Regulation of nucleosome positions and transcription during CTL differentiation

初始CD8 T细胞的激活启动其分化为效应和记忆细胞毒性T淋巴细胞（CTL），利用细胞毒性蛋白穿孔蛋白（Prf1）、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Runx3在CD8 T细胞激活时对Prf1、Gzmb和Ifng基因的转录激活至关重要。此外，我们的研究结果表明，Runx3诱导T-box转录因子Eomesodermin并与之合作。然而，这些因子指导染色质结构编程以建立和维持CTL分化的基本原理尚不清楚。核小体是染色质的基本重复亚基，它们直接结合DNA，这使得编码的转录因子结合基序与同源因子的结合基序模糊。在哺乳动物系统中，很少有证据可以解释转录因子如何侵入核小体DNA并结合其同源位点以改变转录程序；酶促核小体重塑和核小体的热力学：DNA和转录因子：DNA相互作用可能决定转录因子的结合。为了研究这一基本问题，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，并将其应用于高度可处理的细胞培养系统，该系统概括了效应和记忆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发生了显著的占用变化，这些变化在效应CTL和记忆CTL条件下是不同的。值得注意的是，通过染色质免疫沉淀判断，大多数重构的核小体定位于Runx3转录因子物理占据的DNase I超敏（DHS）位点。这表明Runx3结合可能直接调节核小体的占用。在本提案中，我们将验证Runx3结合控制核小体位置和占用的假设，以建立使CTL分化的潜在染色质结构。我们的目标是绘制Runx3结合位点的全基因组图谱，并在CTL分化期间滴定Runx3的表达，以测试Runx3是否在体内与核小体竞争DNA占用（目的1）。接下来，我们将确定在CTL分化过程中使用Brg1 atp酶的染色质重塑复合物的全基因组分布，并阐明Runx3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（目的2）。最后，我们将生成并分析Prf1位点中三个重要DHS位点之一缺失的小鼠，该位点与Runx3结合，并在CTL分化时经历核小体消耗，并确定Runx3缺失如何影响病毒感染期间穿孔素表达和CTL分化（目的3）。这些目标的成功完成将提供决定CTL分化的亲和染色质景观的第一眼，并将有助于澄清与发育调节的转录因子如何获得其在染色质中的结合位点相关的基本问题。

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.