Cytosine methylation is one of the major mechanisms to silence transposons and retroviruses, control genomic imprinting, and regulate reproduction in both mammals and plants. Altered DNA methylation is associated with diseases including leukemia, cancer and congenital syndromes in humans. Arabidopsis thaliana is an excellent system to investigate epigenetic mechanisms, which are evolutionarily conserved between mammals and plants. In Arabidopsis, DEMETER (DME) belongs to a family of DNA glycosylases that excise damaged or mispaired bases in the base excision DNA repair. DME demethylates and activates the maternal allele of imprinted genes. However, one key question is not answered: how do DME or DML DNA glycosylases find their targets to demethylate in the genome? Recently, we isolated DME-INTERACTING PROTEIN 1 (DIP1) in the yeast two-hybrid screen. DIP1 is a putative ubiquitin-conjugating enzyme 7-interacting protein 4 that has a RING domain. Interestingly, we found 50% seed abortion in the self-pollinated progeny of the DIP1/dip1 heterozygous plant. The seed inheriting a maternal dip1 mutant allele is aborted when the DIP1/dip1 female plant was crossed with the wild-type male. To better understand how DIP1 regulates DNA demethylation and imprinting, the following specific aims will be executed: 1) Determine if DIP1 binds to the promoter of imprinted genes and targets DME to demethylate DNA. Using the gel shift assay, we will examine if DIP1 binds to the promoter of imprinted MEDEA (MEA), a Polycomb gene. We will investigate if DIP1 regulates MEA expression, imprinting, and methylation. 2) Analyze molecular functions of DIP1. We will examine if the RING domain protein DIP1 has an E3 ubiquitin ligase activity, and if so, whether DME and DMLs are substrates for the DIP1-mediated ubiquitin proteolytic pathway. 3) Elucidate the mechanism used by DIP1 in regulating DNA demethylation and gene expression. We will explore if DIP1 regulates MEA imprinting through ubiquitination of another DME-interacting protein, histone H1, an event that might lead to DNA methylation and/or histone H3 methylation specifically in the MEA promoter. The proposed experiments carried out using the genetically tractable Arabidopsis system will elucidate mechanisms that link ubiquitination via a RING domain protein to DNA demethylation, gene imprinting, and gene expression - processes that are evolutionarily conserved and integral to the pathology of human diseases such as cancer. Mechanisms amenable to specific targeting of DNA demethylation will provide valuable insights for the development of future gene therapies for targeting specific DNA hypermethylation loci in human diseases. This grant will be used to train undergraduate and graduate students for scientific discovery in epigenetics and molecular biology at Saint Louis University. PI will participate actively in outreach efforts to foster scientific training and education programs in grades K-12 in St. Louis.
The proposed specific aims executed using the epigenetically tractable Arabidopsis thaliana system will elucidate mechanisms that link ubiquitination via a RING domain protein to DNA demethylation, genomic imprinting, and gene expression - processes that are evolutionarily conserved and integral to the pathology of human diseases such as cancer, leukemia, and congenital syndromes. Mechanisms amenable to specific targeting of DNA demethylation will provide significant insights for the development of future gene therapies for targeting specific DNA hypermethylation loci in human diseases.
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