Epigenetic control of Foxp3 expression in induced T regulatory cells

摘要 调节性T细胞(Treg)表达FOXP3，这是一种编码在X染色体上的转录因子。Tregs是 对预防自身免疫和维持免疫动态平衡和耐受性至关重要。幼稚的CD4+T细胞可以 在培养中转化为“诱导”Tregs(ITregs)，但与内源性Tregs相比，iTregs 在细胞分裂或过继转移后，Foxp3的表达迅速丧失。Foxp3基因表达的稳定性 与内含子增强子Foxp3 CNS2的DNA甲基化状态有关：内源性产生 Tregs在CNS2几乎完全没有甲基化，而幼稚的CD4+T细胞和iTregs在体外被 转化生长因子β加维甲酸(RA)几乎完全甲基化。 我们几年前发现，Tet家族的双加氧酶通过氧化5-甲基-4-酮改变DNA修饰状态。 甲基胞嘧啶(5mC)至5-羟甲基胞嘧啶(5hmC)。5HmC可被Tet蛋白进一步氧化为5-HmC。 甲酰胞嘧啶(5fC)和5-羧基胞嘧啶(5caC)。所有三种氧化的甲基胞嘧啶都会干扰主要- DNA甲基转移酶1的维持功能，从而影响“被动”复制依赖的DNA去甲基化。 细胞分裂过程中的分裂。此外，DNA修复酶胸腺嘧啶DNA糖基酶(TDG) 可以切除5fC和5caC，然后通过碱基切除修复将其替换为未修饰的C。 我们最近发现，Tet酶促进了Treg中CNS2的去甲基化。通过激活幼稚的T细胞 在转化生长因子β、RA和四环素激活剂维生素C的存在下，我们可以产生非常稳定的人类 和小鼠iTregs，其中Foxp3表达的稳定性与内源性Tregs相当。 维生素C促进Tet介导的CNS2去甲基化，而RA增加没有CNS2的Foxp3的稳定性 去甲基化。 我们在这项应用中的目标是研究Tet甲基胞嘧啶氧化酶在T细胞谱系中的作用 分子和动力学层面的规范，重点是iTreg的差异化。使用最先进的 在我们实验室开发的技术和基本试剂中，我们将在 ITreg分化的早期和后期(目标1)；研究TDG在DNA去甲基化中的重要性 在iTreg细胞分化过程中(目标2)；并确定RA和维生素C在维持Foxp3稳定性中的作用 在划分iTregs时(目标3)。我们的研究具有很强的临床意义，因为iTreg细胞是在培养中产生的 有可能在移植医学和治疗自身免疫性疾病方面有用。 我们提出的研究将加强我们对DNA如何产生的最基本问题的理解 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，进而决定细胞谱系的可塑性。有可能，我们的 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、移植排斥反应和自身免疫性疾病。

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.