The R-loop-SPT6-SETD2 axis safeguards H3K36me3 to prevent broad H3K4me3 invasion in mouse oocytes.

The orderly establishment of chromatin modifications is essential for oocyte maturation. However, how R-loops contribute to the crosstalk between histone modifications during oocyte development remains unexplored. Here, we demonstrate that R-loop resolution by RNASEH1 significantly compromises the developmental potential of oocytes. R-loops facilitate H3K36me3 deposition by stabilizing histone H3K36 methyltransferase SETD2 via histone chaperone SPT6 in germinal vesicle (GV) oocytes. Loss of R-loops reduces H3K36me3 levels within oocyte-specific gene bodies, triggering broad H3K4me3 invasion into H3K36me3-marked regions and repression of gene expression. dCas9-mediated site-specific restoration of SETD2 recruitment at R-loop-dependent loci effectively rescues local gene expression. Furthermore, Supt6 overexpression substantially rescues the imbalanced deposition between H3K36me3 and H3K4me3 caused by R-loop loss, corrects dysregulated gene expression, and alleviates the meiotic arrest phenotype. Collectively, our findings establish that R-loops act as critical regulators in balancing H3K36me3 and broad H3K4me3 in the maternal genome, thereby safeguarding proper oocyte development.