The vascular endothelial growth factor (VEGF) promoter is capable of forming secondary DNA structures, called G‐quadruplex and i‐motif, in its proximal promoter region (−85 to −50 from the transcription initiation site) that can mask transcription factor binding sites thereby inhibiting transcription. To understand this dynamic region and its implications in transcriptional control we studied protein‐DNA interactions of this structurally versatile region, specifically those involving transcription factors. Our candidate transcription factor, heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a single‐stranded DNA binding protein able to binding to cytosine‐rich sequences such as the VEGF polypurine/polypyrimidine tract. We found that the cytosine‐rich 24‐bp oligomer has the potential of forming an i‐motif at pH 6.5 and formed a complex with hnRNP K as shown by CD spectra analysis and electrophoretic mobility shift assay (EMSA), respectively. The binding of hnRNP K to the VEGF promoter was confirmed through chromatin immunoprecipitation (ChIP) assay, and its effect on transcriptional control was demonstrated through RT/PCR of hnRNP K siRNA KO and overexpression assays. We also observed through EMSA/Footprint that the hnRNP K protein changes the oligo conformation from an i‐motif structure to a more unstructured linear conformation. In summary, this work is the first to show that hnRNP K serves a role in the transcriptional control of VEGF and that a bromine solution left to react to the protein‐complex can be used to footprint its binding motif.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A203.