Early Replication Control Elements Are Developmentally Regulated Genetic Determinants of Replication Timing and Require Transcription Factor Activity

During S phase, chromosomes are replicated in a defined temporal order called the replication timing (RT) program. RT programs are evolutionarily conserved, cell-type specific, and particularly resistant to a wide-range of protein depletions, knockdowns, knockouts, and inhibitors, suggesting tight and extremely robust control. However, the mechanisms controlling RT have remained elusive for >60 years. We previously reported large (>30kb) deletions that, when combined, cause a domain-wide shift from early- to late-replicating that is concomitant with a loss of all gene transcription and A- to B-compartment switch, but those deletions contained many different chromatin features that make those changes difficult to interpret and uncouple. Here, we show that the previously-reported elements B and C in the Dppa2/4 domain are dependent upon Oct4, Sox2, Nanog co-binding sites and independent of transcriptional control, while the sequences at A remain a mystery. Further, we show that sequences derived from the Dppa2/4 domain carry the information required to promote early RT by inserting a bacterial artificial chromosome containing Dppa2/4 domain sequences into a late-replicating domain, and then further show that the ectopic insertion is developmentally regulated like the endogenous site, suggesting that RT is controlled by DNA sequence and that these elements work as enhancer-like elements for replication initiation in a cell-type specific context. Next, we predicted active ERCEs in neural precursor cells based on publicly-available NPC ChIP-seq data and show that deletions of these elements cause delays in RT similar to the phenomena observed at the Dppa2/4 domain, confirming developmental control of replication timing cis-elements. Lastly, we use a wide-range of inducible-degron cell lines and commercially-available small-molecule inhibitors to interrogate ERCE function genome-wide. These results suggest that ERCEs are intimately intertwined with developmental regulation of transcriptional networks, although their functions can be separated from transcription in specific contexts.