AbstractsBiology & Animal Science

In eukaryotes, Origin recognition complex (ORC) proteins establish the pre-replicative complex (pre-RC) at the origins and this is essential for the initiation of DNA replication. In human cells, ORC is a highly dynamic complex with many separate functions attributed to sub-complexes or individual subunits of ORC including heterochromatin organization, telomere and centromere function, centrosome duplication and cytokinesis. Heterochromatic domains are enriched with repressive histone marks, including histone H3 lysine 9 methylation, written by lysine methyltransferases (KMTs). ORC along with the pre-RC protein Origin Recognition Complex-Associated (ORCA/LRWD1), preferentially localizes to heterochromatic regions in post-replicated cells. The role of ORCA and ORC in heterochromatin organization remained elusive. In Chapter II, I describe my efforts to understand the significance of ORCA-ORC’s association with heterochromatin. 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 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 affects replication timing, preferentially at the late-replicating regions. I 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. 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. ORCA associates with constitutive and facultative heterochromatin in human cells and binds to repressive histone marks. In Chapter III, I show 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. I therefore 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. Open chromatin structures regulate the efficiency of preRC formation and replication initiation. However, the molecular mechanisms that affect chromatin structure and how the preRC components establish… Advisors/Committee Members: Prasanth, Supriya G (advisor), Prasanth, Supriya G (Committee Chair), Stubbs, Lisa J (committee member), Belmont, Andrew S (committee member), Freeman, Brian C (committee member), Ha, Taekjip (committee member).