Regulation of the Genome by Cytosine Modification
Epigenetic regulation involves reversible but heritable modifications to chromosomes. Not only are histones modified to dynamically control gene expression, but DNA is modified as well. The most common DNA modification in vertebrate systems is methylation of the 5 carbon of cytosines in CpG dinucleotides. CpG methylation is also a precursor to hydroxylation of methylCpG to hydroxymethylCpG. In general, CpG methylation is a repressive mark, whereas CpG hydroxymethylation is activating. These modifications are developmentally programmed and are dysregulated in cancer and other diseases. Dnmt1, the enzyme primarily responsible for maintenance of DNA methylation, is a genetically validated drug target in carcinogenesis. Thus, dissection of the endogenous regulation of Dnmt1 is warranted and a screen for specific inhibitors of this enzyme is well justified.
Using a fluorigenic oligonucleotide substrate in which methylation is linked to restriction enzyme activity, we discovered that one of the N-terminal domains of Dnmt1, termed replication foci targeting sequence (RFTS), is an endogenous inhibitor of DNA-binding (14). This led us to suggest a model of Dnmt1 regulation in which an accessory protein would have to bind RFTS in a manner that activates Dnmt1 for DNA-association and catalyis. Moreover, we have used this activated form of Dnmt1 to conduct a high throughput screen for novel compounds that specifically inhibit the enzyme. Currently, we are characterizing known inhibitors of Dnmt1, searching for new inhibitors, and exploiting cellular systems to dissect endogenous regulation of Dnmt1 by RFTS and other proteins. Our long term goal is to use new molecular probes to manipulate DNA methylation in living systems and to test the hypothesis that titration of Dnmt1 can retard and/or reverse malignant processes by re-activation of silenced tumor suppressor genes, such as the FHIT gene.
F. Syeda, R.L. Fagan, M. Wean, G.V. Awakumov, J.R. Walker, S. Xue, S. Dhe-Paganon, & C. Brenner, "The RFTS Domain is a DNA-competitive Inhibitor of Dnmt1," JBC, v.286, pp. 15344-15351 (2011).
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R.L. Fagan, D.E. Cryderman, L. Kopelovich, L.L.Wallrath & C. Brenner, "Laccaic Acid A is a Direct, DNA-Competitive Inhibitor of DNA Methyltransferase 1," J Biol Chem, v.288, pp. 23858-23867, 2013. DOI: 10.1074/jbc.M113.480517. Download pdf reprint.
J. Huang, A. Stewart, B. Maity, J. Hagen, R.L. Fagan, J. Yang, D.E. Quelle, C. Brenner & R.A.Fisher, "RGS6 Suppresses Ras-inducded Cellular Transformation by Facilitating Tip60-mediated Dnmt1 Degradation and Promoting Apoptosis," Oncogene, in press, 2013. DOI:10.1038/onc.2013.324. Download pdf reprint.