Selection and analysis of a mutant cell line defective in the hypoxia-inducible factor-1 alpha-subunit (HIF-1alpha). Characterization of hif-1alpha-dependent and -independent hypoxia-inducible gene expression.

The Journal of Biological Chemistry
S M WoodP J Ratcliffe

Abstract

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 termed hypoxia-inducible factor-1 (HIF-1). To analyze this response further, Chinese hamster ovary cells were stably transfected with plasmids bearing HREs linked to genes encoding immunoselectable cell surface markers, and clones that showed reduced or absent hypoxia-inducible marker expression were selected from a mutagenized culture of cells. Analysis of these cells revealed several clones with transacting defects in HRE activation, and in one the defect was identified as a failure to express the alpha-subunit of HIF-1. Comparison of hypoxia-inducible gene expression in wild type, HIF-1alpha-defective, and HIF-1alpha-complemented cells revealed two types of response. For some genes (e.g. glucose transporter-1), hypoxia-inducible expression was critically dependent on HIF-1alpha, whereas for other genes (e.g. heme oxygenase-1) hypoxia-inducible expression appeared largely independent o...Continue Reading

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