R-loops orchestrate RNAPII transcriptional reprogramming for the maternal-to-zygotic transition

R-loops are pervasive genomic structures that link epigenetic modification and transcriptional regulation. However, the functional roles and regulatory mechanisms of R-loops during preimplantation development in mammals remain unexplored. Here, we reveal that the reprogramming of R-loops across developmental stages depends on CG density, with CG-poor R-loops more stage specific and strongly associated with early embryonic development. Loss of CG-poor R-loops causes severe defects in the maternal-to-zygotic transition (MZT) and preimplantation embryo development. This abnormal maintenance of CG-poor R-loops promotes premature activation of major zygotic genome activation (ZGA) genes. CG-poor R-loops inhibit DDX21 helicase activity on the 7SK/HEXIM1 snRNP complex, restricting CDK9 release and subsequent phosphorylation of Ser2 at the C-terminal domain of RNA polymerase II (RNAPII S2p) - the biochemical hallmark of pause release - thus enforcing RNAPII accumulation at major ZGA gene promoters to ensure productive transcription. These findings establish R-loops as direct modulators of RNAPII pause release, promoting the temporal fidelity of gene expression during the MZT.