Regulation and function of active DNA demethylation in Cellular Differentiation

胞嘧啶5'端甲基化在细胞分化和胚胎发育中起着重要作用。尽管其重要性，5 mC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，5 mC到胸腺嘧啶突变的发生率比其他碱基转换高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力学的分子机制。最近的研究发现了一种新型的DNA去甲基化途径，其中5 mC在十十一易位（泰特1-3）的催化下氧化为5羟甲基胞嘧啶（5 hmC）。5 hmC进一步被TcR代谢为5甲酰胞嘧啶（5 fC）和5羧基胞嘧啶（5caC）。5 fC和5caC被碱基切除修复酶胸腺嘧啶DNA糖基化酶（TDG）识别和切除，以恢复未修饰的胞嘧啶。涉及泰特/TDG途径的主动去甲基化的发现代表了一个变革性的发现，极大地推进了我们对DNA甲基化动力学的理解。研究表明，5 mC衍生物在干细胞分化、基因组印记和神经功能中具有重要作用。此外，已经鉴定了每种衍生物的特异性相互作用蛋白，从而反映了5 hmC/5 fC/5caC的独特生物学特性。这些氧化碱基究竟如何发挥其功能尚不清楚，需要更好地了解其沉积机制，以及全面分析哺乳动物基因组中5 mC衍生物分布的能力。该建议侧重于两个互补的策略来测试这一假设，即主动去甲基化是必要的基因转录响应分化信号。首先使用小鼠胚胎干细胞，它有能力进行神经谱系分化，以响应视黄酸，我们将定量地图的变化5 fC/5caC使用一种新的技术称为甲基化辅助亚硫酸氢盐测序（MABseq）。MABseq在碱基分辨率下提供了泰特/TDG介导的活性去甲基化活性的直接读数。第二，为了研究主动去甲基化的分子机制，我们将使用被称为邻近依赖性生物素鉴定（BioID）的蛋白质亲和纯化方法结合质谱，以鉴定细胞分化期间泰特/TDG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新范式，在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主动去甲基化的基因组位点，并与具体目标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.*****