Spatiotemporal mapping of ORC1 reveals its chromatin binding landscape and function in endogenous retrovirus silencing
Abstract
Origin Recognition Complex subunit 1 (ORC1) is essential for ORC assembly at sequence-independent mammalian origins and regulates diverse epigenetic functions. Thus, mapping its genomic distribution is critical to elucidate the mechanisms underlying mammalian replication origin selection and its broader epigenetic roles. However, existing genomic profiles exhibit considerable variation due to inherent technical challenges. To address this, we generated a mouse embryonic stem (mES) cell line with an HA-MNase-mAID tag knocked into the endogenous Orc1 locus and optimized a ChEC-seq approach to map ORC1 chromatin binding with high specificity and reproducibility. Our profiling identified approximately 70,000 ORC1 binding sites broadly distributed across euchromatin and heterochromatin, spanning both early- and late-replicating regions. Intriguingly, cell-cycle-resolved profiling revealed that the ORC1 binding landscape remains remarkably stable, including in S phase. Through repli-ChEC-seq, we found that ORC1 rapidly recovers chromatin binding following DNA replication. Despite this persistent chromatin occupancy, ORC1 is dispensable for S phase progression in mES cells. Instead, we uncovered that ORC1 is involved in the epigenetic inheritance of H3K9me3 and the transcriptional repression of endogenous retroviruses (ERVs). Together, these findings provide a comprehensive spatiotemporal binding map of ORC1 in mES cells, and support the expansion of ORC1’s functional repertoire ranging from canonical DNA replication to heterochromatin maintenance and ERV silencing.
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