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.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Scientist Development Award - Research & Training (K01)
Project #
5K01HG006699-02
Application #
8476242
Study Section
Ethical, Legal, Social Implications Review Committee (GNOM)
Program Officer
Schloss, Jeffery
Project Start
2012-06-01
Project End
2017-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
2
Fiscal Year
2013
Total Cost
$140,495
Indirect Cost
$10,370
Name
University of Chicago
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
005421136
City
Chicago
State
IL
Country
United States
Zip Code
60637
Zhang, Liang; Chen, Weizhong; Iyer, Lakshminarayan M et al. (2014) A TET homologue protein from Coprinopsis cinerea (CcTET) that biochemically converts 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine. J Am Chem Soc 136:4801-4
Wang, Xiao; Lu, Zhike; Gomez, Adrian et al. (2014) N6-methyladenosine-dependent regulation of messenger RNA stability. Nature 505:117-20
Liu, Jianzhao; Yue, Yanan; Han, Dali et al. (2014) A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. Nat Chem Biol 10:93-5
Clark, Tyson A; Lu, Xingyu; Luong, Khai et al. (2013) Enhanced 5-methylcytosine detection in single-molecule, real-time sequencing via Tet1 oxidation. BMC Biol 11:4
Zhang, Liang; Szulwach, Keith E; Hon, Gary C et al. (2013) Tet-mediated covalent labelling of 5-methylcytosine for its genome-wide detection and sequencing. Nat Commun 4:1517
Zheng, Guanqun; Dahl, John Arne; Niu, Yamei et al. (2013) ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol Cell 49:18-29