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

描述（由申请人提供）：本提案的长期目标是描述生理穿孔素基因转录的机制。这些知识是缺乏的，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。穿孔蛋白是天然杀伤（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需的成孔分子。天然和实验性穿孔素缺乏使人类和小鼠易患自发性肿瘤、病毒感染和致命的炎性疾病。穿孔素在NK细胞和CTL中表达的基础是穿孔素基因的转录。生理人类穿孔素转录所需的整个基因组区域跨越150 kb，包括16个DNA酶I超敏位点（DHS）;四个远端DHS包括其基因座控制区（LCR），并在转基因中需要，以驱动生理转录。这个建议的目的是阐明这些远程域，特别是LCR，控制穿孔素的详细机制。为了实现这一目标，将表征在初级CTL分化期间跨穿孔素发生的长距离染色质重塑（目标1）。将产生转基因小鼠，其中人穿孔素LCR可以条件性缺失;小鼠LCR也将被靶向条件性缺失，并且在穿孔素翻译终止后插入内部核糖体进入位点-红色荧光蛋白报告基因，以标记其mRNA并追踪个体活细胞中的内源性穿孔素表达。LCR如何控制转录的机制将通过结合LCR的消融与染色质可及性、组蛋白修饰以及穿孔素启动子处RNA pol II的募集和活性的生化分析来确定（Aim 2）。最后，在人类穿孔素缺乏症患者中，将研究从无效穿孔素等位基因中删除的远端非编码序列的顺式作用要求，以继续确定穿孔素转录在健康和疾病中的重要作用（目的3）。在免疫反应过程中，特殊的免疫细胞通过一种叫做穿孔素的分子杀死恶性或感染的体细胞。某些免疫细胞不能制造穿孔素的人会很快患上癌症，通常会在婴儿或儿童时期死亡。免疫细胞制造穿孔素的能力取决于激活它的基因。在这项研究中，我们将确定免疫细胞如何激活穿孔素基因，并将惰性免疫细胞转化为可以根除癌症的强效杀手。这些知识将指导疫苗设计，以及刺激患者自身免疫细胞杀死癌症或病毒感染的方法。

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.