Fig. 1: Profiling histone modifications and epigenetic factors present on pre-ZGA embryo chromatin.
a Schematic highlighting early developmental stages of interest in this study and their transcriptional states with a focus on ZGA and the methods used in this figure. b Heatmap representing the percent abundance of histone modifications on each respective peptide relative to the total abundance of the corresponding peptide, obtained by PTM analysis of LC-MS/MS. Undetectable modifications are highlighted in red text. Each column represents a single biological replicate of pre-ZGA or mid-ZGA embryos. c Time-series Western blot showing the occurrence of H3K4me3 and H3 in embryos. d Western blots of histone modifications in pre-ZGA and post-ZGA embryos using chromatin-bound protein isolation. Western blots were independently repeated 2–3 times with consistent results. e Representative images of H3K4me3 levels in control and transcription-inhibited mid-ZGA embryos. Nascent transcripts are labeled using 5-EU. Overlay of H3K4me3 (cyan) and H4 (magenta) is represented. Scale bars: 50 µm. Nuclei are segmented using H4, and H3K4me3 values are measured. (Right) Quantification of H3K4me3 immunofluorescence in H4-segmented nuclei across Z-stacks (N = 3 embryos per condition, n = 2 independent experiments). Box plots show the median (center line), 25–75th percentiles (box), whiskers extending to 1.5× the interquartile range, and outliers as individual points. Statistical test: one-sided Wilcoxon rank-sum test with the alternative hypothesis that the values from the amanitin-injected condition are lower than controls; p: (****) ≤ 0.0001,; n.s. are p > 0.05. Exact p: H₂O vs 5EU: 0.2; H₂O vs 5EU+Ama: 2.2 × 10–16; 5EU+Ama vs Ama: 2.2 × 10–16. f IGV tracks displaying H3K4me3 ChIP-seq, CATaDa, and MBD-seq data for pre-ZGA embryos with CpG regions at representative promoters (TSS ± 1kb). g Heatmap showing H3K4me3, accessibility, DNAme, CpG and GATC density across promoters (TSS ± 1kb) in pre-ZGA embryos. Accessibility enrichment is normalized to GATC frequency within the promoter region. Clusters are identified by k-means clustering on the z-scored data, excluding GATC density (k = 6; Hartigan-Wong algorithm).
