Investigating the role of TET deficiency in promoting T cell expansion and inflammation

摘要 Dna胞嘧啶甲基化(以下简称dna甲基化)在细胞谱系中起着至关重要的作用。 抑制基因组中的重复和转座元件。DNA甲基转移酶将甲基 产生5-甲基胞嘧啶(5mC)；Tet甲基胞嘧啶双加氧酶通过以下方式引起DNA去甲基化 将5mC的甲基氧化为5-羟甲基胞嘧啶(5HmC)及以上。我们已经证明了Tet- 缺陷细胞类型不仅表现出启动子和增强子DNA甲基化的预期增加，而且 也是基因组异染色质区域DNA甲基化的矛盾减少。后果是什么？ 其中的分子特征尚不清楚，但在全基因组DNA甲基化中类似的变化 在癌症和衰老中已经观察到了这种模式。 通过研究几个Cre重组酶可诱导表达的小鼠品系的表型 在免疫/造血细胞类型中，我们发现两个或更多Tet基因的缺失 相关细胞类型中的细胞谱系承诺倾斜，其方式与细胞谱系的变化相关 承诺。然而，更引人注目的表型是，TET2/3fl/fl CD4Cre小鼠表现出大量的TCR- INKT细胞的依赖扩增；TET2/3fl/fl Foxp3Cre小鼠发展为显性促炎 杂合子雌性小鼠、混合骨髓嵌合体和免疫活性小鼠的表型观察 受者注射TET2/3fl/fl Foxp3Cre小鼠的全部CD4+T细胞。这种表型与 在杂合子Foxp3+/-雌性和注射Foxp3缺陷的免疫活性小鼠中观察到 细胞，这些细胞不会发展成疾病。在目标1中，我们将阐述惊人扩张背后的机制 TET2/3基因缺陷的iNKT细胞体内过继转移和新近发展的细胞培养 重现体外扩张过程的系统。在目标2中，我们将询问主导的自身免疫/ TET2/3缺陷的T调节细胞的炎性表型直接或间接要求DNA减少 到目前为止，在每一种Tet缺乏的细胞类型中都观察到了异染色质的甲基化。DNA减少 异染色质中的甲基化导致“异染色质功能障碍”，这是一种与 自身免疫性/炎症性疾病、癌症、衰老和神经退行性疾病，源于异常 转座元件(TES)的表达和由此导致的DNA损伤。DNA损伤引发“不育” 炎症“：RNA和DNA先天免疫感知通路的激活，从而导致表达上调 I型干扰素、干扰素诱导基因和促炎细胞因子(如IL-1b、IL-6、IFNG、IL-17) 我们提议的实验将增加我们关于Tet蛋白如何影响T细胞增殖和T细胞的知识 监管职能。更广泛地说，它们将增进我们对连接TET的链接的总体理解 自身免疫性/炎症性疾病、克隆性造血、癌前病变 与炎症、心血管疾病和癌症相关的老年人的综合症。

Abstract DNA cytosine methylation (hereafter, DNA methylation) has a critical role in cell lineage specification as well as suppression of repetitive and transposable elements in the genome. DNA methyltransferases attach a methyl group to generate 5-methylcytosine (5mC); TET methylcytosine dioxygenases cause DNA demethylation by oxidizing the methyl group of 5mC to 5-hydroxymethylcytosine (5hmC) and beyond. We have shown that TET- deficient cell types display not only the expected increase in DNA methylation at promoters and enhancers, but also a paradoxical decrease in DNA methylation in heterochromatic regions of the genome. The consequences of these molecular features remain to be understood, but similar alterations in genome-wide DNA methylation patterns have been observed in cancer and aging. By studying the phenotypes of several mouse strains in which Cre recombinase was expressed either inducibly or developmentally in immune/ hematopoietic cell types, we showed that deletion of two or more Tet genes skewed cell lineage commitment in the relevant cell type, in a manner that correlated with changes in cell lineage commitment. More striking phenotypes, however, were that Tet2/3 fl/fl CD4Cre mice displayed massive TCR- dependent expansion of iNKT cells; and that Tet2/3 fl/fl Foxp3Cre mice developed a dominant proinflammatory phenotype observed in heterozygous female mice, in mixed bone marrow chimaeras, and in immunocompetent recipients injected with total CD4+ T cells from Tet2/3 fl/fl Foxp3Cre mice. This phenotype differs markedly from that observed in heterozygous Foxp3+/- females and in immunocompetent mice injected with Foxp3-deficient cells, which do not develop disease. In Aim 1, we will address the mechanisms underlying the striking expansion of Tet2/3-deficient iNKT cells by using adoptive transfer approaches in vivo and recently-developed cell culture systems that recapitulate the expansion in vitro. In Aim 2, we will ask whether the dominant autoimmune/ inflammatory phenotype of Tet2/3-deficient T regulatory cells requires, directly or indirectly, the decreased DNA methylation in heterochromatin observed in every TET-deficient cell type examined so far. Decreased DNA methylation in heterochromatin results in “heterochromatin dysfunction”, an aberrant cellular condition linked to autoimmune/ inflammatory disorders, cancer, aging, and neurodegenerative diseases, that stems from aberrant expression of transposable elements (TEs) and resulting DNA damage. DNA damage provokes “sterile inflammation”: activation of innate immune sensing pathways for RNA and DNA with consequent upregulation of type I interferons, interferon-induced genes and proinflammatory cytokines (e.g. IL-1b, IL-6, IFNg, IL-17) Our proposed experiments will add to our knowledge of how TET proteins influence T cell expansion and T regulatory function. More broadly, they will enhance our general understanding of the links connecting TET deficiency and TE expression with autoimmune/ inflammatory diseases, clonal hematopoiesis, a premalignant syndrome of older individuals associated with inflammation and cardiovascular disease, and cancer.