HISTONE HYPERACETYLATION IN REPLICATING SV40 CHROMOSOMES

描述(申请人提供)：使用猿猴病毒40(SV40)染色体作为真核细胞染色质的模型，拟议的研究将探讨组蛋白超乙酰化在活跃的复制分叉和复制后染色质成熟过程中的作用，该染色体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细胞复制的机制以及真核细胞复制与基因调控之间的关系的基本问题，这将有可能应用于包括病毒感染和癌症在内的各种疾病的治疗。该应用的具体目的是1.组蛋白在复制叉处附近的超乙酰化的特征和2.与SV40启动子/调控区内复制后染色质重塑相关的组蛋白超乙酰化的特征。复制叉附近的组蛋白超乙酰化的特征是复制SV40染色体，要么用识别仅在复制叉上发现的蛋白质的抗体免疫选择，要么用生物素标记的脱氧核糖核苷酸标记。在这两种策略中，将结合生物物理分离、化学物质和siRNA对复制的抑制、染色质碎裂以及染色质免疫沉淀分析来确定复制叉前和复制后不同位置的组蛋白超乙酰化状态。染色质免疫沉淀分析包括两种分析方法：免疫选择和碎片化(ISF)和免疫选择碎片化和免疫沉淀(ISFIP)。调控区域内的复制后染色质成熟将在SV40染色体中表征，并在携带SV40调控区域部分的复制能力质粒中得到证实。复制过程中发生的成熟将在复制中间产物中确定，而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具有后期转录和封装能力的染色体中存在的高乙酰化来确定。带有复制、转录和包裹染色体的特异性抗体的免疫选择将与各种芯片技术结合使用，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细胞复制过程中组蛋白超乙酰化的第一个详细表征，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。所获得的结果将大大增加我们对复制在真核基因调控中的作用的了解，并可能有助于开发治疗病毒感染和癌症的药物。

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.