Regulation of protein production is vital in maintaining healthy cellular function. Misregulation can lead to excessive cell divisions and eventually tumorigenesis. There are many mechanisms in place to maintain healthy gene regulation. One of these mechanisms involves modifying the DNA of individual genes with a small methyl group. This modification acts as a flag identifying it as DNA that should be condensed and silenced, and recruits other proteins required for this process. In this way, DNA methylation starts a cascade of events that lead to gene silencing. I intend to study a family of proteins, called the YDG-PHDs, required for the maintenance of this DNA methylation at sites that are designated to be silenced. Besides gaining basic knowledge about gene regulation in plants and mammals, these proteins have a direct link to cancer. Increased expression of the mammalian version of this protein is correlated with cancerous characteristics in tissue culture cells and breast cancer tissue. With this study, I propose to confirm the role of the YDG-PHD proteins in maintaining methylation at silent loci. Primarily, I intend to determine the role of a section within the protein called the YDG domain. Preliminary data indicates that this domain binds to the methyl group present on DNA from silent loci within the genome. I will further the characterization of this domain by determining the specificity of this binding. In addition, I will develop plants with a loss-of-function mutation in each of the YDG-PHD genes individually and, if necessary because of redundancies in function, plants will be produced with mutations in several of these genes at the same time. Performing molecular analysis of these lines, I will confirm that methylation patterns are decreased, and that this leads to aberrant expression of previously silenced genes. Finally, I will explore the relationship between the YDG-PHD proteins and the enzymes involved in adding methyl groups to DNA. These experiments are important for understanding methylation-based gene regulation in Arabidopsis, but because of the parallels between plants and humans this will increase knowledge about a central process in cellular transformation. Misregulation of DNA methylation leads to misexpression of genes and a wide variety of known cancers. For example, multiple tumor suppressor genes, including pRB, APC, BRCA, and VHL, have been shown to be hypermethylated and therefore ectopically silenced in cancer cells. A clear understanding the mechanisms of the maintenance of CG methylation is vital in discerning how these processes go awry in cancer cells.
|Feng, Suhua; Cokus, Shawn J; Zhang, Xiaoyu et al. (2010) Conservation and divergence of methylation patterning in plants and animals. Proc Natl Acad Sci U S A 107:8689-94|
|Kraft, Edward; Bostick, Magnolia; Jacobsen, Steven E et al. (2008) ORTH/VIM proteins that regulate DNA methylation are functional ubiquitin E3 ligases. Plant J 56:704-15|