Abstract A203: Heterogeneous nuclear ribonucleoprotein K (hnRNP K) binds to the vascular endothelial growth factor (VEGF) promoter and plays a role in its transcriptional control

Abstract

Abstract 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.