Single-cell replication profiling reveals stochastic regulation of the mammalian replication-timing program

Abstract In mammalian cells, distinct replication domains (RDs), corresponding to structural units of chromosomes called topologically-associating domains (TADs), replicate at different times during S-phase 1–4 . Further, early/late replication of RDs corresponds to active/inactive chromatin interaction compartments 5,6 . Although replication origins are selected stochastically, such that each cell is using a different cohort of origins to replicate their genomes 7–12 , replication-timing is regulated independently and upstream of origin selection 13 and evidence suggests that replication timing is conserved in consecutive cell cycles 14 . Hence, quantifying the extent of cell-to-cell variation in replication timing is central to studies of chromosome structure and function. Here we devise a strategy to measure variation in single-cell replication timing using DNA copy number. We find that borders between replicated and un-replicated DNA are highly conserved between cells, demarcating active and inactive compartments of the nucleus. Nonetheless, measurable variation was evident. Surprisingly, we detected a similar degree of variation in replication timing from cell-to-cell, between homologues within cells, and between all domains genome-wide regardless of their replication timing. These results demonstrate that stochastic variation in replication timing is independent of elements that dictate timing or extrinsic environmental variation.