Labeling and sequencing of 5-hmC 5-caC and 5-fC in genomic DNA - Resubmission 0

描述(申请人提供)：5-羟甲基胞嘧啶(5-HMC)是新发现的哺乳动物基因组DNA的一种碱基修饰。在某些组织或细胞中，它可以积累到相对较高的水平。由于目前的测序方法不能区分5-MC和5-HMC，迫切的挑战是开发可靠的方法来确定5-HMC在哺乳动物基因组中的位置，这一问题最好通过采用我们发明的新的化学标记技术来解决。我们发现5-羟甲基纤维素的羟甲基可以选择性地被化学修饰的葡萄糖利用-葡萄糖基转移酶(GT)标记。这种糖基化提供了一种安装官能团的策略，如 作为生物素结合到5-羟甲基纤维素上。通过这种方法，我们可以亲和捕获含有修饰的5-HMC的DNA片段，并建立测序方法来确定5-HMC的准确位置。利用这一新方法，我们首次获得了哺乳动物基因组中5-羟甲基纤维素的全基因组分布图。在我们早期成功的基础上，我们建议开发单碱基分辨率检测和测序方法来揭示5-HMC在哺乳动物基因组中的确切位置。我们提出了不同但相互补充的方法，以确保在不久的将来生物界能够获得有效的方法。获得的全基因组信息可以用来帮助探索5-HMC的功能作用，例如可能识别特定序列内容中的5-HMC的潜在蛋白质和/或转录因子。我们还发现，5-羟甲基纤维素可以被Tet家族的酶进一步氧化为5-FC和5-CAC。值得注意的是，当与正常G配对时，5-FC和5-CAC修饰可以被人胸腺嘧啶DNA糖基酶(TDG)切除，而不需要碱基脱氨。碱基切除修复(BER)过程在主动去甲基化过程中有效地将这些碱基修饰转化回C。我们计划开发选择性的5-FC和5-CAC标记和测序方法，以分别获得这些有趣的碱基修饰的全基因组分布图。在TDG缺陷细胞中，我们认为5-FC/5-CAC的全基因组分布信息可能揭示了特定细胞阶段的活性去甲基化位点。这项拟议的研究将为PI的团队和广泛的生物界开发迫切需要的工具，以研究生命科学研究的最前沿之一：这些新发现的DNA碱基修饰在表观遗传学、发育和各种人类疾病中的潜在功能作用。

DESCRIPTION (provided by applicant): 5-Hydroxymethylcytosine (5-hmC) is a newly identified base modification in mammalian genomic DNA. In certain tissues or cells it can accumulate to relatively high levels. Because current sequencing methods cannot differentiate 5-mC from 5-hmC, the immediate challenge is to develop robust methods to ascertain the positions of 5-hmC within the mammalian genome, a problem best addressed by adapting a new chemical labeling technology that we have invented. We show that the hydroxymethyl group of 5-hmC can be selectively labeled with chemically modified glucoses using -glucosyltransferase (GT). This glycosylation offers a strategy of installing functional groups such as biotin onto 5-hmC. In this way, we can affinity capture DNA fragments containing the modified 5-hmC and develop sequencing methods to determine the precise locations of 5-hmC. Using this new method, we have obtained the first genome-wide distribution map of 5-hmC in the mammalian genome. Building on our early successes, we propose to develop single-base resolution detection and sequencing methods to reveal the exact locations of 5-hmC in mammalian genomes. We propose different but complementary approaches in order to ensure that effective methods will be available to the biological community in the near future. The genome-wide information obtained can be used to help probe the functional roles of 5-hmC, for instance potential proteins and/or transcriptional factors that may recognize 5-hmC in specific sequence contents. We have also shown that 5-hmC can be further oxidized to 5-fC and 5-caC by the TET family enzymes. Significantly, when paired with a normal G, the 5-fC and 5-caC modification can be excised by the human thymine DNA glycosylase (TDG) without the need for base deamination. The base excision repair (BER) process effectively converts these base modifications back to C in an active demethylation process. We plan to develop selective 5-fC and 5-caC labeling and sequencing methods to obtain a genome-wide distribution map of these intriguing base modifications, respectively. In TDG-deficient cells, we believe the genome-wide distribution information of 5-fC/5-caC may reveal active demethylation sites in the specific cell stages. The proposed research will develop urgently needed tools for the PI's group and the broad biology community to study one of the most cutting-edge frontiers of life sciences research: the potential functional roles of these newly discovered DNA base modifications in epigenetics, development, and various human diseases.