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. 1998;7(3):205-13.

Molecular characterization and chromosomal localization of a third alpha-class hypoxia inducible factor subunit, HIF3alpha

Y Z Gu  1 , S M Moran, J B Hogenesch, L Wartman, C A Bradfield
Affiliations

Molecular characterization and chromosomal localization of a third alpha-class hypoxia inducible factor subunit, HIF3alpha

Y Z Gu et al. Gene Expr. 1998.

Abstract

Hypoxia inducible factors (HIFs) are heterodimeric transcription factors that regulate a number of adaptive responses to low oxygen tension. They are composed of alpha- and beta-subunits that belong to the basic helix-loop-helix-PAS (bHLH-PAS) superfamily. In our efforts to identify new bHLH-PAS proteins, we cloned a cDNA encoding a novel alpha-class hypoxia inducible factor, HIF3alpha. The HIF3alpha open reading frame encodes a 662-amino acid protein with a predicted molecular weight of 73 kDa and is expressed in adult thymus, lung, brain, heart, and kidney. The N-terminal bHLH-PAS domain of this protein shares amino acid sequence identity with that of HIF1alpha and HIF2alpha (57% and 53% identity, respectively). The C-terminus of HIF3alpha contains a 36-amino acid sequence that shares 61% identity with the hypoxia responsive domain-1 (HRD1) of HIF1alpha. In transient transfections, this domain confers hypoxia responsiveness when linked to a heterologous transactivation domain. In vitro studies reveal that HIF3alpha dimerizes with a prototype beta-class subunit, ARNT, and that the resultant heterodimer recognizes the hypoxia responsive element (HRE) core sequence, TACGTG. Transient transfection experiments demonstrate that the HIF3alpha-ARNT interaction can occur in vivo, and that the activity of HIF3alpha is upregulated in response to cobalt chloride or low oxygen tension.

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Figures

FIG. 1
FIG. 1
Cloning of HIF3α. The positions of the original EST clone (AA028416) and the RACE products are shown as dark lines with the mHIF3α ORF shown as an open box. The PCR primers used are posted below the corresponding fragments and the plasmid number are marked on the side. The GenBank accession number for mHIF3α cDNA is AF060194.
FIG. 2
FIG. 2
Alignment of three HIFs. The amino acid sequence of mouse HIF3α is aligned with that of mouse HIF1α and HIF2α using the CLUSTAL method. The positions of amino acids are shown on the left side. The basic region (BR), helix–loop–helix (HLH), PAS, and the hypoxia-responsive domains (HRDs) are marked with dark lines above the sequence. Conserved residues are boxed and in light gray.
FIG. 3
FIG. 3
Northern Blot analysis of HIF3α. Poly(A)+ (2 μg) RNA from each mouse tissue was loaded in each lane and hybridized with either HIF3α (upper panel) or β-actin (lower panel) probe as described in Materials and Methods. The molecular weight marker is shown on the left side.
FIG. 4
FIG. 4
The splicing sites within mHIF3α ORF are compared with those previously reported for mHIF1α and hHIF2α. The numbers of amino acids at which the splicing occurs are marked underneath the sequence. The conserved splicing sites are defined as the splicing sites of HIF1α and HIF2α that are within one amino acid of the corresponding HIF3α splicing sites on the aligned sequence map using CLUSTAL method. These sites are marked with lines between different ORFs (see GenBank accession number AF079140-079153 for detailed sequences of mHIF3α splice sites).
FIG. 5
FIG. 5
Chromosomal localization of HIF3α. Top: Chromosomal distribution in the somatic cell hybrid panel (BIOS Laboratories, CT). Cell lines and chromosomal number are marked on the axes. A “+” indicates > 30% of the cells contain the given chromosome; “#” indicates 5–30% of the cells contain the given chromosome; “D” indicates multiple deletions on the chromosome. The cell lines from which human HIF3α-specific PCR reaction are positive are also marked “+” in the last row. Bottom: HIF3α gene chromosomal localization via human HIF3α-specific PCR reaction. Human HIF3α-specific primers OL1548 and OL1698 were used for PCR reaction against genomic DNA from somatic cell hybrids. The expected 136-bp human HIF3α PCR product is marked. “NS” indicates nonspecific bands generated from the hamster genome. “+” and “−” indicate human and hamster genomic DNA served as positive and negative controls, respectively.
FIG. 6
FIG. 6
Gel-shift analysis of HIF3α. Radiolabeled oligonucleotide probe containing the HRE core sequence TACGTG was incubated with HIF3α and/or ARNT in the absence or presence of preimmune (PI) or immune (Imm) anti-ARNT antibody as described in Materials and Methods. For competition experiments, 400-fold of either wild-type (TACGTG) or mutant (AACGTG or GACGTG) oligonucleotides was added into each reaction. Specific (HIF3α/ARNT) and nonspecific (NS) complexes are marked.
FIG. 7
FIG. 7
Transient transfection of HIF3α. (A) Interaction of HIF3α and ARNT in transiently transfected COS-1 cells. COS-1 cells were cotransfected with plasmids expressing HIF3α and/or ARNT and a luciferase reporter driven by six HRE containing the TACGTG core sequence (inset). (B) Induction of HIF3α HRD1 domain by cobalt chloride and hypoxia treatment. Hep3B cells were transfected with plasmids expressing the GAL4/HRD/TAD fusion protein and a luciferase reporter driven by five GAM binding sites. The cells were treated without or with 100 μM CoCl2 or hypoxia (1% O2) for 20 h prior to harvest. Relative light units (RLU) were measured as described in Materials and Methods. Transfected expression plasmids and reporters are illustrated in the insets. The white, black, and striped columns represent control (C), cobalt chloride (Co), and hypoxia (H) treatments, respectively.
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