ACTIVATION OF HYPOXIA-INDUCIBLE FACTOR 1-ALPHA - POSTTRANSCRIPTIONAL REGULATION AND CONFORMATIONAL CHANGE BY RECRUITMENT OF THE ARNT TRANSCRIPTION FACTOR

In response to hypoxia the hypoxia-inducible factor-1 (HIF-1) mediates transcriptional activation of a network of genes encoding erythropoie tin, vascular endothelial growth factor, and several glycolytic enzyme s, HIF-1 consists of a heterodimer of two basic helix-loop-helix PAS ( Per/Arnt/Sim) proteins, HIF-1 alpha and Arnt, HIF-1 alpha and Arnt mRN As are constitutively expressed and were not altered upon exposure of HeLa or HepG2 cells to hypoxia, suggesting that the activity of the HI F-1 alpha-Arnt complex may be regulated by some as yet unknown posttra nscriptional mechanism, In support of this model, we demonstrate here that Arnt protein levels were not increased under conditions that indu ce an hypoxic response in HeLa and HepG2 cells, However, under identic al conditions, HIF-1 alpha protein levels were rapidly and dramaticall y up-regulated, as assessed by immunoblot analysis, In addition, HIF-1 alpha acquired a new conformational state upon dimerization with Arnt , rendering HIF-1 alpha more resistant to proteolytic digestion irt vi tro, Dimerization as such was not sufficient to elicit the conformatio nal change in HIF-1 alpha, since truncated forms of Arnt that are capa ble of dimerizing with HIF-1 alpha did not induce this effect, Moreove r, the high affinity DNA binding form of the HIF-1 alpha-Arnt complex was only generated by forms of Arnt capable of eliciting the allosteri c change in conformation, In conclusion, the combination of enhanced p rotein levels and allosteric change by dimerization defines a novel me chanism for modulation of transcription factor activity.