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. 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 used this activated form of Dnmt1 to conduct a high throughput screen for novel compounds that specifically inhibit the enzyme. We discovered laccaic acid A as a direct, potent inhibitor of Dnmt1. Furthermore we found that TET1 is a KRAS and m1R-29b-repressed factor that must be removed from Dnmt1-targeted promoters for tumor suppressor gene methylation.
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.
R.L. Fagan, M. Wu, F. Chédin & C. Brenner, "An Ultrasensitive High Throughput Screen for DNA Methyltransferase 1-targeted Molecular Probes"PLoS One, v. 11,e78752, 2013. Download pdf reprint.
B.-K. Wu, S.-C. Mei & C. Brenner, "RFTS-deleted DNMT1 enhances tumorigenicity with focal hypermethylation and global hypomethylation," Cell Cycle, v. 13, pp. 3222-3231 (2014). DOI:10.4161/15384101.2014.950886. Download pdf reprint.
B.-K. Wu & C. Brenner, “Suppression of TET1-Dependent DNA Demethylation is Essential for KRAS-Mediated Transformation,” Cell Reports, v. 9, pp. 1827-1840 (2014). DOI:10.1016/j.celrep.2014.10.063. Download pdf reprint.
S.Thakur & C. Brenner, “KRAS-driven miR-29b Expression is Required for Tumor Suppressor Gene Silencing,” Oncotarget, v.8(43), pp. 74755-74766 (2017). DOI: 10.18632/oncotarget.20364. Download PDF reprint.