Epigenetic changes caused by DNA methylation, histone modifications, and histone variants play crucial roles in the regulation of chromatin structure and genome stability, developmental specific gene expression, genomic imprinting, and transcriptional silencing of transgenes and other foreign DNA. Cancers and many other diseases are associated with epigenetic alterations. The status of DNA methylation is determined by both methylation and demethylation reactions. DNA methylation by methyltransferase enzymes and the interplay between DNA methylation, non-coding RNAs, and histone modification patterns have been studied extensively. In contrast, the mechanism of active DNA demethylation and its relation with non-coding RNAs and histone modifications are less well understood. Our long-term goal is to understand the dynamic regulation of DNA methylation and its role in development and in responses to the environment. We have developed a unique system in the model organism Arabidopsis thaliana for dissecting active DNA demethylation and RNA-directed DNA methylation (RdDM). In this system, an active transgene (RD29A-LUC) and a homologous endogenous gene become silenced when cellular ROS (repressor of silencing) factors are mutated. ROS1 encodes a 5-methylcytosine-specific DNA glycosylase/lyase that prevents the hypermethylation and silencing of specific loci by initiating a base excision repair pathway for active DNA demethylation. ROS3 encodes a small non-coding RNA-binding protein that regulates active DNA demethylation. We have also discovered several new components of the RdDM pathway using ros1 suppressor mutants, and we have developed a new genetic screen to identify DNA hypermethylation mutants. Our working model is that the RD29A-LUC transgene and some endogenous loci are dynamically regulated by RdDM and active DNA demethylation pathways, and that these marker loci can serve as powerful tools for discovering new components of the DNA methylation and demethylation pathways, i.e., these markers can be used to screen Arabidopsis mutants for altered DNA methylation and expression. This renewal will vigorously test our working model by using the mutant screens and biochemical approaches to discover additional enzymes in the active DNA demethylation pathway and regulatory factors of the pathway. The proposed work will advance new concepts and fill major gaps in our understanding of the dynamic control of DNA methylation, and thus will contribute significantly to elucidating the molecular basis of epigenetic regulation.

Public Health Relevance

Cancers and many other diseases are associated with aberrant DNA methylation and histone modification patterns, but the mechanistic basis of these epigenetic changes is poorly understood. Supported by NIH funding, we have broken new grounds in understanding the control of DNA methylation by discovering a DNA glycosylase-based mechanism of active DNA demethylation and its regulation by non-coding RNAs. The research proposed here will continue to generate exciting new knowledge on the mechanisms and function of active DNA demethylation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM070795-10A1
Application #
8371550
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Carter, Anthony D
Project Start
2004-05-01
Project End
2016-04-30
Budget Start
2012-07-23
Budget End
2013-04-30
Support Year
10
Fiscal Year
2012
Total Cost
$339,570
Indirect Cost
$119,070
Name
Purdue University
Department
Miscellaneous
Type
Schools of Earth Sciences/Natur
DUNS #
072051394
City
West Lafayette
State
IN
Country
United States
Zip Code
47907
Qian, Weiqiang; Miki, Daisuke; Lei, Mingguang et al. (2014) Regulation of active DNA demethylation by an ?-crystallin domain protein in Arabidopsis. Mol Cell 55:361-71
Yamamuro, Chizuko; Miki, Daisuke; Zheng, Zhimin et al. (2014) Overproduction of stomatal lineage cells in Arabidopsis mutants defective in active DNA demethylation. Nat Commun 5:4062
Zhang, Huiming; Tang, Kai; Qian, Weiqiang et al. (2014) An Rrp6-like protein positively regulates noncoding RNA levels and DNA methylation in Arabidopsis. Mol Cell 54:418-30
Lei, Mingguang; La, Honggui; Lu, Kun et al. (2014) Arabidopsis EDM2 promotes IBM1 distal polyadenylation and regulates genome DNA methylation patterns. Proc Natl Acad Sci U S A 111:527-32
Zhang, Huiming; Wang, Bangshing; Duan, Cheng-Guo et al. (2013) Chemical probes in plant epigenetics studies. Plant Signal Behav 8:
Ben Chaabane, Samir; Liu, Renyi; Chinnusamy, Viswanathan et al. (2013) STA1, an Arabidopsis pre-mRNA processing factor 6 homolog, is a new player involved in miRNA biogenesis. Nucleic Acids Res 41:1984-97
Li, Lixin; Atef, Ahmed; Piatek, Agnieszka et al. (2013) Characterization and DNA-binding specificities of Ralstonia TAL-like effectors. Mol Plant 6:1318-30
Zhang, Heng; Ma, Ze-Yang; Zeng, Liang et al. (2013) DTF1 is a core component of RNA-directed DNA methylation and may assist in the recruitment of Pol IV. Proc Natl Acad Sci U S A 110:8290-5
Huang, Chao-Feng; Miki, Daisuke; Tang, Kai et al. (2013) A Pre-mRNA-splicing factor is required for RNA-directed DNA methylation in Arabidopsis. PLoS Genet 9:e1003779
Wang, Xingang; Duan, Cheng-Guo; Tang, Kai et al. (2013) RNA-binding protein regulates plant DNA methylation by controlling mRNA processing at the intronic heterochromatin-containing gene IBM1. Proc Natl Acad Sci U S A 110:15467-72

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