Cytosine DNA methylation is an epigenetic mark for gene silencing that is important in many gene regulatory systems including mammalian imprinting, X-chromosome inactivation, and the silencing of transposons and other DNA sequences containing either direct or inverted repeats. Methylation is important in cancer biology, as tumors often show both genome wide demethylation and hypermethylation of specific tumor suppressor genes. We are studying the mechanisms of DNA methylation control in the model plant Arabidopsis, which has methylation systems that have much in common with mammalian systems. Both forward and reverse genetics can be performed, and Arabidopsis can tolerate mutations that virtually eliminate methylation, allowing for detailed analysis. In Arabidopsis, the initial establishment of methylation requires the activity of DRM2, a homolog of mammalian Dnmt3, which is guided to appropriate targets, at least in part, by small interfering RNAs. Once established, DNA methylation is maintained by three different systems for methylation of cytosines in three different sequence contexts, CG, CHG and CHH (asymmetric). CG methylation is maintained by MET1, a homolog of mammalian Dnmt1. CHG methylation is maintained by the CMT3 DNA methyltransferase, which is guided to appropriate targets by the methylation of histone H3. CHH methylation is maintained by the persistent activity of DRM2, which is targeted by small interfering RNAs. In this proposal, we plan to further study these DNA methylation control mechanisms. First, we plan to study the poorly understood process by which DNA methylation is initially established, a process called de novo methylation. We have developed assays that allow us to perform both reverse and forward genetic screens to identify genes required for proper de novo DNA methylation. These mutant screens have already yielded several interesting genes, and a major part of this proposal is to perform a detailed molecular genetic characterization of these genes to understand more about the mechanism by which they contribute to de novo methylation. We also propose to continue additional genetic screens, and to characterize a set of 35 new mutants we have isolated that block de novo methylation. The second major aim of this proposal is to further study the mechanisms by which CG DNA methylation is maintained, by studying a new factor called UHRF1 in mammals and ORTH/VIM in Arabidopsis, which appears to work by binding directly to previously methylated CG sites and recruiting the CG methyltransferase to further maintain methylation. We propose to study the function of six Arabidopsis ORTH/VIM genes by using loss of function genetic alleles, understanding the specificity of chromatin interactions for each gene, and performing molecular and genetic tests of the function of the different domains of each protein. These studies should shed new light on DNA methylation systems in Arabidopsis and have implications for related systems in mammals and other eukaryotes.

Public Health Relevance

Project Narrative This research aims at understanding the basic mechanisms that control DNA methylation, which is a chemical modification that occurs on certain genes and prevents these genes from functioning in a particular tissue. When DNA methylation patterns are not properly maintained, this can cause inappropriate regulation of genes and is a major cause of cancer. Thus a further understanding of the DNA methylation may someday lead to methods for correcting DNA methylation patterning defects.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM060398-13
Application #
8206658
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Carter, Anthony D
Project Start
2000-01-01
Project End
2013-12-31
Budget Start
2012-01-01
Budget End
2012-12-31
Support Year
13
Fiscal Year
2012
Total Cost
$298,091
Indirect Cost
$102,071
Name
University of California Los Angeles
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Feng, Suhua; Cokus, Shawn J; Schubert, Veit et al. (2014) Genome-wide Hi-C analyses in wild-type and mutants reveal high-resolution chromatin interactions in Arabidopsis. Mol Cell 55:694-707
Du, Jiamu; Johnson, Lianna M; Groth, Martin et al. (2014) Mechanism of DNA methylation-directed histone methylation by KRYPTONITE. Mol Cell 55:495-504
Johnson, Lianna M; Du, Jiamu; Hale, Christopher J et al. (2014) SRA- and SET-domain-containing proteins link RNA polymerase V occupancy to DNA methylation. Nature 507:124-8
Zhong, Xuehua; Du, Jiamu; Hale, Christopher J et al. (2014) Molecular mechanism of action of plant DRM de novo DNA methyltransferases. Cell 157:1050-60
Moissiard, Guillaume; Bischof, Sylvain; Husmann, Dylan et al. (2014) Transcriptional gene silencing by Arabidopsis microrchidia homologues involves the formation of heteromers. Proc Natl Acad Sci U S A 111:7474-9
Stroud, Hume; Do, Truman; Du, Jiamu et al. (2014) Non-CG methylation patterns shape the epigenetic landscape in Arabidopsis. Nat Struct Mol Biol 21:64-72
Huang, Yun; Chavez, Lukas; Chang, Xing et al. (2014) Distinct roles of the methylcytosine oxidases Tet1 and Tet2 in mouse embryonic stem cells. Proc Natl Acad Sci U S A 111:1361-6
Law, Julie A; Du, Jiamu; Hale, Christopher J et al. (2013) Polymerase IV occupancy at RNA-directed DNA methylation sites requires SHH1. Nature 498:385-9
Li-Byarlay, Hongmei; Li, Yang; Stroud, Hume et al. (2013) RNA interference knockdown of DNA methyl-transferase 3 affects gene alternative splicing in the honey bee. Proc Natl Acad Sci U S A 110:12750-5
Stroud, Hume; Greenberg, Maxim V C; Feng, Suhua et al. (2013) Comprehensive analysis of silencing mutants reveals complex regulation of the Arabidopsis methylome. Cell 152:352-64

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