Master transcription factor binding sites are necessary for early replication control element activity

Eukaryotic genomes are replicated in a defined temporal order called the replication timing (RT) program. RT is developmentally regulated, and has potential to drive cell fate transitions, but mechanisms controlling RT remain elusive. We previously identified Early Replication Control Elements (ERCEs) necessary for chromosome domain early replication, transcription, 3D chromatin architecture and compartmentalization in mouse embryonic stem cells (mESCs). Deletions of these elements were too large to identify functional elements. Here, we performed deletion analyses of three ERCEs within one domain that is early replicating uniquely in pluripotent mammalian cells. All three of these ERCEs required one or more sites for co-binding of the pluripotency transcription factors Oct4, Sox2, Nanog (OSN) for early replication. Deletion of TSSs throughout the domain eliminated transcription without affecting RT, indicating that early replication activity of ERCEs is independent of transcription. However, in the absence of all ERCE OSN sites, a TSS was sufficient to maintain mid-late replication. ERCE OSN sites do not align with initiation sites, suggesting that they function as replication enhancers. We conclude that pluripotency transcription factor binding sites ensure early replication independent of transcription, suggesting a means for co-regulation of RT with cell fate transitions during development.