DNA cytosine methylation (5-methylcytosine or 5mC) is the main epigenetic mechanism in human gene expression regulation. This methylation is oxidized by the human TET family enzymes to 5-hydroxymethylcytosine (5hmC) in an active demethylation process. While 5mC is a mark for gene repression in general and is also used to suppress repetitive elements in the human genome, recent studies showed that 5hmC tends to mark active loci, as gene activation requires removal of 5mC in gene bodies, promoters, and enhancers. The presence of 5hmC indicates open chromatin loci, whereas heavy 5mC methylation often marks heterochromatin regions. Therefore, genomic locations of 5hmC and 5mC provide genome-wide, comprehensive information on chromatin activation and repression, which are suitable marks for classifying individual human cells. The locations of 5hmC and 5mC can also store information of gene activation in frozen or fragmented genomic samples, such as DNA from biopsy samples or cell- free DNA (cfDNA), providing additional advantages in clinical diagnosis and prognosis of various human diseases. Building on our previous successes in inventing enabling technologies that label and sequence 5hmC and 5mC, we have recently developed robust procedures to map 5hmC and related cytosine modifications genome-wide using 1,000 cells. In this application we propose new approaches that can map both 5hmC and 5hmC at base-resolution down to the single-cell level. We plan to test and validate these new methods by investigating cell-cell heterogeneity using Tet2-mutant acute myeloid leukemia stem cells versus controls. We will also validate the new methods to map 5hmC and 5mC in other systems with limited DNA. In addition, we propose a new and highly useful method for single-cell bisulfite sequencing of 5mC in DNA by pre-amplify the limited DNA samples to much higher abundances compatible with most downstream analysis methods, with 5mC sites replicated faithfully during the amplification. The proposed research will provide urgently needed tools for the PI's group and the broader scientific community to study a range of questions in biomedical research and clinical biomarker discoveries using limited input DNA and at the single-cell level.

Public Health Relevance

The proposed research will develop highly sensitive and selective chemical methods to label and map 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) in DNA at the base- resolution and single-cell level. The new methods could find wide applications in basic biomedical research as well as clinical biomarker studies.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
5R01HG006827-08
Application #
9692739
Study Section
Synthetic and Biological Chemistry A Study Section (SBCA)
Program Officer
Smith, Michael
Project Start
2012-08-07
Project End
2022-05-31
Budget Start
2019-06-01
Budget End
2020-05-31
Support Year
8
Fiscal Year
2019
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
Yu, Miao; Han, Dali; Hon, Gary C et al. (2018) Tet-Assisted Bisulfite Sequencing (TAB-seq). Methods Mol Biol 1708:645-663
Ji, Lexiang; Jordan, William T; Shi, Xiuling et al. (2018) TET-mediated epimutagenesis of the Arabidopsis thaliana methylome. Nat Commun 9:895
Luo, Guan-Zheng; Hao, Ziyang; Luo, Liangzhi et al. (2018) N6-methyldeoxyadenosine directs nucleosome positioning in Tetrahymena DNA. Genome Biol 19:200
Li, Wenshuai; Zhang, Xu; Lu, Xingyu et al. (2017) 5-Hydroxymethylcytosine signatures in circulating cell-free DNA as diagnostic biomarkers for human cancers. Cell Res 27:1243-1257
Ma, Lijia; Zhao, Boxuan; Chen, Kai et al. (2017) Evolution of transcript modification by N6-methyladenosine in primates. Genome Res 27:385-392
Wang, Wei; Xu, Liang; Hu, Lulu et al. (2017) Epigenetic DNA Modification N6-Methyladenine Causes Site-Specific RNA Polymerase II Transcriptional Pausing. J Am Chem Soc 139:14436-14442
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
Luo, Guan-Zheng; Wang, Fang; Weng, Xiaocheng et al. (2016) Characterization of eukaryotic DNA N(6)-methyladenine by a highly sensitive restriction enzyme-assisted sequencing. Nat Commun 7:11301
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
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

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