bZIP proteins, NFAT, and lymphocyte gene induction

bZIP 蛋白、NFAT 和淋巴细胞基因诱导

基本信息

批准号：
8761495
负责人：
Patrick Hogan
金额：
$ 67.89万
依托单位：
LA JOLLA INSTITUTE FOR IMMUNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-01 至 2019-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8761495
关键词：
Address Affect Antigens Antiviral Response Autoimmunity B-Lymphocytes Binding Binding Sites Biological Biological Assay Biological Models Boron Brain CD28 gene CD3 Antigens CD8B1 gene Calcineurin Calcineurin inhibitor Calcium Calmodulin Cell Culture Techniques Cell Differentiation process Cell Nucleus Cell physiology Cells Cellular biology Chromatin Chronic Clinical Collaborations Complementary DNA Complex Cultured Cells Cyclosporine Cytotoxic T-Lymphocytes DNA DNA Binding DNA-Binding Proteins Data Development Drosophila genus Elements Employee Strikes Equilibrium FK506 Family Family member Fibroblasts Fluorescence Resonance Energy Transfer Funding Gene Activation Gene Expression Gene Expression Profile Gene Targeting Genes Genome Genomic DNA Homologous Gene Human IRF4 gene Immediate-Early Genes Immune Immune System Diseases Immune response Immune system Immunologic Deficiency Syndromes Immunosuppressive Agents In Vitro Indium Inherited Interleukin-2 Ionophores Laboratories Leucine Zippers Light Lymphocyte Mus Mutation Myocardium National Cancer Institute Nature Neurons Nuclear Nucleic Acid Regulatory Sequences Organ Output Pancreas Paper Pathway interactions Pattern Peptides Pharmaceutical Preparations Phase Phenotype Phorbol Esters Play Positioning Attribute Procedures Proteins RNA Interference RNA Sequences Reagent Role Signal Transduction Site Skeletal Muscle Skin Smooth Muscle Structure T cell anergy T cell response T-Cell Activation T-Cell Receptor T-Lymphocyte Testing Th1 Cells Time Transcription Factor AP-1 Transformed Cell Line Tumor-Infiltrating Lymphocytes Variant Virus Diseases bZIP Protein base bone calcineurin phosphatase cancer immunotherapy cell type chemokine chemokine receptor chromatin immunoprecipitation clinical efficacy cytokine design dimer embryonic stem cell exhaustion gene induction genome-wide high throughput screening in vivo inhibitor/antagonist medical schools member muscular system mutant new technology next generation sequencing novel nuclear factor of activated T-cells, cytoplasmic 2 protein nuclear factors of activated T-cells programs promoter public health relevance research study response small molecule text searching transcription factor transcription factor NF-AT c3 transcriptome sequencing tumor

项目摘要

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.

描述（由申请人提供）：钙/钙调神经/ NFAT途径对于自适应免疫反应至关重要，这是由钙调蛋白抑制剂环孢菌素A（CSA）和FK506的临床疗效强调的点。 NFAT在许多其他器官和细胞类型的发育和功能中也起着重要作用 - 神经元，心脏，骨骼和平滑肌，骨骼，皮肤，胰腺以及脉管系统。然而，令人惊讶的是，尚未在全基因组水平上进行详细检查NFAT转录因子如何结合顺式调节DNA元件并改变细胞的转录谱。在此应用中，我们解决了CD4+ TH1和CD8+细胞溶解T细胞（CTL）的这一点。 NFAT蛋白的惊人特征是它们能够与无关转录因子（例如AP-1）形成紧密的复合物（fos-jun）在DNA中的“复合”元素上。我们已经表明，在没有伴侣的情况下，NFAT诱导了与NFAT：AP-1复合物引起的T细胞激活程序不同的基因表达（T细胞“ Anergy”或“ Enterion”）的负调节程序。实际上，我们的初步数据表明，在没有AP-1的情况下，NFAT可能充当CD8+ T细胞耗尽的“主调节剂”，这是一个假设，将在此处进行检验。在应用程序的目标1中，我们将在与AP -1合作或缺乏合作的条件下，研究三种免疫相关NFAT蛋白-NFAT1，NFAT2和NFAT4-与CTL基因组DNA和CTL基因组DNA中的调节区域的结合。我们将执行染色质免疫沉淀（CHIP），然后进行下一代测序（CHIP-SEQ），以及一种称为CHIP-EXO的变体过程，更紧密地定义了DNA中这些转录因子的结合位点。我们还将通过对表达蛋白质表达蛋白质的细胞进行稳态或“新生”（染色质相关的）RNA测序来定义由这些野生型和突变NFAT蛋白调节的基因表达模式。在AIM 2中，我们将在人类T细胞中重复这些实验。在AIM 3中，我们将实现一个新颖的高通量屏幕，以识别阻断NFAT：AP-1相互作用的化合物，而不会影响NFAT与DNA的结合或NFAT复合物与其他伴侣（例如Foxp3）的结合。在AIM 4中，我们将将AIM 1和2中在体外的AIM 2中定义的基因表达的模式与体内免疫反应过程中获得的模式相关联，并询问表达无法与AP-1合作的突变NFAT蛋白的T细胞是否诱导更深刻的T细胞表型在病毒感染期间T细胞厌食/精疲力尽。从临床角度来看，我们的项目与癌症免疫疗法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CTL和慢性病毒感染期间的T细胞反应。