bZIP proteins, NFAT, and lymphocyte gene induction

说明(申请人提供)：钙/钙调神经磷酸酶/NFAT途径对于适应性免疫反应是必不可少的，钙调神经磷酸酶抑制剂环孢素A(CsA)和FK506的临床疗效强调了这一点。NFAT还在许多其他器官和细胞类型的发育和功能中发挥重要作用--神经元、心脏、骨骼和平滑肌、骨骼、皮肤、胰腺和血管系统。然而，令人惊讶的是，还没有在全基因组水平上详细研究NFAT转录因子如何结合顺式调节DNA元件和改变细胞的转录图谱。在本应用中，我们针对CD4+Th1和CD8+细胞溶解T细胞(CTL)解决这一点。NFAT蛋白的一个显著特征是能够与AP-1等无关的转录因子形成紧密的复合体 (FOS-Jun)对DNA中的“复合”元素的研究。我们已经证明，在没有其伴侣AP-1的情况下，NFAT诱导了一种不同于NFAT：AP-1复合体诱导的T细胞激活程序的基因表达负调控程序(T细胞无能或耗竭)。事实上，我们的初步数据表明，在缺乏AP-1的情况下，NFAT潜在地发挥CD8+T细胞耗竭的‘主调节’功能，这一假说将在这里得到验证。在应用的目的1中，我们将检测三种免疫相关的NFAT蛋白-NFAT1、NFAT2和NFAT4-在与AP-1合作或不合作的情况下与Th1细胞和CTL基因组DNA调控区的结合。我们将进行染色质免疫沉淀(CHIP)，然后进行下一代测序(CHIP-SEQ)，以及一种称为CHIP-EXO的变体程序，该程序更接近地定义这些转录因子在DNA中的结合位点。我们还将定义这些野生型和突变型NFAT蛋白调控的基因表达模式，方法是对表达这些蛋白的细胞进行RNA测序(RNA-seq)，对表达这些蛋白的细胞进行稳态或新生的(染色质相关)RNA测序。在目标2中，我们将在人类T细胞中重复这些实验。在目标3中，我们将实施一种新型的高通量筛选，以确定阻止NFAT：AP-1相互作用的化合物，而不影响NFAT与DNA的结合或与其他伙伴(如FOXP3)形成NFAT复合体。在目标4中，我们将把AIMS 1和AIMS 2中定义的体外基因表达模式与体内免疫反应中获得的模式联系起来，并询问表达突变的NFAT蛋白的T细胞在病毒感染过程中是否会诱导更深刻的T细胞无能/耗竭表型。从临床角度来看，我们的项目与癌症免疫治疗和病毒感染的治疗非常相关，因为CD8+T细胞耗尽限制了肿瘤浸润性CTL和慢性病毒感染期间的T细胞反应。

DESCRIPTION (provided by applicant): The calcium/ calcineurin/ NFAT pathway is essential for the adaptive immune response, a point underscored by the clinical efficacy of the calcineurin inhibitors cyclosporin A (CsA) and FK506. NFAT also plays an important role in the development and function of many other organs and cell types - neurons, heart, skeletal and smooth muscle, bone, skin, pancreas, and the vasculature. Surprisingly, however, there has not yet been a detailed examination, at a genome-wide level, of how NFAT transcription factors bind cis-regulatory DNA elements and change the transcriptional profiles of cells. In this application, we address this point for CD4+ Th1 and CD8+ cytolytic T cells (CTL). A striking feature of NFAT proteins is their ability to form tight complexes with unrelated transcription factors such as AP-1 (Fos-Jun) on "composite" elements in DNA. We have shown that NFAT in the absence of its partner AP-1 induces a negative regulatory programme of gene expression (T cell 'anergy' or 'exhaustion') that is distinct from the programme of T cell activation induced by NFAT: AP-1 complexes. Indeed, our preliminary data suggest that in the absence of AP-1, NFAT potentially functions as a 'master regulator' of CD8+ T cell exhaustion, a hypothesis that will be tested here. In Aim 1 of the application, we will examine the binding of the three immune-related NFAT proteins - NFAT1, NFAT2 and NFAT4 - to regulatory regions in genomic DNA of Th1 cells and CTL, under conditions of cooperation or lack of cooperation with AP-1. We will perform chromatin immunoprecipitation (ChIP) followed by next-generation sequencing (ChIP-seq), and a variant procedure known as ChIP-exo that more closely defines the binding sites for these transcription factors in DNA. We will also define the gene expression patterns regulated by these wild type and mutant NFAT proteins, by performing RNA-sequencing (RNA-seq) for steady-state or 'nascent' (chromatin-associated) RNA-sequencing on cells expressing the proteins. In Aim 2, we will repeat these experiments in human T cells. In Aim 3, we will implement a novel high-throughput screen to identify compounds that block the NFAT: AP-1 interaction without affecting the binding of NFAT to DNA or the formation of NFAT complexes with other partners such as FOXP3. In Aim 4, we will relate the patterns of gene expression defined in vitro in Aims 1 and 2 to the patterns obtained during immune responses in vivo, and ask whether T cells expressing mutant NFAT proteins that cannot cooperate with AP-1 induce a more profound phenotype of T cell anergy/ exhaustion during viral infections. From a clinical perspective, our project is very relevant to cancer immunotherapy and the treatment of viral infections, since CD8+ T cell exhaustion limits T cell responses in tumour-infiltrating CTL and during chronic viral infections.