Developmental control of replication timing defines a new breed of chromosomal domains with a novel mechanism of chromatin unfolding.

We recently identified a set of chromosome domains that are early replicating uniquely in pluripotent cells. Their switch from early to late replication occurs just prior to germ layer commitment, associated with a stable form of gene silencing that is difficult to reverse. Here, we discuss results demonstrating that these domains are among the least sensitive regions in the genome to global digestion by either MNase or restriction enzymes. This inaccessible chromatin state persists whether these regions are in their physically distended early replicating or compact late replicating configuration, despite dramatic changes in 3D chromatin folding and long-range chromatin interactions, and despite large changes in transcriptional activity. This contrasts with the strong correlation between early replication, accessibility, transcriptional activity and open chromatin configuration that is observed genome-wide. We put these results in context with findings from other studies indicating that many structural (DNA sequence) and functional (density and activity of replication origins) properties of developmentally regulated replication timing ("switching") domains resemble properties of constitutively late replicating domains. This suggests that switching domains are a type of late replicating domain within which both replication timing and transcription are subject to unique or additional layers of control not experienced by the bulk of the genome. We predict that understanding the unusual structure of these domains will reveal a novel principle of chromosome folding.