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: 10683275
• 负责人: Matthew Eugene Pipkin
• 金额: $ 67.84万
• 依托单位: UNIVERSITY OF 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 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10683275
• 关键词: Acute; Address; Adoptive Transfer; Affect; Alleles; Automobile Driving; Binding Sites; Biochemical; 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; Immunotherapy; Individual; Infection; Infiltration; Lead; Link; Lymphocytic choriomeningitis virus; Maps; Mass Spectrum Analysis; Mediating; Memory; Memory impairment; Molecular; Mutate; NuRD complex; Nucleic Acid Regulatory Sequences; Nucleosomes; Pathway interactions; Peptide Initiation Factors; Phase; Phenotype; Physical condensation; Process; Proteins; RNA Interference; RNA interference screen; RUNX3 gene; Regulation; Reporter; Repression; Role; Site; T-Cell Development; T-Cell Receptor; T-Lymphocyte; 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

项目总结/摘要 项目2（Pipkin） 细胞内感染期间初始CD 8 T细胞的活化迅速诱导染色质重塑， 转录重编程，导致记忆（TMEM）CTL的分化， 免疫力我们发现，转录因子（TF）Runx 3可促进TMEM-1的染色质可及性。 在初始T细胞受体（TCR）刺激期间，初始细胞中的相关顺式调节区，并且是必需的 用于区分循环和组织驻留TMEM CTL。Runx 3激活转录回路 其建立新生CTL，抑制替代细胞命运并阻止终末CTL分化。使用 通过质谱分析，我们鉴定了与Runx 3相关的NuRD/HDAC复合物的所有亚基。此外，本发明还提供了一种方法， 我们在CD 8 T细胞中进行了一项合并的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。这揭示了 多个单独的NuRD/HDAC复合物亚单位的缺陷损害了记忆前体CTL分化， 类似于Runx 3功能丧失。此外，BAF家族的核小体重塑和 它的合作者Chd 7在人类免疫缺陷中发生突变， CTL分化和驱动终末分化方面。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 这些分析确定了共同的，然后从幼稚的CD 8细胞分支的发育途径， 效应细胞和记忆细胞的命运在某种程度上，我们使用Blimp 1-YFP在操作上证实了这些途径。 报告等位基因和过继转移实验。在当前的应用中，我们建议建立在这些结果的基础上 阐明CRF和TF如何在幼稚细胞活化和早期细胞凋亡过程中重编程染色质结构， 效应器和记忆样发育路径的建立。具体来说，我们将定义Runx 3和 NuRD/HDAC复合物重塑染色质结构以建立初始TMEM转录程序（目的1）。 我们将阐明Runx 3、Ets 1、Blimp 1和Bcl 6的分级功能如何调节Blimp 1hi的趋异 和Blimp 1 lo效应和记忆样发育途径，并使用体内条件性RNAi方法， 幼稚CD 8筛选所有T细胞表达的TF（1，751个基因），以确定它们在病毒感染过程中在这一过程中的作用。 感染（目标2）。最后，我们将把这些分析与BAF和Chd 7改造如何治理 在早期CTL过程中控制转录重编程的顺式调节区中的核小体组织 差异化（目标3）。这些研究与类似的方法协同整合， CRF和TF在TFH分化和功能中的作用（Crotty，项目1），以及它们在以后的时间如何发挥作用， 维持特异性CD 4和CD 8 TMEM细胞亚群的分化和功能， 感染和浸润肿瘤（Goldrath，项目3）。 1