Nakamura, T.; Furuta, A.; Nakatani, T.; Nakano, T.

The paternal chromatin undergoes extensive reprogramming, characterized by the loss of 5-methylcytosine (5mC) through Ten-eleven translocation 3 (Tet3)-mediated oxidation to 5-hydroxymethylcytosine (5hmC). Given the role of DNA methylation in gene silencing, it has long been posited that this loss of paternal DNA methylation facilitates zygotic genome activation (ZGA), which occurs predominantly on the paternal genome. However, recent evidence indicates that Tet3-mediated 5mC oxidation alone does not influence global transcription in zygotes, leaving the molecular mechanisms governing ZGA largely elusive. Here, we identify the functional significance of O-linked N-acetylglucosamine (O-GlcNAc) modification of histone H2B at Serine 112 (H2BS112GlcNAc), catalyzed by O-GlcNAc transferase (OGT), within the paternal chromatin of mouse zygotes. We demonstrate that OGT is selectively recruited to the paternal chromatin because Stella (also known as PGC7 or Dppa3) inhibits its binding to the maternal chromatin, a recruitment mechanism analogous to that of Tet3. Although Tet3 and OGT associate with the paternal chromatin independently, both Tet3-dependent 5hmC formation and OGT-mediated H2BS112GlcNAcylation are indispensable for successful ZGA. Together, our findings reveal that a dual epigenetic signature, the simultaneous coordination of DNA hydroxymethylation and histone O-GlcNAcylation by Tet3 and OGT, is essential for initiating transcriptional reprogramming during the maternal-to-zygotic transition.