Dissecting Gene Regulatory Roles of TET Enzymes and 5-hydroxymethylcytosine in Mammalian Active DNA Demethylation

项目摘要 表观遗传修饰在细胞功能和细胞谱系特化中具有重要作用。在DNA上， 表观遗传修饰发生在胞嘧啶核碱基的5位上。最常见的修改是5- 甲基胞嘧啶（5 mC）和TET-Eleven-易位（泰特）蛋白通过酶促作用去除DNA甲基化， 将5 mC反复氧化为5-羟甲基胞嘧啶（5 hmC）、5-甲酰胞嘧啶（5 fC）和5-羧基胞嘧啶 （5caC）。在哺乳动物中，TET介导的主动DNA去甲基化可以通过复制依赖性稀释来实现 或胸腺嘧啶DNA糖基化酶（TDG）介导的5 fC/5caC的碱基切除修复（BER）来再生 未修饰C.然而，这些氧化修饰（ox-mC）的功能意义和机制， 不同的活性DNA去甲基化途径仍然知之甚少，排除了我们对如何去甲基化的理解。 DNA甲基化失调会导致疾病。事实上，ox-mC与关键的生物学过程有关， 例如基因转录。然而，将ox-mC的功能作用归因于ox-mC是具有挑战性的， 从5 fC/5caC和未改性C中分离5 hmC的产生在技术上具有挑战性。阐明 ox-mC的功能作用将为开发各种疾病的新疗法建立基础性认识。 病理学为此，我开发了一个CRISPR/dCas 9平台，该平台招募了5 hmC停滞的TET变体， 研究5 hmC、5 fC/5caC和TDG/BER在哺乳动物系统中基因调控作用。 从综合表观遗传测序（亚硫酸氢盐测序（BS-PCR））产生的初步结果 Seq）/亚硫酸氢盐辅助的APOBEC偶联表观遗传测序（bACE-Seq）/甲基化酶辅助的亚硫酸氢盐 测序（MAB-Seq））显示，5 hmC单独不能重新激活高甲基化的基因启动子。 增殖的人细胞，并且需要产生5 fC/5caC。这些结果首次表明， ox-mC之间的功能区别。目前还不清楚下游更高的ox-mC途径如何能够 重新激活基因表达。我假设5 fC/5caC沉积耗尽核小体占用，以促进 转录。在目的1中，我将评估5 hmC/5 fC/5caC/C和TDG在Tet 1 -3三重敲除（TKO）中的作用， Tet 1 -3/Tdg四重敲除（QKO）小鼠胚胎干细胞（mESC）基因表达和局部 染色质结构我的研究结果还表明，单独的5 hmC不能通过复制依赖性恢复未修饰的C 5 hmC的稀释表明这种活性DNA去甲基化的机制比 此前预计。全基因组定量分析ox-mC碱基如何在有丝分裂中遗传 目前，该司缺乏。我假设5 hmC是有丝分裂遗传到新生链，而5 fC/5caC是 通过TDG/BER快速去除。在目标2中，我将开发技术来定量和分析ox-mC有丝分裂， 遗传工程改造的胚胎干细胞中单碱基分辨率的遗传。 通过完成所提出的目标，我将提供前所未有的深入了解主动DNA去甲基化 ox-mC的通路和功能作用，这将有助于我们理解疾病的开始。

Project Summary Epigenetic modifications have important roles in cellular functions and in specialization of cell lineages. On DNA, epigenetic modification occurs on the 5-position of cytosine nucleobases. The most common modification is 5- methylcytosine (5mC), and TET-Eleven-Translocation (TET) proteins enzymatically remove DNA methylation by iteratively oxidizing 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC). In mammals, TET-mediated active DNA demethylation can be achieved by replication-dependent dilution of 5hmC, or thymine DNA glycosylase (TDG) mediated base excision repair (BER) of 5fC/5caC to regenerate unmodified C. However, the functional significance of these oxidized modifications (ox-mC) and mechanistically distinct active DNA demethylation pathways remains poorly understood, precluding our comprehension on how dysregulated DNA methylation contributes to disease. Indeed, ox-mC has been implicated in crucial biological processes such as gene transcription. However, it has been challenging to ascribe functional roles to ox-mC as it was technically challenging to decouple generation of 5hmC from 5fC/5caC, and unmodified C. Elucidating functional roles of ox-mC will establish a foundational understanding for developing novel therapeutics for various pathologies. To this end, I developed a CRISPR/dCas9 platform that recruits 5hmC-stalling TET-variants to interrogate gene regulatory roles of 5hmC, 5fC/5caC, and TDG/BER in mammalian systems. Preliminary results generated from comprehensive epigenetic sequencing (bisulfite sequencing (BS- Seq)/Bisulfite-assisted APOBEC-Coupled Epigenetic sequencing (bACE-Seq)/Methylase-Assisted Bisulfite sequencing (MAB-Seq)) revealed 5hmC alone could not reactivate a hypermethylated gene promoter in proliferative human cells, and generation of 5fC/5caC was requisite. These results show for the first time, functional distinction between ox-mC. It remains ambiguous how downstream higher ox-mC pathways could reactivate gene expression. I hypothesize 5fC/5caC deposition depletes nucleosome occupancy to facilitate transcription. In aim 1, I will evaluate the role of 5hmC/5fC/5caC/C and TDG in Tet1-3 triple knock out (TKO) and Tet1-3/Tdg quadruple knockout (QKO) mouse embryonic stem cells (mESCs), on gene expression and local chromatin structure. My results also reveal 5hmC alone could not restore unmodified C by replication-dependent dilution of 5hmC suggesting this mechanism of active DNA demethylation is more tightly regulated than previously anticipated. Quantitative genome-wide analysis of how ox-mC bases is mitotically inherited across division is currently lacking. I hypothesize 5hmC is mitotically inherited to nascent strands, while 5fC/5caC is rapidly removed by TDG/BER. In aim 2, I will develop technologies to quantify and profile ox-mC mitotic inheritance at single-base resolution in genetically engineered mESCs. By completing the proposed aims, I will afford unprecedented insight into active DNA demethylation pathways and functional roles of ox-mC that will contribute to our understanding of disease inception.