Broad H3K4me3 domains support oocyte genome silencing and maturation but are dispensable for repression in early embryos

To investigate whether demethylation of broad histone H3 lysine 4 trimethyl (H3K4me3) domains is sufficient to trigger premature embryonic genome activation (EGA), we overexpressed the H3K4me3 demethylase KDM5B in mouse zygotes and early 2-cell (2C) embryos via mRNA injection. We performed single-cell RNA sequencing (scRNA-seq) using the SMART-Seq Stranded Kit (Takara Bio) on individual Kdm5b-yfp–injected and water-injected control zygotes and 2C embryos. Differential expression analysis revealed minimal transcriptional changes, and developmental stage, rather than KDM5B overexpression, accounted for most gene expression variance. Our findings demonstrate that wide-scale removal of H3K4me3 in zygotes or early 2C embryos is not sufficient to induce premature EGA, suggesting that genome silencing at these stages does not depend on broad H3K4me3 domains. Overall design: S-phase zygotes or both blastomeres of G1-phase 2C embryos were microinjected with Kdm5b-yfp mRNA; water-injected zygotes or 2C embryos served as controls. The C-terminal YFP tag enabled live-cell confirmation of KDM5B expression. Samples were collected for scRNA-seq ~5–6 h post-injection (late zygotes: 17 h after ovulation, corresponding to late S/G2 phase; early 2C embryos: 5.5–8 h after division, corresponding to late S phase). During collection, 10 single zygotes from each group (Kdm5b-yfp-injected and control) were transferred into separate tubes. For 2C embryos, we included both single intact embryos and pooled blastomeres from partially lysed embryos to maximise sample use (lysed blastomeres were excluded during zona pellucida removal). In the control 2C group, 9 samples were collected: 6 single embryos with both blastomeres intact, one pool of two embryos with only one blastomere intact, and two pools of three such embryos. In the Kdm5b 2C group, 6 samples were collected: 5 single embryos with both blastomeres intact, and one pool of three embryos with only one blastomere intact.