Heterochromatic domains are enriched with repressive bottoms histone marks, including histone H3 lysine 9 methylation, written by lysine methyltransferases (KMTs).The pre-replication complex protein, origin recognition complex-associated (ORCA/LRWD1), preferentially localizes to heterochromatic regions in post-replicated cells.Its role in heterochromatin organization remained elusive.ORCA recognizes methylated H3K9 marks and interacts with repressive KMTs, including G9a/GLP and Suv39H1 in a chromatin context-dependent manner.
Single-molecule pull-down assays demonstrate that ORCA-ORC (Origin Recognition Complex) and multiple H3K9 KMTs exist in a single complex and that ORCA stabilizes H3K9 KMT complex.Cells Fly Rugs lacking ORCA show alterations in chromatin architecture, with significantly reduced H3K9 di- and tri-methylation at specific chromatin sites.Changes in heterochromatin structure due to loss of ORCA affect replication timing, preferentially at the late-replicating regions.We demonstrate that ORCA acts as a scaffold for the establishment of H3K9 KMT complex and its association and activity at specific chromatin sites is crucial for the organization of heterochromatin structure.