Regulation of nucleosome positions and transcription during CTL differentiation

描述（由申请人提供）：概述初始CD 8 T细胞的活化启动其分化为效应和记忆细胞毒性T淋巴细胞（CTL），其使用细胞毒性蛋白穿孔素（Prf 1）、颗粒酶B（Gzmb）和效应细胞因子如Ifng来控制感染和肿瘤。我们发现，转录因子Runx 3是必不可少的Prf 1，Gzmb和Ifng基因的转录激活后，CD 8 T细胞活化。此外，我们的研究结果表明，Runx 3诱导，然后与T-box转录因子Eomesodermin合作。然而，指导这些因子对染色质结构进行编程以建立和维持CTL分化的基本原理是未知的。核小体是染色质的基本重复亚基，并且它们直接结合DNA，这使得编码的转录因子结合基序与它们的同源因子模糊不清。在哺乳动物系统中，很少有证据解释转录因子如何侵入核小体DNA以结合其体内同源位点以改变转录程序;酶促核小体重塑和核小体：DNA和转录因子：DNA相互作用的热力学可能决定转录因子结合。为了研究这个基本问题，我们开发了一种创新的方法，以非常高的分辨率绘制核小体在DNA上的体内位置和占用率，并将其应用于高度易处理的细胞培养系统中，该系统概括了效应子和记忆CTL分化的重要方面。我们的初步研究表明，核小体在分化过程中的占有率发生了显着的变化，这些变化在效应和记忆CTL条件下是不同的。值得注意的是，大多数核小体重塑定位在DNA酶I超敏（DHS）的网站，物理上被Runx 3转录因子占据，通过染色质免疫沉淀判断。这表明Runx 3结合可能直接调节核小体占据。在本提案中，我们将检验Runx 3结合控制核小体位置和占据以建立使CTL分化成为可能的潜在染色质结构的假设。我们的目标是在全基因组范围内定位Runx 3结合位点，并在CTL分化过程中滴定Runx 3表达，以测试Runx 3是否与核小体竞争体内DNA占有率（目的1）。接下来，我们将确定在CTL分化过程中使用Brg 1 ATP酶的染色质重塑复合物的全基因组分布，并阐明Runx 3是否需要其活性来改变它们共同占据的顺式结构域中的核小体位置（Aim 2）。最后，我们将产生和分析小鼠缺乏三个重要的DHS位点之一的Prf 1基因座结合Runx 3和经历核小体耗竭后，CTL分化，并确定Runx 3缺陷如何影响穿孔素表达和CTL分化在病毒感染（目的3）。这些目标的成功完成将提供第一次看到的亲和力染色质景观，决定CTL分化，并将有助于澄清有关的基本问题，如何发育调节转录因子获得访问其结合位点的染色质。 公共卫生相关性：疫苗旨在诱导细胞毒性T淋巴细胞（CTL）的产生，后者可杀死病毒感染的细胞和肿瘤。必须表达特异性基因以使CTL完全具有杀伤能力。我们将定义重新格式化染色体结构以激活这种特定基因表达模式的机制。

DESCRIPTION (provided by applicant): Summary Activation of naive CD8 T cells initiates their differentiation into effector and memory cytotoxic T lymphocytes (CTL) that control infections and tumors using the cytotoxic proteins perforin (Prf1), granzyme B (Gzmb), and effector cytokines such as Ifng. We showed that the transcription factor Runx3 is essential for the transcriptional activation of the Prf1, Gzmb, and Ifng genes upon CD8 T cell activation. In addition, our results showed that Runx3 induces and then cooperates with the T-box transcription factor Eomesodermin. However, the basic principles that guide programming of chromatin structure by these factors to establish and maintain CTL differentiation are unknown. Nucleosomes are the fundamental repeating subunit of chromatin, and they directly bind DNA, which obscures encoded transcription factor binding motifs from their cognate factors. There is very little evidence in mammalian systems to explain how transcription factors invade nucleosomal DNA to bind their cognate sites in vivo in order to change transcriptional programs; both enzymatic nucleosome remodeling and the thermodynamics of nucleosome:DNA and transcription factor:DNA interactions are likely to determine transcription factor binding. To study this fundamental problem, we developed an innovative method to map the in vivo positions and occupancy of nucleosomes on DNA at very high resolution and applied it in the context of a highly tractable cell-culture system that recapitulates important aspects of effector and memory CTL differentiation. Our preliminary studies indicate that nucleosomes undergo striking changes in occupancy during differentiation, and these changes are distinct in effector and memory CTL conditions. Notably, most nucleosomes that are remodeled localize in DNase I hypersensitive (DHS) sites that are physically occupied by Runx3 transcription factors, as judged by chromatin immunoprecipitation. This suggests that Runx3 binding might regulate nucleosome occupancy directly. In this proposal we will test the hypothesis that Runx3 binding controls nucleosome positions and occupancy to establish the underlying chromatin structure that enables CTL differentiation. Our goals are to map Runx3 binding sites genome-wide and to titrate Runx3 expression during CTL differentiation to test whether Runx3 competes with nucleosomes for DNA occupancy in vivo (Aim 1). Next we will determine the genome-wide distribution of chromatin remodeling complexes that use the Brg1 ATPase during CTL differentiation, and clarify whether Runx3 requires its activity to alter nucleosome positions in cis-domains that they co-occupy (Aim 2). Lastly, we will generate and analyze mice lacking one of three important DHS sites in the Prf1 locus that binds Runx3 and that undergoes nucleosome depletion upon CTL differentiation, and determine how Runx3 deficiency affects perforin expression and CTL differentiation during viral infection (Aim 3). Successful completion of these Aims will provide the first look at the affinity chromatin landscape that determines CTL differentiation, and will help to clarify the basic problem relating to how a developmentally regulated transcription factor gains access to its binding sites in chromatin. PUBLIC HEALTH RELEVANCE: Vaccines are intended to elicit development of cytotoxic T lymphocytes (CTL), which kill virally infected cells and tumors. Specific genes must be expressed to fully arm CTL with the capacity to kill. We will define the mechanisms that reformat chromosome architecture to activate this specific gene expression pattern.