Characterization of the C-rich secondary DNA structure in the SP1 binding region of the HIF1-alpha promoter

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Hypoxia inducible factor (HIF) is a commonly overexpressed transcription factor in cancer cells that is involved in anaerobic glycolysis, erythropoietin synthesis, angiogenesis, and ultimately aiding in metastasis. A polyguanine/polycytosine (polyG/polyC) tract in the -65 to -85 position of the promoter of HIF1-a was found to contain several overlapping or adjacent putative transcription factor binding sites. We have shown that SP1 is a major transcription factor in this region through EMSA, DNaseI footprinting as well as treatment with SP1 siRNA. Mutation of the polyG/polyC sequence reduces basal expression of the HIF1-a gene by 10 fold, suggesting this region is highly important for transcription regulation. In our previous studies, we have demonstrated that the guanine-rich strand of this polyG/polyC tract is capable of forming a stable G-quadruplex structure in the presence of potassium. Therefore, we hypothesize that the formation of the G-quadruplex on the guanine-rich strand might facilitate the evolution of another secondary structure on the complimentary cytosine-rich strand. To examine the potential formation of a secondary structure in the cytosine-rich strand we designed a series of 27-basepair oligonucleotides representing the wild-type and mutant cytosine-rich strand and studied them through circular dichroism, bromine footprinting, and photo-cleavage assays. Our CD spectroscopy data of the 27bp oligo demonstrate a 285nm signature peak that is characteristic of an i-motif structure and is stable from pH 4.0 to 6.3 and unstable at pH 7 to 8. Chemical footprinting confirm the formation of the i-motif as shown by CD spectroscopy and reveal that the i-motif structure is an intercalating intramolecular structure consisting of three cytosine basepairs with a 3:3:3 loop size. These data suggest that secondary DNA structures can form in the polyG/polyC tract of the HIF1-a proximal promoter and that the formation of the G-quadruplex and i-motif structures may play a role in the transcriptional regulation of this gene.