Epigenetic control of Foxp3 expression in induced T regulatory cells

摘要 调节性T细胞（Tcells）表达FOXP 3，这是一种在X染色体上编码的转录因子。treg是 对于预防自身免疫和维持免疫稳态和耐受性至关重要。幼稚的CD 4 + T细胞可以是 在培养物中转化为“诱导的”T细胞（iT细胞），但与内源性产生的T细胞相比，iT细胞 在细胞分裂时或过继转移后迅速丧失Foxp 3表达。Foxp 3表达的稳定性 已与内含子增强子Foxp 3 CNS 2的DNA甲基化状态相关联：内源性产生 T细胞在CNS 2处几乎完全未甲基化，而初始的CD 4 + T细胞和iT细胞在体外被激活， TGFβ +视黄酸（RA）几乎完全甲基化。 几年前，我们发现TET家族双加氧酶通过氧化5-氨基-1，4-二硫代苯甲酸酯来改变DNA修饰状态。 甲基胞嘧啶（5 mC）至5-羟甲基胞嘧啶（5 hmC）。5 hmC可被泰特蛋白进一步氧化成5- 甲酰胞嘧啶（5 fC）和5-羧基胞嘧啶（5caC）。三种氧化甲基胞嘧啶都会干扰主要的- 维持DNA甲基转移酶1的功能，从而影响“被动”复制依赖性DNA去甲基化， 在细胞分裂过程中。此外，DNA修复酶胸腺嘧啶DNA糖基化酶（TDG） 可以切除5 fC和5caC，然后通过碱基切除修复用未修饰的C替换。 我们最近发现，泰特酶促进CNS 2去甲基化。通过激活幼稚T细胞 在TGFβ、RA和泰特激活剂维生素C的存在下，我们可以产生异常稳定的人 和小鼠iTclA，其中Foxp 3表达的稳定性似乎与内源性TclA的稳定性相当。 维生素C促进TET介导的CNS 2去甲基化，而RA在没有CNS 2的情况下增加Foxp 3稳定性 去甲基化 我们的目的是研究泰特甲基胞嘧啶氧化酶在T细胞谱系中的作用 在分子和动力学水平上的特异性，重点是iTreg分化。使用最先进的 技术和必要的试剂在我们的实验室开发，我们将探讨泰特蛋白的作用， iTreg分化的早期和晚期阶段（目的1）;研究TDG在DNA去甲基化中的重要性 在iTreg细胞分化过程中（Aim 2）;并确定RA和维生素C在维持Foxp 3稳定性中的作用 在划分iTterm（目标3）方面。我们的研究具有很强的临床相关性，因为iTreg细胞在培养中产生， 有可能在移植医学和治疗自身免疫性疾病中发挥作用。 我们提出的研究将增强我们对DNA如何 修饰调节基因转录，特别是关于谱系表达的稳定性， 决定转录因子，这反过来又决定了细胞谱系的可塑性。潜在地，我们 这些数据还将有助于确定免疫相关疾病治疗干预的新候选者，包括 癌症免疫治疗、移植排斥和自身免疫性疾病。

Abstract Regulatory T cells (Tregs) express FOXP3, a transcription factor encoded on the X-chromosome. Tregs are critical to prevent autoimmunity and maintain immune homeostasis and tolerance. Naïve CD4+ T cells can be converted into “induced” Tregs (iTregs) in culture, but compared to endogenously generated Tregs, iTregs rapidly lose expression of Foxp3 upon cell division or after adoptive transfer. The stability of Foxp3 expression has been linked to the DNA methylation status of an intronic enhancer, Foxp3 CNS2: endogenously generated Tregs are almost fully unmethylated at CNS2, whereas naïve CD4+ T cells and iTregs activated in vitro with TGFβ plus retinoic acid (RA) are almost fully methylated. We discovered several years ago that TET-family dioxygenases alter DNA modification status by oxidizing 5- methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). 5hmC can be further oxidized by TET proteins to 5- formylcytosine (5fC) and 5-carboxylcytosine (5caC). All three oxidized methylcytosines interfere with the main- tenance function of DNA methyltransferase 1, thus effecting “passive” replication-dependent DNA demethy- lation during the course of cell division. Additionally, the DNA repair enzyme thymine DNA glycosylase (TDG) can excise 5fC and 5caC, which are then replaced with unmodified C through base excision repair. We have recently shown that TET enzymes promote CNS2 demethylation in Tregs. By activating naïve T cells in the presence of TGFβ, RA and the TET activator Vitamin C, we can generate exceptionally stable human and mouse iTregs, in which the stability of Foxp3 expression appears equivalent to that of endogenous Tregs. Vitamin C promotes TET-mediated CNS2 demethylation, whereas RA increases Foxp3 stability without CNS2 demethylation. Our goal in this application is to investigate the role of TET methylcytosine oxidases in T cell lineage specification at a molecular and kinetic level, with a focus on iTreg differentiation. Using state-of-the-art technologies and essential reagents developed in our laboratory, we will explore the role of TET proteins at early and later stages of iTreg differentiation (Aim 1); investigate the importance of TDG in DNA demethylation during iTreg cell differentiation (Aim 2); and define the roles of RA and Vitamin C in maintaining Foxp3 stability in dividing iTregs (Aim 3). Our studies have strong clinical relevance, since iTreg cells generated in culture have the potential to be useful in transplant medicine and to cure autoimmune disease. Our proposed studies will enhance our understanding of the very fundamental question of how DNA modification regulates gene transcription, particularly with respect to the stability of expression of lineage- determining transcription factors, which in turn determines the plasticity of cellular lineages. Potentially, our data will also help identify novel candidates for therapeutic intervention in immune-related disorders, including cancer immunotherapy, transplant rejection and autoimmune disease.