Human ORC/MCM density is low in active genes and correlates with replication time but does not delimit initiation zones

Abstract

Eukaryotic DNA replication initiates during S phase from origins that have been licensed in the preceding G1 phase. Here, we compare ChIP-seq profiles of the licensing factors Orc2, Orc3, Mcm3, and Mcm7 with gene expression, replication timing and fork directionality profiles obtained by RNA-seq, Repli-seq and OK-seq. ORC and MCM are significantly and homogeneously depleted from transcribed genes, enriched at gene promoters, and more abundant in early- than in late-replicating domains. Surprisingly, after controlling these variables, no difference in ORC/MCM density is detected between initiation zones, termination zones, unidirectionally replicating and randomly replicating regions. Therefore, ORC/MCM density correlates with replication timing but does not solely regulate the probability of replication initiation. Interestingly, H4K20me3, a histone modification proposed to facilitate late origin licensing, was enriched in late replicating initiation zones and gene deserts of stochastic replication fork direction. We discuss potential mechanisms specifying when and where replication initiates in human cells.

Replication Stress, Genomic Instability, and Replication Timing: A Complex Relationship

“…It was also recently revealed that ORC density is correlated with replication timing. ORC was observed to preferentially bind to early-replicating chromatin regions and transcription start sites of actively transcribed genes [ 99 ]. Furthermore, the proteins DONSON and FANCM are associated with euchromatin replicated early in the S-phase and heterochromatin replicated in the late S-phase, respectively [ 100 ].…”

“…Similar works need to extend these studies to mammalian cells. Nonetheless, a recent genome-wide analysis of the ORC/MCM distribution performed in human cells has weakened the role of ORC and MCM proteins in the regulation of the RT program [ 99 ]. It would thus be informative to observe the RT profiles of human cells with mutations/deregulation in/of ORC and MCM genes to directly assess their roles in the RT program, especially since ORC and MCM mutations have been observed in human diseases including cancers (reviewed in [ 139 , 140 ]).…”

The consequences of differential origin licensing dynamics in distinct chromatin environments

“…As a result, heterochromatin loading lags behind euchromatin in early G1, but differential acceleration allows heterochromatin to “catch up” with euchromatin by the end of G1. Other studies of MCM binding sites in mammalian cells also report similar overall loading in both transcriptionally active and repressed genomic loci which generally correlate with euchromatin and heterochromatin respectively 18, 60 .…”

“…In many species, replication timing in S phase shows a general (though not exclusive) pattern of early euchromatin replication and late heterochromatin replication 71, 72 . Differences in the density of G1 MCM loading at different individual loci have been implicated in these S phase replication timing differences; more ORC or MCM loading in G1 correlates with early firing origins 17, 60, 73 . Early firing in S and early G1 origin licensing may both be consequences of general DNA accessibility in euchromatin.…”

“…Loaded MCM complexes are very stably-associated with DNA and are essentially only unloaded in S phase 57 or very locally displaced by active transcription [58][59][60] . The remarkable stability of loaded MCM means that licensing in G1 is largely unidirectional, and the loaded MCM we detect in late G1 is the sum of all the loading that has occurred since early G1.…”