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的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苷酸碱基的5位上。最常见的修改是5- 甲基胞嘧啶(5mC)和Tet-11转位(Tet)蛋白通过以下途径酶促DNA甲基化 将5mC反复氧化为5-羟甲基胞嘧啶(5hmC)、5-甲酰胞嘧啶(5fC)和5-羧基胞嘧啶 (5caC)。在哺乳动物中，Tet介导的活性DNA去甲基化可以通过复制依赖的稀释来实现 5hmC或胸腺嘧啶DNA糖基酶(TDG)介导的5fC/5caC碱基切除修复(BER)再生 然而，这些氧化修饰(OX-MC)的功能意义和力学意义 不同的活性DNA去甲基化途径仍然知之甚少，使我们无法理解 DNA甲基化异常导致疾病。事实上，OX-MC与关键的生物学作用有关 基因转录等过程。然而，将功能角色归因于OX-MC AS一直是一个挑战 将5hmC的产生与5fC/5caC和未修饰的C分离在技术上是具有挑战性的。 OX-MC的功能作用将为开发治疗多种疾病的新疗法奠定基础 病理学。为此，我开发了一个CRISPR/dCas9平台，它招募了5hmC-stating Tet-Variants来 探讨5hmC、5fC/5caC和TDG/BER在哺乳动物系统中的基因调控作用。 综合表观遗传测序(亚硫酸氢盐测序(BS- SEQ)/亚硫酸盐辅助APOBEC偶联表观遗传测序(BACE-SEQ)/甲基酶辅助亚硫酸盐 测序(MAB-Seq)显示，5hmC单独不能重新激活高甲基化的基因启动子 增殖的人细胞，5fC/5caC的产生是必需的。这些结果首次表明， OX-MC之间的功能差异。目前还不清楚下游更高的OX-MC通路如何 重新激活基因表达。I假设5fC/5CaC沉积耗尽核小体占有率 抄写。在目标1中，我将评估5hmC/5fC/5caC/C和TDG在Tet1-3三重敲除(TKO)中的作用和 Tet1-3/TDG四基因敲除小鼠胚胎干细胞对基因表达和局部表达的影响 染色质结构。我的结果还表明，仅靠5hmC不能通过依赖复制恢复未修改的C 稀释5hmC表明这种活性DNA去甲基化的机制比 之前已经预料到了。对OX-MC碱基如何跨有丝分裂遗传的全基因组定量分析 目前缺乏部门。我假设5hmC是有丝分裂遗传给新生的链，而5fC/5caC是 由TDG/BER快速去除。在目标2中，我将开发技术来量化和描述ox-MC有丝分裂 遗传工程mESCs中单碱基分辨率的遗传。 通过完成提议的目标，我将对活跃的dna去甲基化提供前所未有的洞察力。 OX-MC的通路和功能作用将有助于我们理解疾病的发生。

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.