Fig. 5: Promoter H3K4me3 during early cell divisions is required for proper ZGA. | Nature Communications

Fig. 5: Promoter H3K4me3 during early cell divisions is required for proper ZGA.

From: Pre-marking chromatin with H3K4 methylation is required for accurate zygotic genome activation and development

Fig. 5: Promoter H3K4me3 during early cell divisions is required for proper ZGA.The alternative text for this image may have been generated using AI.

a Experimental setup for window-specific and persistent H3K4me3 depletion. b Western blot of cytoplasmic fractions from pre- and mid-ZGA embryos showing ectopic HA-tagged Kdm5bwt + H3.3K4M (treatment) or Kdm5bci + H3.3 wt (control) at pre-ZGA, and their auxin-induced (IAA) degradation at mid-ZGA. Tubulin represents the loading control; “NI” denotes non-injected. Blots were independently repeated 2–3 times with consistent results. c H3K4me3 intensity relative to H4 in treated and control pre-ZGA embryos after chromatin-bound protein isolation. d Genome browser snapshots for sox2.L showing E. coli spike-in normalized coverage for control and treated samples under window and persistent conditions, and wildtype and IgG controls (biological replicate 2). e MA-plot comparing H3K4me3 levels in treated versus control samples at promoter peaks for window (top) and persistent (bottom) conditions. Gray: all promoter peaks subset from the consensus peak set in (b). Blue: significantly changed (p < 0.05). f Volcano plots displaying log2 fold change and adj. p-value (cutoff: 0.05) at 7.5 hpf, colored by RNA dynamics groups for (top) window and (bottom) persistent conditions (biological replicate 1). g Zygotic gene expression (three technical replicates per sample), clustered by H3K4me3 dynamics groups (SHARED, GAINED, ABSENT) (biological replicate 1). Log2 fold changes are relative to the mean control TPM at each time point. Genes are annotated for differential expression in each condition and by RNA dynamics group. The rightmost column displays triptolide-induced log2 fold changes at 7 hpf (adapted from Phelps et al., 2022), representing the expected maximal transcriptional downregulation at this stage. h Log2 fold change of zygotic SHARED (n = 473) and GAINED (n = 121) genes over the mean TPM of respective control technical replicates in window (left) and persistent (right) conditions at 7.5 hpf in biological replicate 1. One-sided Wilcoxon rank-sum test with alternative hypothesis that values of the left distribution tend to be larger than the right distribution; p: (****) ≤ 0.0001, (***) ≤ 0.001, (**) ≤ 0.01, (*) ≤' 0.05; n.s. >0.05. Box plots show the median (center line), 25–75th percentiles (box), whiskers extending to 1.5× the interquartile range, and outliers as individual points. i Log2 fold change expression of pou5f3.2 and sox3 homeologs over mean TPM of respective controls at 7.5 hpf (n = 12); both biological replicates shown (distinct point styles). Error bars indicate mean ± SD.

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