Effects of 5-Hydroxymethylcytosine Epigenetic Modifications within the VEGF Promoter Region on G-Quadruplex and I-Motif DNA Structure and Stability

Abstract

Epigenetic modifications to DNA base sequences may regulate gene expression. CpG islands can contain methylated (5mC) or hydroxymethylated (5hmC) cytosine. Most CpG islands are found primarily in promoter regions that may also contain a high number of repeated cytosines and/or guanines. G-quadraplexes (G4) and i-motifs (iM) are two unique DNA secondary structures that can form in repeating sequences of either guanine or cytosine, respectively. Both G4 and iM sequences may contain CpG sequences that can be methylated or hydroxymethylated. The effects of CpG islands on DNA secondary structures were determined by incorporating a single 5hmC at varying positions in the Vascular Endothelial Growth Factor (VEGF) G4 and iM sequences. An Olis DSM-20 spectropolarimeter and a Cary 100 UV-visible spectrometer were used to monitor the effect of 5hmC on G4 and iM thermal stability. Two of the three 5hmC-containing loops showed a notable decrease in stability for G4's and increased intermolecular structure formation. Contrastingly, the iM stability increased when 5hmC was incorporated into its sequence. Additionally, there was little change in the iM pka. In summary, our results suggest the 5hmC has little effect on iM structures, but can destabilize the G4's.