Abstract
Heterochromatic domains are enriched with repressive 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 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.
Highlights
Origin recognition complex-associated (ORCA/LRWD1), a protein required for the initiation of DNA replication, preferentially localizes to heterochromatic regions in post-replicated cells (Bartke et al, 2010; Shen et al, 2010; Vermeulen et al, 2010)
By using a highly sensitive and quantitative single-molecule pull-down (SiMPull) approach (Jain et al, 2011; Shen et al, 2012), we demonstrate that ORCA preferentially binds to H3K9me3 and ORCA-ORC, and multiple H3K9 KMTs exist in a single complex
In order to address if ORCA interacts with the machinery that causes the establishment of heterochromatin, we used a candidate approach to investigate the interaction of ORCA with individual H3K9 KMTs that catalyze H3K9 repressive modifications
Summary
Origin recognition complex-associated (ORCA/LRWD1), a protein required for the initiation of DNA replication, preferentially localizes to heterochromatic regions in post-replicated cells (Bartke et al, 2010; Shen et al, 2010; Vermeulen et al, 2010). We and others have demonstrated that ORCA and ORC associate with centromeric and telomeric heterochromatin in mammalian cells (Shen et al, 2010). Using a stable isotope labeling by amino acids in cell culture (SILAC)-based proteomic approach, ORCA-ORC complex has been shown to bind the repressive histone lysine methylation marks, H3K9me, H3K27me, and H4K20me (Bartke et al, 2010; Vermeulen et al, 2010) that are known to be enriched at heterochromatic sites. ORCA is critical for stabilizing ORC binding to chromatin
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