Histone H3K36me3 regulates E2F transcription as part of the DNA replication stress checkpoint response. Histone H3K36me3 regulates E2F transcription as part of the DNA replication stress checkpoint response

DNA replication stress drives genomic instability, thus contributing to the rapid evolution of tumours. Sustained E2F-dependent transcription, which is actively maintained in a checkpoint-dependent manner, is required for replication stress tolerance and is a key mechanism preventing the generation of DNA damage under these conditions. However, the activation and regulation of the E2F response remains poorly understood. Here, we establish a role for SETD2-dependent H3K36 trimethylation in facilitating E2F target gene expression in S-phase and promoting efficient DNA replication under both normal and replication stress conditions in human cells. Loss of SETD2 results in reduced E2F1-binding to its target genes, causing expression defects in almost all E2F transcripts, including CDT1, CDC6, and MCM2-7. Further, we find that E2F target gene expression following hydroxyurea-induced replication fork stalling requires ATR-dependent H3K36 trimethylation. Accordingly, SETD2 loss results in reduced replication fork progression and increased levels of replication stress-induced DNA damage, indicative of reduced replication stress tolerance. Together, these findings establish a central role for SETD2-dependent H3K36me3 in the replication stress checkpoint, thereby ensuring genomic integrity. Overall design: RNA-seq of U2OS cell lines treated with either non-targeting control siRNA or SETD2 siRNA, followed by control, hydroxyurea (HU), ATR inhibitor (VE-822), or HU+VE-822 48 hours post-transfection (n=2)