Role of ORCA in heterochromatin organization and replication

ORCA 在异染色质组织和复制中的作用

• 批准号: 8718491
• 负责人: Sumanprava Giri
• 金额: $ 4.27万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-16 至 2016-08-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8718491
• 关键词: Affinity; Binding; Biochemical; Biological Assay; Bromodeoxyuridine; Cell Cycle; Cell Survival; Cell division; Cells; Chromatin; Co-Immunoprecipitations; Complex; DNA Sequence; DNA biosynthesis; DNA replication origin; Data; Disease; Epigenetic Process; Eukaryota; Event; G1 Arrest; G1 Phase; Geminin; Genetic Materials; Genome; Genomics; Glycerol; Heterochromatin; Histone-Lysine N-Methyltransferase; Histones; Human; Leucine-Rich Repeat; Light; Link; Maintenance; Malignant Neoplasms; Methylation; Modification; Molecular; Nature; Organism; Peptides; Photobleaching; Play; Pre-Replication Complex; Process; Proteins; Reader; Reading; Recruitment Activity; Regulation; Replication Initiation; Replication Origin; Role; S Phase; Scaffolding Protein; Sedimentation process; Specific qualifier value; Staging; Testing; Therapeutic; Time; Transgenes; Work; Yeasts; combat; domain mapping; histone methyltransferase; histone modification; in vivo; inhibitor/antagonist; insight; novel; origin recognition complex; public health relevance; research study; scaffold; single molecule; stoichiometry

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：遗传物质和表观遗传遗传的准确复制对于生物的生存至关重要。原点识别复合物（ORC）在真核生物中的DNA复制中起着必不可少的作用。 Origin识别复合物相关（ORCA）是最近确定的ORC交互者，并且有可能成为异染色质组织和复制的主要参与者。 ORCA在染色质上的稳定性是必需的。同样，逆戟检察到在G1/s边界处导致细胞停滞。最后，已显示兽人与ORCA一起结合抑制性组蛋白标记。我的目的是剖析ORCA在调节异染色质组织和复制中的功能。虽然DNA复制的起源是由其在酵母中的序列定义的，但它们在后生动物中的规范尚未清楚地理解。已经提出了染色质环境，表观遗传标记和新因素来影响该过程。由于其能够读取抑制性组蛋白修饰的能力，因此可能是确定异质素中特殊的原始规范的因素。该项目的目的是阐明Orca在后生动物中的两个关键过程中的作用，即异染色质组织和复制。由于Orca与抑制性组蛋白标记相互作用，因此Orca与差异修饰的组蛋白以及组蛋白赖氨酸甲基转移酶（KMT）的关联将通过不同的生物化学和单个分子pollldown（Simpull）方法的组合来检查产生这些修饰的组蛋白。将进行肽下拉和Simpull分析，以准确确定ORCA结合的特定修饰。将进行共免疫沉淀，甘油梯度沉积和SIMPULL实验，以了解含有Orca和KMT的复合物的性质。特别是，ORCA与H3K9 KMTS G9A和SUV39H1 WRIL的互动将进行详细分析。甘油梯度沉积分析和SIMPULL和共定位实验将分析ORCA-ORC-G9A-SUV39H1复合物的组成，以确定所有这些参与者是否存在于单个复合物中。将进行测定，以确定KMT与染色质和其他H3K9 KMT的关联是否在逆逆碳酸检检测后会受到损害。为了了解Orca如何调节异氯丁蛋白的复制，将在S期的各个阶段进行BRDU染色质免疫沉淀（CHIP），并且将评估Orca敲低复制的对晚期复制异染料素的影响。此外，将进行ORCA芯片，并将分析Orca的基因组结合曲线，以确定ORCA是否与异染色质特别结合。拟议的项目将阐明多唑异染色质维持的新型调节机制。由于癌症是一种异常细胞分裂的疾病，因此了解在表观遗传和复制控制水平上调节细胞周期的各种调节机制对于制定更好的治疗策略以对抗疾病。