HISTONE HYPERACETYLATION IN REPLICATING SV40 CHROMOSOMES

SV40 染色体复制中的组蛋白高度乙酰化

• 批准号: 7462223
• 负责人: Barry Ira Milavetz
• 金额: $ 6.75万
• 依托单位: UNIVERSITY OF NORTH DAKOTA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-15 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7462223
• 关键词: Address; Affect; Antibodies; Biological; Biological Process; Biotin; Chemicals; Chromatin; Chromatin Structure; Chromosomes; Complement; DNA; DNA biosynthesis; DNA chemical synthesis; Daughter; Deacetylation; Deoxyribonucleotides; Development; Disease; EP300 gene; Eukaryota; Eukaryotic Cell; Event; Gene Expression Regulation; Generations; Genes; Genetic Transcription; Goals; Histones; Immune; Immunoprecipitation; Infection; Investigation; Knowledge; Label; Life; Link; Malignant Neoplasms; Modeling; Nucleic Acid Regulatory Sequences; Organism; Plasmids; Play; Positioning Attribute; Principal Investigator; Proteins; Public Health; Replication-Associated Process; Research; Role; Side; Simian virus 40; Site; System; Techniques; Therapeutic; Virus Diseases; chromatin immunoprecipitation; chromatin remodeling; novel; programs; promoter

DESCRIPTION (provided by applicant): Using the Simian Virus 40 (SV40) chromosome as a model for eukaryotic chromatin, the proposed research will address the role of histone hyperacetylation at an active replication fork and during post-replication chromatin maturation within the regulatory region of a chromosome whose biological function changes over the course of infection. The studies will answer fundamental questions concerning the mechanism of eukaryotic replication and the relationship between eukaryotic replication and gene regulation which will be potentially applicable to the therapy of a wide variety of diseases including viral infections and cancer. The specific aims of the application are 1. Characterization of histone hyperacetylation in the vicinity of a replication fork and 2. Characterization of histone hyperacetylation associated with post-replicative chromatin remodeling within the SV40 promoter/regulatory region. Histone hyperacetylation in the vicinity of a replication fork will be characterized in replicating SV40 chromosomes either immune selected with antibodies which recognize proteins found only at the fork or labeled with biotin conjugated deoxyribonucleotides. In both strategies the status of histone hyperacetylation at various sites on the pre- and post-replicative sides of the replication fork will be determined using a combination of biophysical separation, inhibition of replication by chemicals and siRNAs, and chromatin fragmentation in combination with chromatin immunoprecipitation analyses including two which we have developed: Immune Selection and Fragmentation (ISF) and Immune Selection Fragmentation and Immunoprecipitation (ISFIP). Post-replication chromatin maturation within the regulatory region will be characterized in SV40 chromosomes and confirmed in replication-competent plasmids carrying portions of the SV40 regulatory region. Maturation occurring during replication will be determined in replication intermediates, while maturation occurring after replication will be determined by comparing histone hyperacetylation present in terminating replicating chromosomes to hyperacetylation present in chromosomes competent for late transcription and encapsidation. Immune selection with antibodies specific for replicating, transcribing, and encapsidating chromosomes will be used in conjunction with various ChIP techniques to determine the status of histone hyperacetylation in the chromosomes and plasmids. PUBLIC HEALTH RELEVANCE: The proposed studies will be the first detailed characterization of histone hyperacetylation during the eukaryotic replication process and will directly link changes in histone hyperacetylation to specific events occurring during the replication process. The results obtained will significantly add to our knowledge of the role of replication in eukaryotic gene regulation and be potentially useful in the development of therapeutics for viral infections and cancers.

描述（由申请人提供）：使用猿猴病毒40 （SV40）染色体作为真核染色质模型，拟议的研究将解决组蛋白超乙酰化在活跃复制叉和复制后染色质成熟过程中的作用，染色体的调节区域的生物学功能在感染过程中发生变化。这些研究将回答有关真核复制机制以及真核复制与基因调控之间关系的基本问题，这将潜在地适用于包括病毒感染和癌症在内的各种疾病的治疗。该应用程序的具体目标是：1。在复制叉和2附近组蛋白超乙酰化的表征。SV40启动子/调控区内与复制后染色质重塑相关的组蛋白超乙酰化特征在复制叉附近的组蛋白超乙酰化将在SV40染色体的复制中被表征，要么被免疫选择的抗体识别仅在叉上发现的蛋白质，要么被生物素结合的脱氧核糖核苷酸标记。在这两种策略中，将使用生物物理分离、化学物质和sirna的复制抑制、染色质碎片化以及染色质免疫沉淀分析(包括我们开发的两种：免疫选择和碎片化（ISF）和免疫选择碎片化和免疫沉淀（ISFIP）相结合的方法来确定复制叉复制前和复制后各个位点的组蛋白超乙酰化状态。调控区域内的复制后染色质成熟将在SV40染色体中被表征，并在携带SV40调控区域部分的复制能力质粒中得到证实。在复制过程中发生的成熟将在复制中间体中确定，而在复制后发生的成熟将通过比较终止复制染色体中存在的组蛋白超乙酰化和在负责晚期转录和衣壳化的染色体中存在的超乙酰化来确定。针对复制、转录和封装染色体的特异性抗体的免疫选择将与各种ChIP技术结合使用，以确定染色体和质粒中组蛋白超乙酰化的状态。公共卫生相关性：拟议的研究将是真核生物复制过程中组蛋白超乙酰化的第一个详细特征，并将直接将组蛋白超乙酰化的变化与复制过程中发生的特定事件联系起来。获得的结果将显著增加我们对真核生物基因调控中复制作用的认识，并可能对开发病毒感染和癌症的治疗方法有用。