STRUCTURAL AND FUNCTIONAL-ANALYSIS OF HYPOXIA-INDUCIBLE FACTOR-1

Hypoxia-inducible factor 1 (HIF-1) is a basic helix-loop-helix protein that activates transcription of hypoxia-inducible genes, including th ose encoding: erythropoietin, vascular endothelial growth factor, heme oxygenase-1, inducible nitric oxide synthase, and the glycolytic enzy mes aldolase A, enolase 1, lactate dehydrogenase A, phosphofructokinas e L, and phosphoglycerate kinase 1. Hypoxia response elements from the se genes consist of a HIF-1 binding site (that contains the core seque nce 5'-CGTG-3') as well as additional DNA sequences that are required for function, which in some elements include a second HIF-1 binding si te. HIF-1 is a heterodimer. The HIF-1 alpha subunit is unique to HIF-1 , whereas HIF-1 beta (ARNT) can dimerize with other bHLH-PAS proteins. Structural analysis of HIF-1 alpha revealed that dimerization with HI F-1 beta (ARNT) requires the HLH and PAS domains, DNA binding is media ted by the basic domain, and that HIF-1 alpha contains a carboxyl-term inal transactivation domain. Co-transfection of HIF-1 alpha and HIF-1 beta (ARNT) expression vectors and a reporter gene containing a wild-t ype hypoxia response element resulted in increased transcription in no n-hypoxic cells and a superinduction of transcription in hypoxic cells , whereas HIF-1 expression vectors had no effect on the transcription of reporter genes containing a mutation in the HIF-1 binding site. HIF -1 alpha and HIF-1 beta (ARNT) protein levels were induced by hypoxia in all primary and transformed cell lines examined. In HeLa cells, the levels of HIF-1 alpha and HIF-1 beta protein and HIF-1 DNA-binding ac tivity increased exponentially as cellular oxygen tension decreased, w ith maximum values at 0.5% oxygen and half-maximal values at 1.5 to 2% oxygen. HIF-1 alpha and HIF-1 beta (ARNT) mRNAs were detected in all human, mouse, and rat organs assayed and mRNA expression was modestly induced in rodents subjected to hypoxia. HIF-1 alpha protein levels we re induced in vivo when animals were subjected to anemia or hypoxia. T he HIF1A gene was mapped to human chromosome 14q21-q24 and mouse chrom osome 12.