Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.

Abstract

Hypoxia-inducible factor 1 (HIF-1) is found in mammalian cells cultured under reduced 02 tension and is necessary for transcriptional activation mediated by the erythropoietin gene enhancer in hypoxic cells. We (8,9).The molecular mechanisms that mediate genetic responses to hypoxia have been extensively investigated for the EPO gene, which encodes a growth factor that regulates erythropoiesis and, thus, blood 02-carrying capacity (1, 4). Cis-acting DNA sequences required for transcriptional activation in response to hypoxia were identified in the EPO 3' flanking region, and a trans-acting factor that binds to the enhancer, hypoxia-inducible factor 1 (HIF-1), fulfilled criteria for a physiological regulator of EPO transcription: inducers of EPO expression (1% 02, CoC12, and desferrioxamine) also induced HIF-1 DNA-binding activity with similar kinetics, inhibitors of EPO expression (actinomycin D, cycloheximide, and 2-aminopurine) blocked induction of HIF-1 activity, and mutations in the EPO 3' flanking region that eliminated HIF-1 binding also eliminated enhancer function (4). These results also support the hypothesis that 02 tension is sensed by a hemoprotein (10) and suggest that a signal transduction pathway requiring ongoing transcription, translation, and protein phosphorylation participates in the induction of HIF-1 DNA-binding activity and EPO transcription in hypoxic cells (4 Glycerol gradient sedimentation, UV crosslinking, and methylation interference studies suggested that HIF-1 was a heterodimer consisting of the 120-kDa HIF-la subunit complexed with a 91-to 94-kDa HIF-13 subunit and that both subunits contacted DNA in the major groove (11,13,14). Here we show that HIF-la and HIF-13 are basic-helix-loop-helix (bHLH)-PAS proteins whose expression is regulated by cellular 02 tension.t MATERIALS AND METHODSProtein Microsequence Analysis. Purified HIF-1 subunits were fractionated by SDS/PAGE, and the 120-and 94-kDa polypeptides were transferred to poly(vinylidene difluoride) membranes and individually digested with trypsin in situ, and peptides were fractionated by reverse-phase HPLC (13). Protein microsequence analysis was performed at the Wistar Protein Microchemistry Laboratory (Philadelphia) (15).cDNA Library Construction and Screening. Poly(A)+ RNA was isolated from Hep 3B human hepatoma cells cultured for 16 hr at 37°C in a chamber flushed with 1% 02/5% C02/94% N2. cDNA was synthesized by using oligo(dT) and random hexamer primers, and bacteriophage libraries were constructed in Agtl 1 and Uni-ZAP XR (Stratagene). cDNA libraries were screened with 32P-labeled cDNA fragments by plaque hybridization (16).PCR. Degenerate oligonucleotide primers were designed by using codon preference rules (17). aF1 (5'-ATCGGATCCAT-CACIGARCTSATGGGITATA-3') was based upon the amino terminus of HIF-la peptide 87-1 and used as a forward primer. Two nested reverse primers, aRl (5'-ATTAAGCTTT-TGGTSAGGTGGTCISWGTC-3') and aR2 (5'-ATTAAGCT-TGCATGGTAGTAYTCATAGAT-3'), were based upon the carboxyl terminus of peptide ...

The TWIST1 oncogene is a direct target of hypoxia-inducible factor-2α

“…Among HIF target genes are glycolytic genes (for example Phosphoglycerate Kinase 1 (PGK1)) and genes involved in angiogenesis (for example Vascular Endothelial Growth Factor (VEGF)), which are key players in the hypoxic response (Greijer et al, 2005). The human genome encodes three homologous HIFa proteins, respectively HIF-1, -2 and -3a (Wang et al, 1995;Ema et al, 1997;Gu et al, 1998) and three prolyl hydroxylases (Epstein et al, 2001;Semenza, 2001). The mechanism of oxygen sensing via the HIF pathway is conserved throughout evolution.…”

“…To determine whether HIF induces TWIST1 directly, we screened the genomic TWIST1 sequence for consensus HIF binding elements or HREs (Wang et al, 1995). We identified two potential HREs, 1(CGCGTG) and 2(TGCGTG), within 6 bp proximity within the only intron of TWIST1, located immediately downstream of the coding region that terminates in exon 1 (Figure 5a).…”

Hypoxia upregulates hypoxia inducible factor (HIF)-3α expression in lung epithelial cells: characterization and comparison with HIF-1α

“…Both subunits are constitutively expressed and belong to a family of basic helix-loop-helix-Per-Arnt-Sim (bHLH-PAS) transcriptional factors that are required both for DNA binding and for transactivation of an array of target genes. Whereas HIF-1β , also termed the aryl hydrocarbon nuclear receptor translocator (ARNT), can heterodimerize with other proteins containing the bHLH-PAS domain [7]. HIF-1α is the specific and oxygen-regulated subunit of the HIF-1 complex and determines the level of HIF-1 activity [8,9].…”

“…Under normoxia, the HIF-1α subunit is rapidly degraded by the ubiquitin-proteasome system, thus hampering the heterodimeric HIF-1 activity [10]. By contrast, under hypoxia or in the presence of iron chelators, the degradation of HIF-1α is prevented [7,11,12]. As a result, this stabilization initiates a multi-step pathway of activation of HIF-1α that includes hypoxia-dependent nuclear translocation and dimerization with ARNT to interact with hypoxia responwww.cell-research.com | Cell Research Qi Fang Li et al 549 npg sive element (HRE) of target genes such as erythropoietin (EPO) [13], vascular endothelial growth factor (VEGF) [14], inducible nitric-oxide synthase [15], heme oxygenase 1 [16], and so on.…”

Assessment of the gene expression profile of differentiated and dedifferentiated human fetal chondrocytes by microarray analysis

“…Since we were primarily interested in gene regulatory factors that were differentially regulated by the two culture conditions, we proceeded to document HIF-1␣ protein levels and binding activity in freshly isolated chondrocytes. We hypothesized that as chondrocytes form clusters in poly-HEMA culture, they become hypoxic and should therefore express HIF-1␣ (46). In order to address this hypothesis, we performed an EMSA to detect binding of HIF-1, which is a dimer of HIF-1␣ and HIF-1␤ (also known as aryl hydrocarbon receptor nuclear translocator [ARNT]).…”