DNA methylation is a crucial regulator of mammalian development. Mutations in DNA methyltransferases and methylcytosine binding protein genes cause genetic disease, and disrupted DNA methylation is a hallmark of cancer. Defects in DNA demethylation are associated with disorders of the central nervous system, tumor development, and failure to establish embryonic pluripotency. In addition, defective DNA demethylation prevents the in vitro derivation of induced pluripotent stem cells, a technology with the potential to be used in the clinic for the regeneration of cells, tissues and organs. Whereas most common model organisms lack DNA methylation, flowering plants, including Arabidopsis thaliana, share highly conserved methylation and demethylation pathways with mammals. Both plants and mammals use DNA methylation to regulate genomic imprinting - epigenetic marking and resultant gene expression only from an allele inherited from either the male or the female parent. We discovered that DNA demethylation catalyzed by the Arabidopsis DME DNA glycosylase occurs exclusively in companion cells, the central cell and the vegetative cell, which are adjacent to the gametes, the egg and sperm, respectively. One sperm fertilizes the central cell to generate the endosperm, a nutritive extra-embryonic tissue. A second sperm fertilizes the egg to form the embryo. We showed that gene imprinting occurs in the endosperm, which is established by DNA demethylation in the central cell. Moreover, our recent results suggest that DNA demethylation of central cell and vegetative cell genomes generates small RNAs that move to the adjacent gametes and silence transposable elements by the RNA-directed DNA methylation pathway.
In Aim 1, we will test our model that DME-mediated DNA demethylation in companion cells functions to reinforce TE silencing in gametes.
In Aim 2, we will elucidate the critical relationship between chromatin structure and targeted DNA demethylation.
In Aim 3, we will identify new genes required for DNA demethylation. Our studies will lead to a detailed understanding of the epigenetic reprogramming catalyzed by DNA demethylation during plant sexual reproduction.

Public Health Relevance

DNA methylation is an important mechanism for regulating human genes, the disruption of which contributes to the development of many cancers, congenital disorders, and syndromes associated with defects in gene imprinting. In early mammalian development, DNA demethylation is required for acquiring pluripotency, generating stem cells, and for gene imprinting. Defects in DNA demethylation are associated with disorders in the central nervous system associated with stress, as well as the development and progression of cancer. DNA demethylation is thought to be the rate-limiting step in generating induced pluripotent stem (iPS) cells, an emerging medical technology that offers the promise of patient-specific stem cell implantations that can potentially regenerate cells, tissues and organs. We are studying how DNA is actively demethylated by the DME DNA glycosylase;this will support the development of diagnostic and therapeutic interventions, detecting and potentially reversing the onset and progression of disease-causing aberrant DNA methylation. As described in a letter of support from Dr. George Martin (Director of Technology, Chief Technology Office, Roche Diagnostics), continued research into DME is invaluable for the study of epigenetics in human disease, and complements efforts to develop reagents to detect aberrant DNA methylation in clinical samples.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM069415-09A1
Application #
8437797
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Carter, Anthony D
Project Start
2004-02-01
Project End
2017-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
9
Fiscal Year
2013
Total Cost
$328,476
Indirect Cost
$118,476
Name
University of California Berkeley
Department
Other Basic Sciences
Type
Schools of Earth Sciences/Natur
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
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Mérai, Zsuzsanna; Chumak, Nina; García-Aguilar, Marcelina et al. (2014) The AAA-ATPase molecular chaperone Cdc48/p97 disassembles sumoylated centromeres, decondenses heterochromatin, and activates ribosomal RNA genes. Proc Natl Acad Sci U S A 111:16166-71
Yu, Agnes; Lepere, Gersende; Jay, Florence et al. (2013) Dynamics and biological relevance of DNA demethylation in Arabidopsis antibacterial defense. Proc Natl Acad Sci U S A 110:2389-94
Rodrigues, Jessica A; Ruan, Randy; Nishimura, Toshiro et al. (2013) Imprinted expression of genes and small RNA is associated with localized hypomethylation of the maternal genome in rice endosperm. Proc Natl Acad Sci U S A 110:7934-9
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Hsieh, Tzung-Fu; Shin, Juhyun; Uzawa, Rie et al. (2011) Regulation of imprinted gene expression in Arabidopsis endosperm. Proc Natl Acad Sci U S A 108:1755-62
Bauer, Matthew J; Fischer, Robert L (2011) Genome demethylation and imprinting in the endosperm. Curr Opin Plant Biol 14:162-7
Jeong, Cheol Woong; Roh, Hyungmin; Dang, Tuong Vi et al. (2011) An E3 ligase complex regulates SET-domain polycomb group protein activity in Arabidopsis thaliana. Proc Natl Acad Sci U S A 108:8036-41
Mosher, Rebecca A; Tan, Ek Han; Shin, Juhyun et al. (2011) An atypical epigenetic mechanism affects uniparental expression of Pol IV-dependent siRNAs. PLoS One 6:e25756

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