HIF-1 was originally identified as a nuclear factor that was induced by hypoxia and bound to a site in the EPO hypoxia-response element. Nucleotide substitutions in the hypoxia-response element that eliminated HIF-1 binding also eliminated transcriptional activation in response to hypoxia. HIF-1 DNA-binding activity was shown to be tightly regulated by cellular 02 tension. Biochemical purification by ion exchange and DNA affinity chromatography revealed that HIF-1 was a heterodimer consisting of HIF-la and HIF-ip subunits.
Protein microsequence analysis and complementary DNA (cDNA) cloning revealed the primary structure of both subunits. The amino terminal half of both subunits contained two important protein motifs (Fig 2). The basic helix-loop-helix (bHLH) motif is found in a large number of transcription factors. Protein dimerization is mediated by the helix-loop-helix domain that juxtaposes basic amino acids from each monomer to form an intact DNA-binding domain. In a subset of bHLH proteins, a second domain is required for efficient dimerization, which was named the PAS domain by virtue of its identification within the PER, ARNT, and SIM proteins. canadian health and care mall
Analysis of protein and cDNA sequences revealed that the HIF-lp subunit was identical to ARNT, the aryl hydrocarbon receptor nuclear translocator protein, a subunit of the aryl hydrocarbon receptor complex (dioxin receptor), a ligand-activated transcription factor involved in cellular responses to xenobiotic agents. ARNT was shown to be synthesized as isoforms of 774 and 789 amino acids based on alternative splicing of the primary RNA transcript. In contrast to HIF-1 p, HIF-la was shown to represent a novel bHLH-PAS protein of 826 amino acids. The helix-loop-helix-PAS domains of HIF-la were required for dimerization to HIF-lp, which was a necessary prerequisite for DNA binding via the basic domains of HIF-la and HIF-lp.
Figure 2. Structure of HIF-1. The HIF-la and HIF-ip subunits are shown schematically. The amino terminal half of each polypeptide includes the bHLH domain (stippled box) and PAS domain (hatched box) which contains internal 50-amino-acid homology units, the A and B repeats (filled boxes). The 789-amino-acid (aa) isoform of HIF-1 (3 contains a region of 15 aa immediately preceding the bHLH domain that is absent from the 774-aa isoform.
Tags: dna-binding activity, hypoxia-response element, protein hif-1