Chromatin and transcriptional regulatory factors that initiate and stabilize memory CD8 T cell development

启动和稳定记忆 CD8 T 细胞发育的染色质和转录调节因子

基本信息

批准号：
10024588
负责人：
Matthew Eugene Pipkin
金额：
$ 59.43万
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：
2020
资助国家：
美国
起止时间：
2020-08-01 至 2021-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10024588
关键词：
Acute Address Adoptive Transfer Affect Alleles Automobile Driving Binding Sites Biochemical Biological CD8-Positive T-Lymphocytes CD8B1 gene CHD7 gene Cells Chromatin Chromatin Remodeling Factor Chromatin Structure Complex Cytotoxic T-Lymphocytes Development Ensure Exhibits Family Genes Genetic Transcription Goals Half-Life Histone Acetylation Histone Deacetylase Hour Human Immunity Immunologic Deficiency Syndromes Individual Infection Lead Link Lymphocytic choriomeningitis virus Maps Mass Spectrum Analysis Mediating Memory Memory impairment Molecular Mutate NuRD complex Nucleic Acid Regulatory Sequences Nucleosomes Pathway interactions Phase Phenotype Physical condensation Process Proteins RNA Interference RNA interference screen RUNX3 gene Regulation Reporter Role Site T-Cell Development T-Cell Receptor T-Lymphocyte Time Tissues Transcriptional Regulation Vaccines Virus Diseases chromatin remodeling cytotoxic CD8 T cells effector T cell experimental study in vivo insight loss of function prevent programs recruit single-cell RNA sequencing synergism transcription factor tumor

项目摘要

PROJECT SUMMARY / ABSTRACT Project 2 (Pipkin) Activation of naive CD8 T cells during intracellular infections rapidly induces chromatin remodeling and transcriptional reprogramming that results in the differentiation of memory (TMEM) CTLs that provide long-term immunity. We discovered that the transcription factor (TF) Runx3 instigates chromatin accessibility of TMEM- associated cis-regulatory regions in naive cells during initial T cell receptor (TCR) stimulation, and is essential for the differentiation of both circulating and tissue resident TMEM CTLs. Runx3 activates transcriptional circuits that establishes nascent CTLs, represses alternative cell fates and prevents terminal CTL differentiation. Using mass spectrometry we identified all subunits of the NuRD/HDAC complex in association with Runx3. In addition, we conducted a pooled, RNA interference (RNAi) -mediated, loss-of-function screen in CD8 T cells responding to viral infection that targeted all mammalian chromatin regulator factors (CRFs, 312 genes). This revealed that deficiency in multiple individual NuRD/HDAC complex subunits impaired memory precursor CTL differentiation, similar to Runx3 loss-of-function. In addition, multiple subunits of the BAF-family of nucleosome remodelers and its collaborator Chd7, which is mutated in a human immunodeficiency, were essential for initiating very early aspects of CTL differentiation and driving terminal differentiation. To gain insight into early aspects of CTL development, we used a single cell RNA-seq (scRNA-seq) approach and performed computational trajectory analyses which identified common, and then branching developmental pathways from naive CD8s that lead toward effector and memory cell fates. In part, we confirmed these pathways operationally using Blimp1-YFP reporter alleles and adoptive transfer experiments. In the current application, we propose to build on these results to elucidate how CRFs and TFs reprogram chromatin structure during naive cell activation and early establishment of effector and memory-like developmental paths. Specifically, we will define how Runx3 and NuRD/HDAC complexes remodel chromatin structure to establish initial TMEM transcriptional programs (Aim 1). We will elucidate how hierarchical functions of Runx3, Ets1, Blimp1 and Bcl6 regulate the divergence of Blimp1hi and Blimp1lo effector- and memory-like developmental paths, and use an in vivo conditional RNAi approach in naive CD8s to screen all T cell-expressed TFs (1,751 genes) to identify their roles in this process during viral infection (Aim 2). Finally, we will integrate these analyses with how BAF and Chd7 remodelers govern nucleosome organization in cis-regulatory regions that control transcriptional reprograming during early CTL differentiation (Aim 3). These studies integrate synergistically with analogous approaches addressing the roles of CRFs and TFs in TFH differentiation and function (Crotty, Project 1), and how they function at later times to maintain the differentiation and function of specific CD4 and CD8 TMEM cell subsets that persist following infections, and that infiltrate tumors (Goldrath, Project 3). 1

项目概要/摘要项目2（皮普金）细胞内感染过程中幼稚 CD8 T 细胞的激活迅速诱导染色质重塑和转录重编程导致记忆 (TMEM) CTL 的分化，从而提供长期的免疫。我们发现转录因子 (TF) Runx3 可以促进 TMEM- 的染色质可及性在初始 T 细胞受体 (TCR) 刺激过程中，幼稚细胞中存在相关的顺式调节区域，并且是必不可少的用于区分循环和组织驻留 TMEM CTL。 Runx3 激活转录电路建立新生 CTL、抑制细胞命运的替代并防止最终 CTL 分化。使用通过质谱分析，我们鉴定了与 Runx3 相关的 NuRD/HDAC 复合物的所有亚基。此外，我们对 CD8 T 细胞反应进行了汇总、RNA 干扰 (RNAi) 介导的功能丧失筛选针对所有哺乳动物染色质调节因子（CRF，312 个基因）的病毒感染。这表明多个单独的 NuRD/HDAC 复合体亚基的缺陷损害了记忆前体 CTL 的分化，类似于 Runx3 功能丧失。此外，核小体重塑剂 BAF 家族的多个亚基和它的合作者 Chd7 在人类免疫缺陷中发生突变，对于早期启动至关重要 CTL 分化和驱动终端分化的方面。深入了解 CTL 的早期方面开发中，我们使用了单细胞 RNA-seq (scRNA-seq) 方法并进行了计算轨迹分析确定了来自幼稚 CD8 的共同的、然后分支的发育途径，这些途径导致影响效应细胞和记忆细胞的命运。在某种程度上，我们使用 Blimp1-YFP 确认了这些通路的可操作性报告等位基因和过继转移实验。在当前的应用中，我们建议以这些结果为基础阐明 CRF 和 TF 如何在幼稚细胞激活期间和早期重新编程染色质结构建立效应器和类似记忆的发展路径。具体来说，我们将定义 Runx3 和 NuRD/HDAC 复合物重塑染色质结构以建立初始 TMEM 转录程序（目标 1）。我们将阐明 Runx3、Ets1、Blimp1 和 Bcl6 的层次功能如何调节 Blimp1hi 的发散和 Blimp1lo 效应器和记忆样发育路径，并使用体内条件 RNAi 方法初始 CD8 筛选所有 T 细胞表达的 TF（1,751 个基因），以确定它们在病毒传播过程中的作用感染（目标 2）。最后，我们将把这些分析与 BAF 和 Chd7 重塑者的治理方式结合起来顺式调控区的核小体组织在早期 CTL 期间控制转录重编程差异化（目标 3）。这些研究与解决角色问题的类似方法协同整合 CRF 和 TF 在 TFH 分化和功能中的作用（Crotty，项目 1），以及它们后来如何发挥作用维持特定 CD4 和 CD8 TMEM 细胞亚群的分化和功能，这些细胞亚群持续存在感染，并浸润肿瘤（Goldrath，项目 3）。 1