This proposal aims at an understanding of the mechanism of expression of DNA methylated genes. DNA methylation is a key mechanism for gene regulation in most eukaryotes. It is generally associated with gene silencing when found in promoter regions. However, DNA methylation does not always cause complete silencing of a gene. In some cases DNA methylated genes can be actively transcribed suggesting the presence of anti-silencing factors. I performed a genetic screen to look for such anti-silencing factors. I found that mutations in MED12, MED13, and SAC3B were able to repress the expression of a DNA methylated GFP reporter. All of the three protein factors were found previously to work together in gene regulation through a defined network. Moreover, defects in MED12 and MED13 have been previously shown to cause severe diseases in humans, such as cancers and cardiovascular diseases. Preliminary study of SAC3B suggested that it preferentially affects the expression of DNA methylated genes. It is therefore hypothesized that genes isolated from the screen are anti-silencing factors preferentially required for the expression of DNA methylated genes. To provide further support for this hypothesis, the importance of DNA methylation in MED12/13- and SAC3B- mediated gene expression will be determined by using the same GFP reporter without DNA methylation. In addition, the genome-wide dependence of DNA methylation related gene expression on MED12/13 and SAC3B will be tested by using DNA methyltransferase mutants and DNA methyltransferase inhibitors. It is expected that the expression of certain genes will be only affected by these anti-silencing factors when they carry DNA methylation. Next, I plan to determine what sequence context of DNA methylation (CG, CHG, or CHH, where H is not G) is preferentially associated with this phenomenon. In addition to DNA methylation, two other types of epigenetic features may also facilitate the recruitment of the above-mentioned anti-silencing factors. One is histone modification, and the other is chromatin accessibility. I will profile the genome-wide binding sites of these anti-silencing factors, and examine what other histone marks are enriched at these binding sites. ATAC-seq will be performed to determine the chromatin accessibility around their genomic binding sites. Finally, I will tether these anti-silencing factors to an endogenous DNA methylated and silenced locus using a well-tested zinc finger system. If my hypothesis is correct, it is expected that the tethered anti- silencing proteins will activate transcription without affecting DNA methylation. Finally, immunoprecipitation combined with mass spectrometry will be performed to dissect the composition of anti-silencing protein complexes.
Many human diseases are caused by misregulated gene expression. The precise and timely regulation of gene expression is complicated by a particular type of chemical modification of DNA, DNA methylation, that acts to suppress the gene activation. This project aims to help us better understand the mechanisms of activating the expression of DNA methylated genes.
