5-Hydroxymethylcytosine (5-hmC) is a newly identified base modification in mammalian genomic DNA. 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 developed. 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 approach we have mapped the genome-wide distribution of 5-hmC in human ES cells. Building on our early successes here we propose to develop single base-resolution detection and sequencing methods to reveal the distribution of 5-hmC in human ES cells. We propose two different approaches, both utilizing the selective chemical labeling strategy we have developed. In one approach, we will label 5-hmC with bulky groups to hinder ligation and linear PCR reactions, thus achieving single base-resolution detection of 5-hmC. The linear PCR approach can be adapted into Illumina sequencing to perform high throughput determination of 5-hmC in human ES cells. In an alternative approach, we will develop an exonuclease digestion blockage method that detects modified 5-hmC at 3'end of undigested DNA fragments using Illumina sequencing;we have already shown that the chemically modified 5-hmC blocks exonuclease III digestion at 3'end of the modified 5-hmC. The new technologies proposed in this R21 application not only enable us to map 5-hmC at single-base resolution in human ES cells, but also could be applied to other samples to map 5-hmC systemically.
5-Hydroxymethylcytosine (5-hmC) is a newly discovered base modification surprisingly abundant in the genomic DNAs of embryonic stem cells. The proposed work will develop efficient chemical labeling methods to perform single base-resolution detection and sequencing of 5-hmC in human ES cells. The success of the proposed work will reveal the fundamental role(s) of 5-hmC in stem cells.
|Cheng, Ying; Bernstein, Alison; Chen, Dahua et al. (2015) 5-Hydroxymethylcytosine: A new player in brain disorders? Exp Neurol 268:9-Mar|
|Yao, Bing; Jin, Peng (2014) Unlocking epigenetic codes in neurogenesis. Genes Dev 28:1253-71|
|Szulwach, Keith E; Jin, Peng (2014) Integrating DNA methylation dynamics into a framework for understanding epigenetic codes. Bioessays 36:107-17|
|Irier, Hasan; Street, R Craig; Dave, Ronak et al. (2014) Environmental enrichment modulates 5-hydroxymethylcytosine dynamics in hippocampus. Genomics 104:376-82|
|Yao, Bing; Jin, Peng (2014) Cytosine modifications in neurodevelopment and diseases. Cell Mol Life Sci 71:405-18|
|Zhu, Gengzhen; Li, Yujing; Zhu, Fei et al. (2014) Coordination of engineered factors with TET1/2 promotes early-stage epigenetic modification during somatic cell reprogramming. Stem Cell Reports 2:253-61|
|Wang, Tao; Warren, Stephen T; Jin, Peng (2013) Toward pluripotency by reprogramming: mechanisms and application. Protein Cell 4:820-32|
|Song, Chun-Xiao; Szulwach, Keith E; Dai, Qing et al. (2013) Genome-wide profiling of 5-formylcytosine reveals its roles in epigenetic priming. Cell 153:678-91|
|Huang, Hao; Jiang, Xi; Li, Zejuan et al. (2013) TET1 plays an essential oncogenic role in MLL-rearranged leukemia. Proc Natl Acad Sci U S A 110:11994-9|
|Lu, Xingyu; Song, Chun-Xiao; Szulwach, Keith et al. (2013) Chemical modification-assisted bisulfite sequencing (CAB-Seq) for 5-carboxylcytosine detection in DNA. J Am Chem Soc 135:9315-7|
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