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. 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)