Abstract
Abstract CpG islands are prone to epigenetic cytosine modification via methylation (5mC) or 5-hydroxymethylation (5hmC). Although their potential sequences and regions for occurrence in G/C-rich DNA overlap, throughout the genome 5mC and 5hmC, and 5mC and G-quadruplex (G4) and i-motif (iM) structures, are found to not be co-existent. Moreover, in some regions of DNA, 5hmCs and G4/iM forming regions have high potential to form in concurrence. However, minimal research has been done examining the effect of 5hmC incorporation into G4 and iMs in order to determine the effect of cytosine modification on non-B-DNA structures. These higher order secondary DNA formations have been described within the VEGF promoter and have great potential to silence its transcription and prevent subsequent angiogenesis. In addition, there are three cytosines in the promoter on both the guanine- and the cytosine-rich strands found in CpG sequences that have the potential for 5mhC modification. Therefore, the current study was undertaken to examine the whether there is an effect of 5hmC incorporation into G4 loops and iM stems on structure formation and stabilization. Significant changes in structure or stability in buffers with varying pH’s, cationic strength, and molecular crowding conditions were examined by circular dichroism and confirmed by UV-monitored melting. Electromobility shift assays differentiated inter- and intra-molecular structures, further identifying the number of isoforms. Notable changes in G4 dynamics were seen with two of the three loop modifications, which decreased overall stability and increased intermolecular structures. Much less marked effects were discovered for the iM structures. Ongoing studies examining the role of G4 and iM 5hmC incorporation in regulation by endogenous proteins are part of a global effort to best understand the physiological conditions regulating G4 and iM stability and function. These VEGF promoter higher order structures are promising therapeutic targets for anti-angiogenic therapy, and a comprehensive understanding of the physiological principles governing their formation will best inform future drug discovery efforts. Brooks Lab startup funds, R15, University of Mississippi School of Pharmacy Citation Format: Rhianna Morgan, Randy Wadkins, Tracy Brooks. Effects of 5-hydroxymethylcytosine epigenetic modification on G-quadruplex and i-motif structure and stability within the VEGF promoter. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1050. doi:10.1158/1538-7445.AM2015-1050
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