Molecular Biology of Hypoxia-Inducible Factor-1

Summary

The hypoxia-inducible factor-1 (HIF-1) is a basic-helix-loop-helix-PAS heterodimeric transcription factor that confers oxygen-regulated expression to a number of genes involved in oxygen homeostasis including erythropoietin (Epo), transferrin, glycolytic enzymes, and vascular endothelial growth factor (VEGF). Hypoxic exposure stabilizes the HIF-1α protein by a mechanism involving redox processes. Following het- erodimerization with HIF-1β, better known as the aryl hydrocarbon receptor nuclear translocator (ARNT), HIF-1 binds to the DNA consensus sequence CGTG, known as a potential target of CpG methylation in mammalian cells. We showed that CpG methylation blocks HIF-1 DNA-binding as well as transactivation of reporter gene expression. The hypoxia-responsive 3′ enhancer of the Epo gene was found to be methylation-free in Epo-producing cells despite its location outside of a CpG island. Intriguingly, this site was also methylation-free in cells that do not express Epo, indicating a general selective pressure to prevent CpG methylation, even in the absence of HIF-1 under normoxic conditions. We previously identified the constitutively expressed ATF-1/CREB-1 family members as candidate factors capable of binding the HIF-1 site. We cloned the mouse HIF-1α gene (designated Hif1a) and found that it consists of 15 exons dispersed over 45 kb. Interestingly, mouse Hif1acontains two alternative first exons whose expression is driven by a tissue-specific promoter (exon I.1) or a housekeeping-type promoter located within a methylation-free CpG island (exon I.2). The exon I.1-containing mRNA isoform encodes a predicted polypeptide that is 12 amino acids shorter than the exon I.2-derived mRNA isoform. So far, however, we did not find any functional differences between the two isoforms.The genomic Hif1a clone was used to introduce a null mutation into the mouse Hif1a locus by gene targeting in embryonic stem cells. HIF-1α deficiency is embryonic lethal, suggesting that HIF-1 serves as a non-redundant master regulator of oxygen homeostasis.