Defining the Role of Secondary DNA Structures and Transcription Factors on the Transcriptional Control of the HIF-1alpha and VEGF Promoters

Abstract

Angiogenesis is known to be induced and maintained in tumors by the constant expression of the hypoxia inducible factor 1 alpha (HIF-1α) and human vascular endothelial growth factor (VEGF). In fact, tumor recurrence, aggressive metastatic legions and patient mortality rates are known to be positively correlated with overexpression of these two proteins. The HIF-1α and VEGF promoters contain a polypurine/polypyrimidine (pPu/pPy) tract, which are known to play critical roles in their transcriptional regulation, and are structurally dynamic where they can undergo a conformational transition between B-DNA, single stranded DNA and atypical secondary DNA structures such as G-quadruplexes and i-motifs. We hypothesize that the i-motif and G-quadruplex structures can form within the pPu/pPy tracts of the HIF-1α and VEGF proximal promoters, which play important roles in the transcriptional regulation of these genes by acting as scaffolds for alternative transcription factor binding sites. The purpose of this dissertation was to elucidate the transcriptional regulation of the HIF-1α and VEGF genes through the atypical DNA structures that form within the pPu/pPy tracts of their proximal promoters. We investigated the interaction of the C-rich and guanine-rich (G-rich) strands of both of these tracts with transcription factors heterogeneous nuclear ribonucleoprotein (hnRNP) K and nucleolin, respectively, both in vitro and in vivo and their potential role in the transcriptional control of HIF-1α and VEGF. In this dissertation, we demonstrate that both nucleolin and hnRNP K bind selectively to the G- and C-rich sequences, respectively, in the pPu/pPy tract of the HIF-1α and VEGF promoters. Specifically, the small interfering RNA-mediated silencing of either nucleolin or hnRNP K resulted in the down-regulation of basal VEGF gene, and the opposite effect was seen when the transcription factors were overexpressed, suggesting that they act as activators of VEGF transcription. Taken together, the identification of transcription factors that can recognize and bind to atypical DNA structures within pPu/pPy tracts will provide new insight into mechanisms of transcriptional regulation of the HIF-1α and VEGF gene.