Master transcription factor binding sites constitute the core of early replication control elements [Repli-seq]

Eukaryotic genomes replicate in a defined temporal order called the replication timing (RT) program. RT is developmentally regulated with potential to drive cell fate transi-tions, but mechanisms controlling RT remain elusive. We previously identified “Early Replication Control Elements” (ERCEs) necessary for early RT, domain-wide transcrip-tion, 3D chromatin architecture and compartmentalization in mouse embryonic stem cells (mESCs) but, deletions identifying ERCEs were large and encompassed many putative regulatory elements. Here, we show that ERCEs are compound elements whose RT activity can largely be accounted for by multiple sites of diverse master tran-scription factor binding (subERCEs), distinguished from other such sites by their long-range interactions. While deletion of subERCEs had large effects on both transcription and RT, deleting transcription start sites eliminated nearly all transcription with moder-ate effects on RT. Our results suggest a model in which subERCEs respond to diverse master transcription factors by functioning both as transcription enhancers and as el-ements that organize chromatin domains structurally and support early RT, potentially providing a feed-forward loop to drive robust epigenomic change during cell fate transi-tions. Replication timing profiling of hybrid mESCs containing various deletions of suspected cis-elements regulating replication timing