Mechanics of LCR action on perforin to establish cytotoxicity in NK cells and CTL

LCR 对穿孔素作用以在 NK 细胞和 CTL 中建立细胞毒性的机制

• 批准号: 7743840
• 负责人: Matthew Eugene Pipkin
• 金额: $ 1.25万
• 依托单位: IMMUNE DISEASE INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-12-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7743840
• 关键词: Ablation; Alleles; Biochemical; CD4 Positive T Lymphocytes; Cell Culture Techniques; Cell Differentiation process; Cells; Child; Chromatin; Chromatin Structure; Cytokine Gene; Cytoplasmic Granules; Cytotoxic T-Lymphocytes; DNA Polymerase II; Deoxyribonuclease I; Disease; Distal; Doctor of Philosophy; Environment; Epigenetic Process; Event; Family; Functional RNA; Generations; Genes; Genetic; Genetic Transcription; Genomics; Goals; Health; Helper-Inducer T-Lymphocyte; Human; Immune; Immune response; Immunotherapy; Individual; Infant; Infection; Inflammatory; Inherited; Internal Ribosome Entry Site; Knowledge; Laboratories; Link; Locus Control Region; Lymphocyte; Maintenance; Malignant - descriptor; Malignant Neoplasms; Maps; Mechanics; Mediating; Messenger RNA; Methods; Modeling; Modification; Molecular; Monoclonal Antibodies; Mus; Mutant Strains Mice; Mutation; Natural Killer Cells; Pathway interactions; Patients; Phosphorylation; Physiological; RNA; RNA Polymerase II; Recruitment Activity; Regulatory Element; Reporter; Role; Site; T cell differentiation; Training; Transcriptional Regulation; Transgenes; Transgenic Mice; Vaccine Design; Virus Diseases; base; chromatin immunoprecipitation; chromatin remodeling; cytotoxic; cytotoxicity; familial hemophagocytic lymphohistiocytosis; histone modification; human disease; killings; mouse perforin; neuronal cell body; perforin; prevent; promoter; red fluorescent protein; skills; transgene expression; tumor

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to delineate the mechanisms of physiological perforin gene transcription. This knowledge is lacking, but is essential for optimal design of vaccines and immunotherapy that successfully prevent or treat cancer and infection. Perforin is a pore-forming molecule that is required for the ability of natural killer (NK) cells and cytotoxic T-lymphocytes (CTL) to kill malignant or infected cells using cytotoxic granules. Natural and experimental perforin deficiency renders humans and mice susceptible to spontaneous tumors, viral infection and a lethal inflammatory disease. The basis of perforin expression in NK cells and CTL is transcription of the perforin gene. The entire genomic territory required for physiological human perforin transcription spans 150 kb and comprises 16 DNase I hypersensitive sites (DHSs); four distal DHSs comprise its locus control region (LCR) and are required in transgenes, to drive physiological transcription. The objective of this proposal is to elucidate in mechanistic detail how these long-range domains, particularly the LCR, controls perforin. To achieve this objective, the long-range chromatin remodeling that occurs across perforin during primary CTL differentiation will be characterized (Aim 1). Transgenic mice will be generated in which the human perforin LCR can be deleted conditionally; the mouse LCR will also be targeted for conditional deletion, and an internal ribosome entry site-red fluorescent protein reporter will be inserted after the perforin translational stop to tag its mRNA and trace endogenous perforin expression in individual, vital cells. Mechanics for how the LCR functions to control transcription will be determined by combining ablation of the LCR with biochemical analysis of chromatin accessibility, histone modification, and recruitment and activity of RNA pol II, at the perforin promoter (Aim 2). Finally, the cis-acting requirement of distal non-coding sequences that are deleted from null perforin alleles, in patients with human perforin deficiency, will be investigated to continue defining the essential role of perforin transcription in health and disease (Aim 3). During immune responses, special immune cells develop to kill malignant or infected body cells using a molecule called perforin. Certain people whose immune cells cannot make perforin develop cancer quickly, and usually die as infants or children. The ability of immune cells to make perforin depends on activating its gene. In this study, we will determine how immune cells activate the perforin gene and convert inert immune cells into potent killers that can eradicate cancer. This knowledge will guide vaccine design, and methods to stimulate a patients own immune cells to kill their cancer or viral infection.

描述（由申请人提供）：该提案的长期目标是描述生理穿孔基因转录的机制。缺乏这些知识，但对于成功预防或治疗癌症和感染的疫苗和免疫疗法的最佳设计至关重要。孔蛋白是一种孔形成分子，是天然杀伤（NK）细胞和细胞毒性T淋巴细胞（CTL）使用细胞毒性颗粒杀死恶性或感染细胞的能力所必需的。天然和实验性穿孔蛋白缺乏使人类和小鼠容易受到自发肿瘤，病毒感染和致命的炎症性疾病的影响。 NK细胞和CTL表达表达的基础是穿孔蛋白基因的转录。生理人穿孔蛋白转录所需的整个基因组领土跨度为150 kb，包括16个DNase I超敏部位（DHSS）；四个远端DHSS组成其基因座控制区域（LCR），在转基因中需要进行生理转录。该提案的目的是阐明这些远程域，尤其是LCR的机械详细信息如何控制穿孔蛋白。为了实现这一目标，将表征远程染色质重塑，在初级CTL分化过程中跨孔蛋白发生的远距离染色质重塑（AIM 1）。将产生转基因小鼠，其中可以有条件地删除人穿孔蛋白LCR；小鼠LCR还将针对条件缺失，并且在穿孔蛋白转换停止后将插入内部核糖体入口位点红色荧光蛋白报道器，以标记其mRNA和痕量内源性型在个体，生命细胞中。 LCR如何控制转录的力学将通过将LCR的消融与染色质可及性，组蛋白修饰以及RNA POL II在perforin启动子处的染色质可及性，组蛋白修饰以及募集和活性的生化分析来确定（AIM 2）。最后，将研究从无效的穿孔蛋白缺乏素中删除的远端非编码序列的顺式作用要求，该序列将被调查，以继续确定穿孔素转录在健康和疾病中的基本作用（AIM 3）。在免疫反应过程中，特殊的免疫细胞使用称为perforin的分子杀死恶性或感染的身体细胞。某些免疫细胞无法使穿孔蛋白快速发展癌症的人，通常会死于婴儿或儿童。免疫细胞产生穿孔蛋白的能力取决于激活其基因。在这项研究中，我们将确定免疫细胞如何激活穿孔蛋白基因并将惰性免疫细胞转化为可以消除癌症的有效杀手。这些知识将指导疫苗设计，以及刺激患者自己的免疫细胞以杀死其癌症或病毒感染的方法。