How does chromatin dictate pre-replication complex positioning and activation in humans?
With every cell division, the genome needs to be faithfully duplicated. Tens of thousands of DNA replication initiation sites (origins of replication) are involved in replicating the human genome. The pre-replication complex (pre-RC) is the basis for replication initiation and consists of two major subcomponents: the origin recognition complex (ORC) binds DNA and is required for loading of the second component, Mcm2-7 helicases, which initiate DNA replication. Chromatin features driving pre-RC positioning on the human genome remain largely unknown. Genome-wide pre-RC chromatin immunoprecipitation experiments followed by sequencing (ChIP-seq) studies are rare but required for full understanding of DNA replication regulation.
This work presents the first genome-wide ChIP-seq analysis of the two major pre-RC subcomponents ORC and Mcm2-7 in the Epstein-Barr virus (EBV) infected Burkitt’s lymphoma cell line Raji. Pre-RC sequencing results correlate with zones of active replication, which validated the ChIP-seq approach. Pre-RC binding was preferentially found at regions of active transcriptional regulation, presumably determined by chromatin accessibility. In heterochromatin, ORC was enriched at H4K20me3 sites, while Mcm2-7 enrichment was less prominent. Employing a plasmid-based replication system, ORC association to H4K20me3 was proven to promote successful pre-RC assembly and replication initiation, situating direct ORC-chromatin interactions being the major determinant for DNA replication regulation in heterochromatin. Taken together, this study proposes two different modes of pre-RC assembly regulation depending on chromatin environment.