Investigation of 5-hmC, 5fC and 5-caC Modifications in Genome DNA

描述(申请人提供)：5-羟甲基胞嘧啶(5-HMC)是最近在哺乳动物基因组DNA中发现的一种碱基修饰。最近的研究有力地表明，5-HMC是另一个重要的表观遗传标记，在基因调控中发挥着重要和广泛的作用。由于现有的测序方法不能区分5-MEC和5-HMC，我们最近开发了一种5-HMC测序的化学标记策略，通过这种策略，我们成功地获得了第一张哺乳动物基因组中5-HMC的分布图。在这一应用中，我建议开发新的单碱基分辨测序方法来确定5-HMC在基因组DNA中的准确位置，这将使我们能够解决5-HMC在基因调控中的确切作用。此外，以合成的DNA寡核苷酸为模型，我们发现DNA寡核苷酸中的5-HMC修饰在体外可以被Tet蛋白氧化为5-甲酰胞嘧啶(5-FC)和5-羧基胞嘧啶(5-CAC)，而且从Tet高表达细胞中提取的基因组DNA中可以检测到5-FC和5-CAC的修饰。这些发现表明，在某些细胞的基因组DNA中也存在5-FC和5-CAC修饰。在这项应用中，我建议开发新的方法来标记和丰富这些修饰，以便在基因组DNA中检测和测序。这些方法如果成功，将为研究这些修饰在基因调控和发育中的潜在作用奠定基础。在这个项目中，我将利用我在核酸化学方面的专业知识，并与我的导师和合作者密切合作，扩展我在生物化学、细胞生物学和基因组学方面的知识和实验技能，以解决与基因组DNA中5-HMC、5-FC和5-CAC修饰相关的关键问题。这一K01奖项不仅让我能够研究这些基本问题，也为我未来在这一领域的独立研究生涯做好了准备。

DESCRIPTION (provided by applicant): 5-Hydroxymethylcytosine (5-hmC) is a newly discovered base modification in mammalian genomic DNA. Recent studies strongly suggest that 5-hmC is another vital epigenetic mark that plays major and broad roles in gene regulation. Because the sequencing methods currently available cannot differentiate 5-meC from 5-hmC, we recently developed a chemical-labeling strategy for 5-hmC sequencing through which we have successfully obtained the first 5-hmC- distribution map in a mammalian genome. In this application, I propose to develop new single- base resolution sequencing methods to determine the precise locations of 5-hmC in genomic DNA, which will allow us to address the exact roles of 5-hmC in gene regulation. In addition, using the synthesized DNA oligos as models, we have found that the 5-hmC modification in DNA oligos could be oxidized to 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC) by TET proteins in vitro; moreover, both 5-fC and 5-caC modifications could be detected from genomic DNA isolated from TET over-expressed cells. These findings suggest that 5-fC and 5-caC modifications are also present in the genomic DNA of certain cells. In this application, I propose to develop new methods for labeling and enriching these modifications for their detection and sequencing in genomic DNA. These methods, if successful, will lay the groundwork for the study of the potential roles of these modifications in gene regulation and development. During this project, I will make use of my expertise in nucleic acid chemistry and work closely with my mentor and collaborators to extend my knowledge and experimental skills in biochemistry, cell biology and genomics to address key questions related to 5-hmC, 5-fC, and 5-caC modifications in genomic DNA. This K01 award will not only allow me to study these fundamental questions, but also prepare me for an independent career in research in this area in the future.