Regulation and function of active DNA demethylation in Cellular Differentiation

胞嘧啶(5mC)5‘端甲基化在细胞分化和胚胎发育中起重要作用。尽管5mC很重要，但它也通过促进脱氨基形成胸腺嘧啶来增加CpG突变率。在人类中，5mC到胸腺嘧啶突变的发生率是其他碱基转换的10到50倍。由此产生的G：T和G：U(G：T/U)错配可以改变编码和调控序列。因此，有必要了解基因组甲基化动态的分子机制。最近的研究发现了一种新的DNA去甲基化途径，通过10个11位易位(Tet 1-3)将5mC氧化成5-羟甲基胞嘧啶(5hmC)。5HmC被四环素进一步代谢成5甲酰胞嘧啶(5fC)和5羧基胞嘧啶(5caC)。5fC和5caC被碱基切除修复酶胸腺嘧啶DNA糖基酶(TDG)识别和切除，以恢复未修饰的胞嘧啶。涉及Tet/TDG途径的活性去甲基化的发现是一项革命性的发现，极大地促进了我们对DNA甲基化动力学的理解。研究表明，5mC衍生物在干细胞分化、基因组印迹和神经元功能等方面具有重要作用。此外，还鉴定了每个衍生物的特定相互作用蛋白，从而反映了5hmC/5fC/5caC的独特生物学性质。这些被氧化的碱基到底是如何发挥作用的尚不清楚，需要更好地了解它们的沉积机制，以及全面描述5mC衍生物在哺乳动物基因组中分布的能力。这一建议侧重于两种互补的策略来检验这一假说，即主动去甲基化对基因转录响应分化信号是必不可少的。首先使用小鼠胚胎干细胞，它有能力经历神经谱系分化对维甲酸的反应，我们将使用一种名为甲基化辅助亚硫酸盐测序(MABseq)的新技术来定量绘制5fC/5caC的变化图。MABseq提供了Tet/TDG介导的碱基分辨活性的直接读数。其次，为了研究活性去甲基化的分子机制，我们将使用一种被称为邻近依赖的生物素鉴定(BioID)的蛋白质亲和纯化方法结合质谱学，来鉴定Tet/TDG在细胞分化过程中的蛋白质相互作用网络。主动DNA去甲基化代表了基因调控的一种新范式，对细胞生理学的各个方面都有重要的影响。总的来说，我们的研究将确定发生活性去甲基化的基因组位置，并与特定目标2中提出的实验相结合，将为理解动态DNA甲基化提供一个重要的框架。

Methylation at the 5' position of cytosine (5mC) plays an essential role in cell differentiation and embryonic development. Despite its importance, 5mC also increases the CpG mutation rate by promoting deamination to form thymine. In humans the incidence of 5mC to thymine mutations is 10 to 50-fold higher than other base transitions. The resulting G:T and G:U (G:T/U) mispairs can alter coding and regulatory sequences. Consequently, there is a need to understand the molecular mechanisms underlying the dynamics of genome methylation. Recent studies have identified a novel DNA demethylation pathway whereby 5mC is oxidized to 5 hydroxymethylcytosine (5hmC) catalyzed by the Ten Eleven Translocation (TET 1-3). 5hmC is metabolized further by TETs into 5 formylcytosine (5fC) and 5 carboxylcytosine (5caC). 5fC and 5caC are recognized and excised by the base excision repair enzyme thymine DNA glycosylase (TDG) to restore unmodified cytosine. The discovery of active demethylation involving the TET/TDG pathway represents a transformative discovery that has greatly advanced our understanding of the dynamics of DNA methylation. Studies have shown that 5mC derivatives have important roles in stem cell differentiation, genomic imprinting and neuronal function. Furthermore, specific interacting proteins for each derivative have been identified thus reflecting the unique biological properties of 5hmC/5fC/5caC. Exactly how these oxidized bases exert their function is unclear and will require a better understanding of their mechanism of deposition, as well as the ability to comprehensively profile the distribution of 5mC derivatives in the mammalian genome. This proposal focuses on two complimentary strategies to test the hypothesis that active demethylation is essential for gene transcription in response to differentiation signals. First using mouse embryonic stem cells, which have the capacity to undergo neural lineage differentiation in response to retinoic acid, we will quantitatively map changes in 5fC/5caC using a new technique known as methylation assisted bisulphite sequencing (MABseq). MABseq provides a direct readout of TET/TDG mediated active demethylation activity at base resolution. Second, to study the molecular mechanisms of active demethylation we will use a protein affinity purification approach known as Proximity-dependent biotin identification (BioID) in combination with mass spectrometry, to identify the protein interaction network for TET/TDG during cellular differentiation. Active DNA demethylation represents a new paradigm in gene regulation that is having important ramifications in all aspects of cell physiology. Collectively, our studies will identify genomic sites where active demethylation occurs and in combination with the experiments proposed in specific aim 2 will provide an important framework towards understanding dynamic DNA methylation.