5-Hydroxymethylcytosine (5hmC) is a newly identified base modification in mammalian genomic DNA. TET proteins, a group of iron(II)/?KG-dependent dioxygenases, have been shown to utilize dioxygen to oxidize 5-methylcytosine (5mC) to 5hmC, and to further form 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) in the mammalian genome. Both 5fC and 5caC can be recognized and excised by human thymine DNA glycosylase (TDG), followed by base excision repair (BER) to replace the modified cytosine with a normal cytosine in an active demethylation process. The 5mC oxidation derivatives of 5hmC, 5fC, and 5caC may also be passively diluted to the unmethylated stage. In certain tissues or cells, 5hmC can accumulate to relatively high levels, whereas both 5fC and 5caC exist in much lower levels in most cells consistent with their constant removal by TDG through BER. The TET-mediated 5mC oxidation has been shown to play vital functional roles in mammalian early development events such as epigenetic reprogramming following fertilization and differentiations of various mammalian progenitor cells. However, studies of these systems have been significantly hampered due to lack of effective technologies that can map the presence and locations of oxidized 5mC derivatives with limited genomic samples; in many mammalian early development events only hundreds to thousands of cells can be isolated for functional characterizations. Building on our successes in inventing enabling technologies that label and sequence 5hmC and 5fC, we propose several innovative approaches that can robustly map the locations of these cytosine modifications genome-wide with as few as 1,000 cells or less. We further propose to study demethylation following fertilization to validate the new methods we plan to develop. The proposed research will provide urgently needed tools for the PI's group and the broader scientific community to study a range of research questions that should reveal new fundamental knowledge of epigenetics, development, and various human diseases.

Public Health Relevance

5-Hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) are newly discovered base modifications in the genomic DNAs of certain mammalian tissues and cells. The proposed research will develop highly sensitive methods in order to map genome-wide distributions of these base modifications with genomic DNA samples isolated from limited numbers of cells.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
5R01HG006827-05
Application #
9091608
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Smith, Michael
Project Start
2012-06-01
Project End
2018-05-31
Budget Start
2016-06-01
Budget End
2017-05-31
Support Year
5
Fiscal Year
2016
Total Cost
Indirect Cost
Name
University of Chicago
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
005421136
City
Chicago
State
IL
Country
United States
Zip Code
60637
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Ma, Lijia; Zhao, Boxuan; Chen, Kai et al. (2017) Evolution of transcript modification by N6-methyladenosine in primates. Genome Res 27:385-392
Han, Dali; Lu, Xingyu; Shih, Alan H et al. (2016) A Highly Sensitive and Robust Method for Genome-wide 5hmC Profiling of Rare Cell Populations. Mol Cell 63:711-719
Hohos, Natalie M; Lee, Kevin; Ji, Lexiang et al. (2016) DNA cytosine hydroxymethylation levels are distinct among non-overlapping classes of peripheral blood leukocytes. J Immunol Methods 436:1-15
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
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Yang, Yeqing A; Zhao, Jonathan C; Fong, Ka-Wing et al. (2016) FOXA1 potentiates lineage-specific enhancer activation through modulating TET1 expression and function. Nucleic Acids Res 44:8153-64
Liu, Jianzhao; Zhu, Yuanxiang; Luo, Guan-Zheng et al. (2016) Abundant DNA 6mA methylation during early embryogenesis of zebrafish and pig. Nat Commun 7:13052
von Meyenn, Ferdinand; Iurlaro, Mario; Habibi, Ehsan et al. (2016) Impairment of DNA Methylation Maintenance Is the Main Cause of Global Demethylation in Naive Embryonic Stem Cells. Mol Cell 62:848-861
Yu, Ping; Ji, Lexiang; Lee, Kevin J et al. (2016) Subsets of Visceral Adipose Tissue Nuclei with Distinct Levels of 5-Hydroxymethylcytosine. PLoS One 11:e0154949

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