Accurate duplication of genetic material and epigenetic inheritance is crucial for an organism's survival. The Origin Recognition Complex (ORC) plays an indispensable role in DNA replication in eukaryotes. The Origin Recognition Complex Associated (ORCA) is a recently identified interactor of ORC and has the potential to turn out to be a major player in heterochromatin organization and replication. ORCA is required for stabilization of ORC on chromatin. Also, ORCA knockdown has been shown to cause arrest of cells at G1/S boundary. Finally, ORC along with ORCA has been shown to bind to repressive histone marks.
I aim to dissect out the functions of ORCA in regulating heterochromatin organization and replication. While origins of DNA replication are defined by their sequences in yeast, their specification in metazoans is not clearly understood. Chromatin context, epigenetic marks and novel factors have been proposed to influence the process. ORCA, because of its ability to read repressive histone modifications, could turn out to be the factor determining origi specification in metazoans specifically at heterochromatin.
The aim of the project is to elucidate the role of ORCA in in two crucial processes in metazoans, namely heterochromatin organization and replication. Since ORCA interacts with repressive histone marks, ORCA's association with both differentially modified histones and with histone lysine methyltransferases (KMTs) that create those modifications will be examined by a combination of different biochemical and single molecule pulldown (SiMPull) approaches. Peptide pulldown and SiMPull assays will be carried out to accurately determine the specific modifications to which ORCA binds. Co-immunoprecipitation, glycerol gradient sedimentation and SiMPull experiments will be carried out to understand the nature of complexes containing ORCA and KMTs. In particular, ORCA's interaction with the H3K9 KMTs G9a and Suv39H1will be analyzed in great detail. The composition of ORCA-ORC-G9a-Suv39H1 complexes will be analyzed by both glycerol gradient sedimentation analysis and SiMPull and co-localization experiments to determine whether all these players exist in a single complex. Assays will be carried out to determine whether the association of KMTs with chromatin and with other H3K9 KMTs is compromised upon ORCA knockdown. To understand how ORCA regulates heterochromtin replication, BrdU Chromatin Immunoprecipiation (ChIP) will be carried out at various stages of S phase and the effect of ORCA knockdown on replication of late-replicating heterochromatin will be assessed. Also, ORCA ChIP will be carried out and ORCA's genomic binding profile will be analyzed to determine if ORCA binds specifically to heterochromatin. The proposed project will throw light on a novel regulatory mechanism of metazoan heterochromatin maintenance. As cancer is a disease of aberrant cell division, understanding the various regulatory mechanisms that exist to regulate cell cycle both at the level of epigenetic and replicative control is imperative for developing better therapeutic strategies to combat the disease.
My project will help identify the functions of ORCA in heterochromatin organization and replication. Investigating the molecular details of organization and replication of heterochromatin by characterizing the functions of novel players such as ORCA is crucial for understanding and effectively treating diseases of uncontrolled cell division and aberrant heterochromatin regulation such as cancer.