Identification of cis elements for spatio-temporal control of DNA replication

The temporal order of DNA replication (replication timing, RT) is highly coupled with genome architecture, but cis -elements regulating spatio-temporal control of replication have remained elusive. We performed an extensive series of CRISPR mediated deletions and inversions and high-resolution capture Hi-C of a pluripotency associated domain (DppA2/4) in mouse embryonic stem cells. Whereas CTCF mediated loops and chromatin domain boundaries were dispensable, deletion of three intra-domain prominent CTCF-independent 3D contact sites caused a domain-wide early to late switch in RT, shift in sub-nuclear chromatin compartment and loss of transcriptional activity, These early replication control elements (ERCEs) display prominent chromatin features resembling enhancers/promoters and individual and pair-wise deletions of the ERCEs confirmed their partial redundancy and interdependency in controlling domain-wide RT and transcription. Our results demonstrate that discrete cis -regulatory elements mediate domain-wide RT, chromatin compartmentalization, and transcription, representing a major advance in dissecting the relationship between genome structure and function.