SELECTION AND ANALYSIS OF A MUTANT-CELL LINE DEFECTIVE IN THE HYPOXIA-INDUCIBLE FACTOR-I ALPHA-SUBUNIT (HIF-1-ALPHA) - CHARACTERIZATION OF HIF-1-ALPHA-DEPENDENT AND HIF-1-ALPHA-INDEPENDENT HYPOXIA-INDUCIBLE GENE-EXPRESSION

Hypoxia-inducible expression has been demonstrated for many groups of mammalian genes, and studies of transcriptional control have revealed the existence of hypoxia-responsive elements (HREs) in the cis-acting sequences of several of these genes, These sequences generally contain one or more binding sites for a heterodimeric DNA binding complex ter med hypoxia-inducible factor-1 (HIF-1), To analyze this response furth er, Chinese hamster ovary cells were stably transfected with plasmids bearing HREs linked 60 genes encoding imnmunoselectable cell surface m arkers, and clones that showed reduced or absent hypoxia-inducible mar ker expression were selected from a mutagenized culture of cells. Anal ysis of these cells revealed several closes with transacting defects i n HRE activation, and in one the defect was identified as a failure to express the ru-subunit of HIF-1, Comparison of hypoxia-inducible gene expression in wild type, HIF-1 alpha-defective, and HIF-1 alpha-compl emented cells revealed tyro types of response, For some genes (e.g. gl ucose transporter-1), hypoxia-inducible expression was critically depe ndent on HIF-1 alpha, whereas for other genes (e.g. heme oxygenase-1) hypoxia-inducible expression appeared largely independent of the expre ssion of HIF-1 alpha. These experiments show the utility of mutagenesi s and selection of mutant cells in the analysis of mammalian transcrip tional responses to hypoxia and demonstrate the operation of HIF-1 alp ha-dependent and HIF-1 alpha-independent pathways of hypoxia-inducible gene expression in Chinese hamster ovary cells.