Coincident transcription and DNA replication causes replication stress and genome instability. Rapidly dividing mouse pluripotent stem cells are highly transcriptionally active and experience elevated replication stress, yet paradoxically maintain genome integrity. Here, we study FOXD3, a transcriptional repressor enriched in pluripotent stem cells, and show that its repression of transcription upon S phase entry is critical to minimizing replication stress and preserving genome integrity. Acutely deleting Foxd3 leads to immediate replication stress, G2/M phase arrest, genome instability and p53-dependent apoptosis. FOXD3 binds near highly transcribed genes during S phase entry, and its loss increases the expression of these genes. Transient inhibition of RNA polymerase II in S phase reduces observed replication stress and cell cycle defects. Loss of FOXD3-interacting histone deacetylases induces replication stress, while transient inhibition of histone acetylation opposes it. These results show how a transcriptional repressor can play a central role in maintaining genome integrity through the transient inhibition of transcription during S phase, enabling faithful DNA replication
