coefficients are also shown. (E) Scatter plots comparing the H3K4me2 signals with H3K4me3 signals in mouse sperm, oocytes and early embryos. Spearman correlation coefficients are also shown. (F) Hierarchical clustering of H3K4me2 mouse oocytes and early embryos. Pearson correlation was used to measure distances. (G) Scatter plots comparing the H3K4me2 signals between 4-cell and 8-cell in mouse embryos. Spearman correlation coefficients are also shown.

H3K4me2 in mouse gametes.

(A) Heatmaps showing H3K4me2 signals at all promoter regions (TSS ± 2.5kb) in mouse gametes. DNA methylation (TSS ± 2.5kb) is also mapped. DNA methlation data cited in GEO dataset:GSE56697. (B) The box plots showing DNA methylation level at all promoter regions in mouse gametes. Random peaks are shown as a control. DNA methlation data cited in GEO dataset: GSE56697. (C) Heatmaps showing H3K4me2 signals at all distal regions (peak ± 2kb) in mouse gametes. DNA methylation (peak ± 2kb) is also mapped. DNA methlation data cited in GEO dataset: GSE56697. (D) The box plots showing DNA methylation level at all distal regions in mouse gametes. Random peaks are shown as a control. DNA methlation data cited in GEO dataset:GSE56697.

H3K4me2 in late 2-cell embryos.

(A) Heatmaps showing the expression of H3K4me2 related regulators in mouse (based on published RNA-seq data43). (B) The box plots showing DNA methylation level at all promoter regions and distal region in mouse 2-cell stage. Random peaks are shown as a control. (C-D) Venn diagrams showing De novo 4-cell H3K4me2 promoter and distal peaks number.

Cell culture experiment under TCP treatment

(A)Morphological observation of cells under different concentration of TCP culture, scale bar is also shown. (B) Morphological observation of embryo development after 5mM TCP inhibition of LSD2 expression. (C-D)Immunofluorescence images showed the immunofluorescence intensity of H3K4me1 (C) and H3K4me3 (D) of 2-cell embryos in the experimental group (5mM TCP) and control group, scale bar is also shown.

H3K4me2 in mouse 4/8-cell embryos.

(A) Scatter plots comparing the promoter and distal H3K4me2 signals with CpG densities in mouse gametes, 4-cell, 8-cell, ICM and mESC. The Spearman correlation coefficients are also shown. (B) The box plots showing the CpG density and promoter/distal H3K4me2 peaks. Random peaks are shown as a control. (C) The box plot showing DNA methylation in promoter and distal 4-cell H3K4me2 peaks. Random peaks are shown as a control. (D) TF motifs identified from active enhancers in mouse early embryos (8-cell, ICM) and mESC. showing motif enrichment p value < 1e-20 at least at one stage were included. Circle size showing TF enrichment and the expression of the TF is color coded. (E) Heatmaps showing stage-specifically expressed genes further classified by their H3K4me2 states (high, dynamic, and low). RNA-seq, H3K4me3 state, CpG densities and GO analysis results are also mapped and shown.

Chromatin states at specific stages and distal CcREs.

(A) Box plots showing H3K4me2, H3K4me3 and ATAC-seq signals, “All active” and “All inactive” promoters in mouse gametes, embryos and mESC. CpG density for each group is also shown (right). (B) The box plot showing the ATAC-seq, H3K4me2 and H3K27ac signals at putative active, poised or random enhancers in mouse gametes, early embryos or mESC, Mouse histone modified H3K27ac data refer to GEO database : GSE72784.

CUT&RUN experimental sample information