Specification of Origins of DNA Replication Within Distinct Chromatin States in Human Cells
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A comprehensive map of binding sites for the Origin Recognition Complex (ORC) in the human genome reveals relationships among replication licensing, chromatin state, three-dimensional genome organization, location of origins of DNA replication and DNA replication timing. Defining these relationships is essential for understanding how replication licensing is integrated with the broader organization and duplication of the human genome. Using 30 high-quality ORC1 CUT&RUN datasets, an ORC1 three-dimensional binding matrix (ORC1 3DM) was constructed that captures genomic position, binding intensity, and reproducibility across replicates. This analysis reveals considerable heterogeneity in ORC1 binding, and integration with chromatin-state maps shows that ORC1-binding sites separate into distinct epigenomic groups, with high-confidence sites enriched in H2A.Z1- and H3K4me3-associated open chromatin. Hi-C analysis demonstrates preferential localization of ORC1 binding to sub-regions of genomic A compartments and frequent association with loop anchors, supported by a direct interaction between ORC1 and CTCF. Replication-timing analysis shows that ORC1-binding sites are enriched in initiation zones and exhibit subgroup-specific temporal patterns. An integrated analysis combining multiple replication initiation assays and chromatin features further places ORC1 3DM within a unified replication–chromatin framework. Together, these results support a hierarchical, probabilistic model of replication licensing that is embedded within chromatin architecture.
Significance Statement
Understanding how DNA replication licensing relates to chromatin organization requires quantitative mapping and integration of genomic features. We developed an ORC1 three-dimensional binding matrix (ORC1 3DM) from 30 CUT&RUN datasets, quantifying genomic binding coordinates, intensity and reproducibility. We established an integrative framework to compare diverse genomic datasets across 25 chromatin states and a high-resolution approach to characterize replication timing profiles and their relationships with replication initiation. These approaches revealed distinct chromatin-associated ORC1-binding groups, preferential localization of high-confidence ORC1 sites in active chromatin and highly interactive A-compartment subregions, and subgroup-specific relationships with replication initiation and timing. Together, these findings and methodological advances provide a quantitative framework for investigating relationships among replication licensing, chromatin state, three-dimensional genome organization, and replication timing.
