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

• 批准号: 10224893
• 负责人: Matthew Eugene Pipkin
• 金额: $ 30.24万
• 依托单位: SCRIPPS FLORIDA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2022-04-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10224893
• 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; transcriptional reprogramming; tumor

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

项目摘要/摘要 项目2(管道) 初始CD8T细胞在细胞内感染时的激活迅速诱导染色质重塑和 转录重编程导致记忆分化(TMEM)CTL提供长期 豁免权。我们发现转录因子(Tf)Runx3促进了TMEM染色质的可及性。 在初始T细胞受体(TCR)刺激过程中幼稚细胞中相关的顺式调节区，并且是必不可少的 用于区分循环和组织驻留的TMEM CTL。RUNX3激活转录回路 这建立了新生的CTL，抑制了不同的细胞命运，并阻止了终末CTL的分化。vbl.使用 我们鉴定了与Runx3相关的NuRD/HDAC复合体的所有亚单位。此外, 我们在CD8T细胞中进行了集中的RNA干扰(RNAi)介导的功能丧失筛查 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复合亚单位缺陷损害了记忆前体CTL的分化， 类似于Runx3功能丧失。此外，BAF核小体重构体家族的多个亚基和 它的合作者CHD7在人类免疫缺陷的情况下发生突变，对于很早就启动是必不可少的 CTL分化和推动终末分化的方面。要深入了解CTL的早期方面 开发过程中，我们使用了单细胞rna-seq(scrna-seq)方法并进行了计算轨迹。 哪些分析确定了共同的发展途径，然后从幼稚的CD8引导 对于效应器和记忆细胞的命运。在一定程度上，我们使用Blimp1-YFP在操作上确认了这些通路 报告等位基因和采用迁移实验。在当前的应用程序中，我们建议以这些结果为基础 阐明CRF和TFS如何在幼稚细胞激活和早期对染色质结构重新编程 效应器和记忆样发育路径的建立。具体地说，我们将定义Runx3和 NuRD/HDAC复合体重塑染色质结构，以建立初始的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效应器和类似记忆的发育途径，并使用体内条件RNAi方法 天然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重建器的治理方式相结合 控制早期CTL转录重编程的顺式调控区的核小体组织 差异化(目标3)。这些研究与处理角色的类似方法相结合。 CRF和TF在TFH分化和功能中的作用(Crotty，项目1)，以及它们在以后如何发挥作用 维持特定的CD4和CD8 TMEM细胞亚群的分化和功能 感染和浸润性肿瘤(Goldrath，项目3)。 1