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.

Public Health Relevance

5-Hydroxymethylcytosine (5-hmC) is a newly discovered base modification surprisingly abundant in the genomic DNA of certain mammalian tissues and cells. Our recent study showed that 5-hmC could be oxidized to 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC) by TET proteins. The proposed research will develop efficient chemical-labeling methods to perform single-base resolution detection and sequencing of 5-hmC, 5-fC, and 5-caC in genomic DNA. Our success will help reveal the fundamental roles of 5-hmC, 5-fC, and 5-caC in gene regulation.

National Institute of Health (NIH)
National Human Genome Research Institute (NHGRI)
Research Scientist Development Award - Research & Training (K01)
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Ethical, Legal, Social Implications Review Committee (GNOM)
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Schloss, Jeffery
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University of Chicago
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Zhou, Katherine I; Clark, Wesley C; Pan, David W et al. (2018) Pseudouridines have context-dependent mutation and stop rates in high-throughput sequencing. RNA Biol 15:892-900
Shu, Xiao; Dai, Qing; Wu, Tong et al. (2017) N6-Allyladenosine: A New Small Molecule for RNA Labeling Identified by Mutation Assay. J Am Chem Soc 139:17213-17216
Dai, Qing; Moshitch-Moshkovitz, Sharon; Han, Dali et al. (2017) Nm-seq maps 2'-O-methylation sites in human mRNA with base precision. Nat Methods 14:695-698
Dai, Qing; Zheng, Guanqun; Schwartz, Michael H et al. (2017) Selective Enzymatic Demethylation of N2 ,N2 -Dimethylguanosine in RNA and Its Application in High-Throughput tRNA Sequencing. Angew Chem Int Ed Engl 56:5017-5020
Zhou, Katherine I; Parisien, Marc; Dai, Qing et al. (2016) N(6)-Methyladenosine Modification in a Long Noncoding RNA Hairpin Predisposes Its Conformation to Protein Binding. J Mol Biol 428:822-833
Ngo, Thuy T M; Yoo, Jejoong; Dai, Qing et al. (2016) Effects of cytosine modifications on DNA flexibility and nucleosome mechanical stability. Nat Commun 7:10813
Dai, Qing; Sanstead, Paul J; Peng, Chunte Sam et al. (2016) Weakened N3 Hydrogen Bonding by 5-Formylcytosine and 5-Carboxylcytosine Reduces Their Base-Pairing Stability. ACS Chem Biol 11:470-7
Dominissini, Dan; Nachtergaele, Sigrid; Moshitch-Moshkovitz, Sharon et al. (2016) The dynamic N(1)-methyladenosine methylome in eukaryotic messenger RNA. Nature 530:441-6
Bernstein, Alison I; Lin, Yunting; Street, R Craig et al. (2016) 5-Hydroxymethylation-associated epigenetic modifiers of Alzheimer's disease modulate Tau-induced neurotoxicity. Hum Mol Genet 25:2437-2450
Sun, Miao; Song, Mingxi M; Wei, Bin et al. (2016) 5-Hydroxymethylcytosine-mediated alteration of transposon activity associated with the exposure to adverse in utero environments in human. Hum Mol Genet 25:2208-2219

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