KAT5/TIP60 orchestrates H4K16 and H2A.Z acetylation to drive the gonocyte-to-spermatogonia transition.

Spermatogenesis begins with the gonocyte-to-spermatogonia transition (GST) during the perinatal window, yet how chromatin regulation orchestrates this developmental transition remains elusive. Transcriptomic analysis identified histone acetylation as a significantly enriched pathway during the GST, and immunofluorescence revealed a progressive increase in H4K16 acetylation alongside high levels of H2A.Z acetylation. Conditional deletion of the acetyltransferase Kat5/Tip60 drastically reduced both marks, leading to GST failure characterized by impaired prospermatogonial mitotic resumption, migration, and survival. While the loss of either H4K16ac or H2A.Zac alone causes only moderate defects, their combined depletion largely phenocopies Kat5 deficiency, revealing a functional synergy between the two modifications. CUT&Tag and RNA-seq analyses showed that gene-body-enriched H4K16ac and TSS-enriched H2A.Zac cooperate to promote chromatin accessibility and RNA polymerase II recruitment, thereby activating cell-cycle and spermatogonial developmental programs. Notably, we observed a dynamic, KAT5-dependent spatial shift of H4K16ac from the TSS toward the gene body regions during this transition, indicating a distinct role for this spatial redistribution during early germline development. Together, these findings establish KAT5 as an essential epigenetic factor that integrates dual acetylation pathways to coordinate the GST and safeguard male germline development.