HIRA and dPCIF1 coordinately establish totipotent chromatin and control orderly ZGA in Drosophila embryos [RNA-Seq]

Early embryos undergo profound changes in their genomic architecture to establish the totipotent state, enabling pioneer factors to access chromatin and drive zygotic genome activation (ZGA). However, the mechanisms by which the totipotent state is established and properly interpreted by pioneer factors to allow orderly ZGA remain unknown. Here, we identify the H3.3-specific chaperone HIRA as a critical factor involving establishing totipotent-state chromatin in Drosophila early embryos. Through co-phase separation with HIRA, the pioneer factor GAGA factor (GAF) efficiently binds to H3.3-marked nucleosomes to activate major-wave zygotic genes. Importantly, dPCIF1, a chromatin-associated protein, antagonized the GAF–HIRA interaction by competitively binding to HIRA, thereby restricting GAF on earlier chromatin and avoiding premature ZGA. Hence, the coordinated action of HIRA and dPCIF1 ensures sequential ZGA from the minor to major wave in early embryos. This study provides insights into understanding how a totipotent state is established and properly controlled during ZGA. Overall design: To investigate the effect of dPCIF1/H3.3/HIRA/GAF on the zygotic gene activation of drosophila embryo, we established a variety of mutants. We then performed gene expression profilinf analysis using data from RNA-seq of mutant embryos and wild-type embryos at 2-3 time points.