Global Reorganization of Replication Domains During Embryonic Stem Cell Differentiation

Abstract

DNA replication in mammals is regulated via the coordinate firing of clusters of replicons that duplicate megabase-sized chromosome segments at specific times during S-phase. Cytogenetic studies show that these “replicon clusters” coalesce as subchromosomal units that persist through multiple cell generations, but the molecular boundaries of such units have remained elusive. Moreover, the extent to which changes in replication timing occur during differentiation and their relationship to transcription changes has not been rigorously investigated. We have constructed high-resolution replication-timing profiles in mouse embryonic stem cells (mESCs) before and after differentiation to neural precursor cells. We demonstrate that chromosomes can be segmented into multimegabase domains of coordinate replication, which we call “replication domains,” separated by transition regions whose replication kinetics are consistent with large originless segments. The molecular boundaries of replication domains are remarkably well conserved between distantly related ESC lines and induced pluripotent stem cells. Unexpectedly, ESC differentiation was accompanied by the consolidation of smaller differentially replicating domains into larger coordinately replicated units whose replication time was more aligned to isochore GC content and the density of LINE-1 transposable elements, but not gene density. Replication-timing changes were coordinated with transcription changes for weak promoters more than strong promoters, and were accompanied by rearrangements in subnuclear position. We conclude that replication profiles are cell-type specific, and changes in these profiles reveal chromosome segments that undergo large changes in organization during differentiation. Moreover, smaller replication domains and a higher density of timing transition regions that interrupt isochore replication timing define a novel characteristic of the pluripotent state. Microscopy studies have suggested that chromosomal DNA is composed of multiple, megabase-sized segments, each replicated at different times during S-phase of the cell cycle. However, a molecular definition of these coordinately replicated sequences and the stability of the boundaries between them has not been established. We constructed genome-wide replication-timing maps in mouse embryonic stem cells, identifying multimegabase coordinately replicated chromosome segments—“replication domains”—separated by remarkably distinct temporal boundaries. These domain boundaries were shared between several unrelated embryonic stem cell lines, including somatic cells reprogrammed to pluripotency (so-called induced pluripotent stem cells). However, upon differentiation to neural precursor cells, domains encompassing approximately 20% of the genome changed their replication timing, temporally consolidating into fewer, larger replication domains that were conserved between different neural precursor cell lines. Domains that changed replication timing showed a unique sequence composition, a strongly biased directionality for changes in resident gene expression, and altered radial positioning within the three-dimensional space in the cell nucleus, suggesting that changes in replication timing are related to the reorganization of higher-order chromosome structure and function during differentiation. Moreover, the property of smaller discordantly replicating domains may define a novel characteristic of pluripotency.