The primary goal of the research in my laboratory is to understand how DNA demethylation regulates gene imprinting. DNA methyltransferases methylate DNA (5-methylcytosine), which silences transposons, repeated sequences, and genes. In Arabidopsis, the base excision DNA repair pathway is used to actively demethylate DNA. The DEMETER (DME) DNA glycosylase initiates the pathway by removing 5-methylcytosine through cleavage of the N-glycosylic bond (
Choi et al (2002),
Xiao et al (2003),
Choi et al (2004)),
Gehring et al (2006)). AP endonuclease, DNA polymerase, and ligase insert cytosine and seal the DNA, resulting in a loss of DNA methylation.
Gene imprinting is an important process for reproduction in plants (
Huh et al (2008)). Alleles of imprinted genes are expressed differently depending on whether they are inherited from the male or female parent. In mammals, imprinted genes contribute to the control of fetal growth and development, and human diseases are linked to mutations in imprinted genes. In plants, imprinted genes control the growth and development of seeds, which are the primary source of carbon, nitrogen, and energy for humans and domesticated animals.
In both plants and mammals DNA methylation (5-methylcytosine) is a critical regulator of gene imprinting. In mammals, allele-specific silencing by de novo DNA methylation establishes gene imprinting in the embryo. In plants, allele-specific activation by DNA demethylation establishes DNA imprinting in the endosperm (
Gehring et al (2006)). Because endosperm does not contribute to the next generation, the activated allele need not be silenced again. Double fertilization enables plants to use such “one-way” control of imprinting and DNA methylation in endosperm (
Kinoshita et al (2004)).
In Arabidopsis, DME specifically excises 5-methylcytosine at the MEDEA (MEA) Polycomb group gene and activates its transcription in the central cell of the female gametophyte. After fertilization of the central cell, the maternal expression of the MEA Polycomb group protein then maintains the silenced state of its own paternal allele and additional imprinted genes in the endosperm (
Choi et al (2002),
Gehring et al (2006)).
DME related proteins ROS1 (REPRESSOR OF SILENCING1), DML2 and DML3 (DEMETER-LIKE2 and 3) are broadly expressed in vegetative and reproductive tissues. They actively demethylate approximately 200 sites in the Arabidopsis genome (
Penterman et al (2007a)). Their function may be to protect genes from being silenced by preventing the spread of DNA methylation from neighboring transposons and repeated sequences (
Penterman et al (2007b)). This process may prevent the accumulation of excessive DNA methylation over successive generations that could interfere with transcription of genes.
Parent-of-origin-specific (imprinted) gene expression is regulated in Arabidopsis thaliana endosperm by cytosine demethylation of the maternal genome mediated by the DNA glycosylase DEMETER, but the extent of the methylation changes is not known. We found that virtually the entire endosperm genome is demethylated, coupled with extensive local non-CG hypermethylation of small interfering RNA–targeted sequences. Mutation of DEMETER partially restores endosperm CG methylation to levels found in other tissues, indicating that CG demethylation is specific to maternal sequences. Endosperm demethylation is accompanied by CHH hypermethylation of embryo transposable elements. Our findings demonstrate extensive reconfiguration of the endosperm methylation landscape that likely reinforces transposon silencing in the embryo (
Hsieh et al (2009)).
