Association of ORCA/LRWD1 with repressive histone methyl transferases mediates heterochromatin organization

Abstract

Heterochromatin mostly constitutes tightly packaged DNA, decorated with repressive histone marks, including histone H3 methylated at lysine 9, histone H4 methylated at lysine 20 and histone H3 methylated at lysine 27. Each of these marks is incorporated by specific histone lysine methyl transferases. While constitutive heterochromatin enriched with H3K9me3 and H4K20me3 occur within repetitive elements, including centromeres and telomeres, the facultative heterochromatin resides on the inactive X-chromosome and contains H3K27me3 mark. Origin recognition complex-associated (ORCA/LRWD1) protein is required for the initiation of DNA replication and also plays crucial roles in heterochromatin organization. ORCA associates with constitutive and facultative heterochromatin in human cells and binds to repressive histone marks. We demonstrate that ORCA binds to multiple repressive histone methyl transferases including G9a, GLP, Suv39h1 (H3K9me2/3), Suv420h1/h2 (H4K20me2/3) and EZH2 (H3K27me3). Removal of ORCA from human cells causes aberrations in the chromatin architecture. We propose that ORCA acts as a scaffold protein that enables the formation of multiple histone lysine methyltransferase complexes at heterochromatic sites thereby facilitating chromatin organization.

Keywords: ORCA/LRWD1; heterochromatin; histone methyl transferases; replication.

Figure 1.

ORCA interacts with H3K9, K27 and H4K20 histone lysine methyltransferases. (A) U2OS 2–6–3 CLTon cells co-transfected with individual YFP-LacI-KMTs and CFP-ORCA. Inset represents 200% magnification of the boxed region.

Figure 2.

ORCA interacts with Suv420H2. (A) a. Schematic representation of various truncation mutants of ORCA containing a T7-epitope on the N-terminus. The specific domains that can associate with Suv420H2 are depicted as '+'. IP in U2OS cells expressing various T7-ORCA mutants and HA-Suv420H2 using T7 Ab and analysis by T7 and HA- IB. (B) a. Schematic representation of various truncation mutants of Suv420H2 containing a HA-epitope on the N-terminus. The interaction domains of Suv420H2 that interact with ORCA are denoted as '+'. b. IP in U2OS cells expressing various HA-Suv39H1 mutants and T7-G9a using HA Ab and analysis by T7 and HA IB.

Figure 3.

Model depicting the role of ORCA in organizing different chromatin domains. Model representing the mode of regulation of chromatin at constitutive and facultative heterochromatin and at origins by ORCA. Inactive X chromosome is depicted as an example of facultative heterochromatin.