Regulation and function of active DNA demethylation in Cellular Differentiation

细胞分化中主动 DNA 去甲基化的调控和功能

• 批准号: RGPIN-2018-06542
• 负责人: Torchia, Joseph
• 金额: $ 2.86万
• 依托单位: University of Western Ontario
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=687476
• 关键词: Regulation; function; active; DNA; demethylation

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.*****

胞嘧啶5′位置的甲基化在细胞分化和胚胎发育中起着至关重要的作用。尽管它很重要，5mC也通过促进脱胺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背后的分子机制。最近的研究发现了一种新的DNA去甲基化途径，其中5mC被10 - 11易位（TET 1-3）催化氧化为5羟甲基胞嘧啶（5hmC）。5hmC被tet进一步代谢为5甲酰基胞嘧啶（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糖基化酶（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，5mC衍生物在干细胞分化、基因组印迹和神经元功能中具有重要作用。此外，每种衍生物的特异性相互作用蛋白已被确定，从而反映了5hmC/5fC/5caC独特的生物学特性。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的分布的能力。这一建议侧重于两种互补的策略，以验证活性去甲基化是基因转录对分化信号的反应所必需的假设。首先使用小鼠胚胎干细胞，这种干细胞在维甲酸的作用下具有神经谱系分化的能力，我们将使用一种称为甲基化辅助亚硫酸盐测序（MABseq）的新技术定量绘制5fC/5caC的变化。MABseq提供了碱基分辨率下TET/TDG介导的活性去甲基化活性的直接读数。其次，为了研究活性去甲基化的分子机制，我们将使用一种称为接近依赖生物素鉴定（BioID）的蛋白质亲和纯化方法，结合质谱法，确定细胞分化过程中TET/TDG的蛋白质相互作用网络。活性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，我们的研究将确定活跃去甲基化发生的基因组位点，并与特定目标2中提出的实验相结合，将为理解动态DNA甲基化提供重要框架。*****