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的准确复制对于生物体的生存至关重要。起源识别复合物（ORC）在真核生物的DNA复制中起着不可或缺的作用。起源识别复合体相关蛋白 (ORCA) 是最近发现的 ORC 相互作用蛋白，有可能成为异染色质组织和复制的主要参与者。 ORCA 是 ORC 在染色质上的稳定所必需的。此外，ORCA 敲低已被证明会导致细胞在 G1/S 边界停滞。最后，ORC 和 ORCA 已被证明可以与抑制性组蛋白标记结合。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复制的起点是由它们在酵母中的序列定义的，但它们在后生动物中的具体情况尚不清楚。染色质背景、表观遗传标记和新因素被认为会影响这一过程。由于 ORCA 具有读取抑制性组蛋白修饰的能力，因此可能成为决定后生动物特别是异染色质起源规范的因素。该项目的目的是阐明 ORCA 在后生动物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，因此将通过不同生化和单分子下拉 (SiMPull) 方法的组合来检查 ORCA 与差异修饰组蛋白和产生这些修饰的组蛋白赖氨酸甲基转移酶 (KMT) 的关联。将进行肽 Pulldown 和 SiMPull 测定，以准确确定 ORCA 结合的特定修饰。将进行免疫共沉淀、甘油梯度沉降和SiMPull实验来了解含有ORCA和KMT的复合物的性质。特别是，ORCA 与 H3K9 KMT G9a 和 Suv39H1 的相互作用将被详细分析。 ORCA-ORC-G9a-Suv39H1 复合物的组成将通过甘油梯度沉降分析和 SiMPull 和共定位实验进行分析，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 与染色质以及与其他 H3K9 KMT 的关联是否因 ORCA 敲低而受到损害。为了了解 ORCA 如何调节异染色质复制，将在 S 期的各个阶段进行 BrdU 染色质免疫沉淀 (ChIP)，并评估 ORCA 敲低对复制晚期异染色质复制的影响。此外，还将进行 ORCA ChIP，并分析 ORCA 的基因组结合谱，以确定 ORCA 是否与异染色质特异性结合。该项目将揭示后生动物异染色质维持的新调控机制。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开发更好的治疗策略来对抗该疾病至关重要。