Mechanisms Of Age-related Changes in Transcriptional Regulation in lympphocytes

淋巴细胞转录调节与年龄相关的变化机制

• 批准号: 10007357
• 负责人: Nan-ping Peter Weng
• 金额: $ 57.37万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10007357
• 关键词: ATAC-seq; Age; Aging; Antigens; Apoptosis; CD8-Positive T-Lymphocytes; CDKN1C gene; Cell Aging; Cell Cycle; Cell Survival; Cell division; Cell physiology; Cells; Cessation of life; ChIP-seq; Chromatin; Complex; Cyclin-Dependent Kinase Inhibitor; Elderly; Event; FOXP3 gene; Gene Expression; Gene Expression Profiling; Generations; Genes; Genetic Transcription; Goals; Histones; Immune response; Immune system; Immunity; Impairment; In Vitro; Infection; Knowledge; Listeria; Lysine; Malignant Neoplasms; Measures; Medical; Memory; Methyltransferase; Mus; Organism; Play; Polycomb; Population; Regulatory T-Lymphocyte; Reporting; Repression; Research; Rest; Role; Spleen; T memory cell; T-Cell Proliferation; T-Lymphocyte; Thymocyte Development; Time; Tissue-Specific Gene Expression; Transcriptional Regulation; age related; comparative; differential expression; histone methylation; improved; in vivo; inhibitor/antagonist; interest; pathogen; promoter; real-time images; response; transcriptome; transcriptome sequencing

Our goal is to understand how T cell functions and its change with aging. We focus on 1) how memory T cells are generated, maintained, and functioned in an immune response, and 2) identification of age-associated changes in chromatin and transcriptome of CD8 T cells. We are particularly interested in investigating the mechanism of differential transcription at levels of chromatin state. We apply ChIP-Seq, ATAC-seq, microarray, RNA-seq and scRNAseq to identify genes associated subset populations that differentially expressed in nave and memory T cells and to quantitate the chromatin states (measured by ChIP-seq and ATAC-seq) in the corresponding gene loci, and their changes with age. The parallel comparative analyses of gene expression and chromatin states reveal the chromatin basis of the differential gene expression and the role of chromatin in memory T cell functions and in CD8 T cell aging. Transition from resting to cell cycle in response to antigenic stimulation is an essential step for nave CD8+ T cells to differentiate to effector and memory cells. Leaving the resting state requires dramatic changes of chromatin status in the key cell cycle inhibitors but the details of these concerted events are not fully elucidated. Here, we showed that Ezh2, an enzymatic component of polycomb repressive complex 2 (PRC2) catalyzing the trimethylation of lysine 27 on histone 3 (H3K27me3), regulates activation induced nave CD8+ T cells proliferation and apoptosis. Upon deletion of Ezh2 during thymocyte development (Ezh2fl/flCd4Cre+ mice), naive CD8+ T cells displayed impaired proliferation and increased apoptosis in response to antigen stimulation. However, naive CD8+ T cells only had impaired proliferation but no increase in apoptosis when Ezh2 was deleted after activation (Ezh2fl/flGzmBCre+ mice), suggesting cell cycle and apoptosis are temporally separable events controlled by Ezh2. We then showed that deletion of Ezh2 resulted in the increase in expression of cyclin-dependent kinase inhibitors Cdkn2a (p16 and Arf) and Cdkn1c (p57) in activated nave CD8+ T cells as the consequence of reduced levels of H3K27me3 at these two gene loci. Finally, with real time imaging, we observed prolonged cell division times of nave CD8+ T cells in the absence of Ezh2 post in vitro stimulation. Together, these findings reveal that repression of Cdkn1c and Cdkn2a by Ezh2 plays a critical role in execution of activation-induced CD8+ T cell proliferation. Lysine specific methyl-transferase 2D (Kmt2d) catalyzes the mono-methylation of histone 3 lysine 4 (H3K4me1) and play a critical role in regulatory T cell generation via modulating Foxp3 gene expression. Here we report a role of Kmt2d in nave CD8+ T cell survival upon activation. First, we found that the number of CD8+ T cells, in particular nave CD8+ T cells (CD62Lhi/CD44lo), in spleen was greatly decreased in the absence of Kmt2d (Kmt2dfl/flCD4cre+, Kmt2d-/-) compared to wild type (Kmt2dfl/flCD4cre-, Kmt2dWT) mice. Second, we observed significant increase in death of nave CD8+ T cells upon stimulation in vitro. Third, we found reduced H3K4me1 amount in promoters and increased expressions of apoptosis-related genes in activated nave CD8+ T cells in the absence of Kmt2d. Finally, we confirmed increased activation-induced death of antigen specific nave CD8+ T cells in Kmt2d mice in vivo upon Listeria monocytogen infection. These findings reveal that Kmt2d regulates activation induced nave CD8+ T cell survival via modulating H3K4me1 levels in apoptosis-related gene expressions. Ageing has a profound detrimental impact on almost all living organisms. Immune systems play a particularly important role in protection against external challenges (pathogens) and internal insults (cancer) but their protective capacity commonly wanes with advancing age. With the rapid increase in the numbers of older people around the world, research in the field of immunity and ageing is becoming increasingly important. This realization, together with recent and ongoing technical advances in analytical capabilities, is facilitating rapid progress towards a better understanding of immunity and ageing and the resulting anticipated improved application of this knowledge to medical treatments in the years ahead.

我们的目标是了解T细胞的功能及其随衰老的变化。我们专注于1）记忆T细胞如何产生，维持和在免疫反应中发挥作用，以及2）识别CD 8 T细胞染色质和转录组中与年龄相关的变化。我们特别感兴趣的是在染色质状态水平上研究差异转录的机制。我们应用ChIP-Seq、ATAC-seq、微阵列、RNA-seq和scRNAseq来鉴定在原始和记忆T细胞中差异表达的基因相关亚群，并定量相应基因位点中的染色质状态（通过ChIP-seq和ATAC-seq测量）及其随年龄的变化。基因表达和染色质状态的平行比较分析揭示了差异基因表达的染色质基础以及染色质在记忆T细胞功能和CD 8 T细胞老化中的作用。 从静止到细胞周期的转变是原始CD 8 + T细胞分化为效应细胞和记忆细胞的重要步骤。离开静息状态需要关键细胞周期抑制剂中染色质状态的急剧变化，但这些协同事件的细节尚未完全阐明。在这里，我们表明，Ezh 2，多梳抑制复合物2（PRC 2）催化组蛋白3（H3 K27 me 3）上的赖氨酸27的三甲基化的酶组分，调节活化诱导的幼稚CD 8 + T细胞增殖和凋亡。在胸腺细胞发育过程中Ezh 2缺失后（Ezh 2fl/flCd 4Cre+小鼠），初始CD 8 + T细胞响应于抗原刺激显示增殖受损和凋亡增加。然而，当Ezh 2在活化后缺失时（Ezh 2fl/flGzmBCre+小鼠），初始CD 8 + T细胞仅具有受损的增殖，但凋亡没有增加，这表明细胞周期和凋亡是由Ezh 2控制的时间上可分离的事件。然后，我们发现Ezh 2的缺失导致细胞周期蛋白依赖性激酶抑制剂Cdkn 2a（p16和Arf）和Cdkn 1c（p57）在活化的原始CD 8 + T细胞中的表达增加，这是由于这两个基因位点的H3 K27 me 3水平降低。最后，通过真实的时间成像，我们观察到在体外刺激后，在不存在Ezh 2的情况下，幼稚CD 8 + T细胞的细胞分裂时间延长。总之，这些发现揭示了Ezh 2对Cdkn 1c和Cdkn 2a的抑制在激活诱导的CD 8 + T细胞增殖的执行中起着关键作用。 赖氨酸特异性甲基转移酶2D（Kmt 2d）催化组蛋白3赖氨酸4（H3 K4 me 1）的单甲基化，并通过调节Foxp 3基因表达在调节T细胞生成中起关键作用。在这里，我们报告了Kmt 2d在活化后的幼稚CD 8 + T细胞存活中的作用。首先，我们发现，与野生型（Kmt 2dfl/flCD 4cre-，Kmt 2dWT）小鼠相比，在不存在Kmt 2d（Kmt 2dfl/flCD 4cre+，Kmt 2d-/-）的情况下，脾脏中的CD 8 + T细胞，特别是原始CD 8 + T细胞（CD 62 Lhi/CD 44 lo）的数量大大减少。第二，我们观察到在体外刺激时幼稚CD 8 + T细胞的死亡显著增加。第三，我们发现在没有Kmt 2d的情况下，活化的nave CD 8 + T细胞中启动子中H3 K4 me 1的量减少，而凋亡相关基因的表达增加。最后，我们证实了单核细胞增生李斯特菌感染后Kmt 2d小鼠体内抗原特异性原始CD 8 + T细胞的激活诱导死亡增加。这些发现揭示了Kmt 2d通过调节阿尔茨海默病相关基因表达中的H3 K4 me 1水平来调节活化诱导的幼稚CD 8 + T细胞存活。 衰老对几乎所有生物体都有深远的有害影响。免疫系统在抵御外部挑战（病原体）和内部侮辱（癌症）方面发挥着特别重要的作用，但它们的保护能力通常会随着年龄的增长而减弱。随着世界各地老年人数量的迅速增加，免疫和衰老领域的研究变得越来越重要。这一认识，加上分析能力方面最近和正在取得的技术进步，正在促进在更好地了解免疫和衰老方面取得迅速进展，并预期在今后几年中将这一知识更好地应用于医疗。