H3K9 di-methylation dynamics underlies mouse minor zygotic genome activation [spike-in ChIP-seq of mESC]

Minor zygotic genome activation (ZGA) is crucial for early development and totipotency acquisition, yet the regulatory mechanisms driving minor ZGA genes remain elusive. Here we show that dynamic regulation of H3K9me2 is essential for minor ZGA. H3K9me2 levels at minor ZGA gene loci are reduced at early 2-cell and are re-established by morula. Maternal depletion of the H3K9 demethylases KDM3A and KDM3B leads to increased H3K9me2 and impaired minor ZGA in early 2-cell, followed by developmental arrest at 2- to 4-cell. In mESCs, H3K9 at minor ZGA loci is highly di-methylated; combined loss of the H3K9 methyltransferases G9a and SETDB1 leads to synergistic de-repression of minor ZGA genes. Mechanistically, SETDB1 specifically targets Dux, while G9a broadly represses minor ZGA genes through H3K9 di-methylation linked to lamina-associated heterochromatin formation. These findings establish H3K9me2 dynamics as a key regulator for minor ZGA, highlighting the indispensable role of epigenetic control in early embryogenesis.