Alternative titles; symbolsHIF3-ALPHAINHIBITORY PAS DOMAIN PROTEIN; IPASHGNC Approved Gene Symbol: HIF3ACytogenetic location: 19q13.32 Genomic coordinates (G...
Alternative titles; symbols
HGNC Approved Gene Symbol: HIF3A
Cytogenetic location: 19q13.32 Genomic coordinates (GRCh38): 19:46,297,041-46,343,432 (from NCBI)
Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors that regulate adaptive responses to low oxygen tension. HIFs are composed of alpha subunits, such as HIF3A, and beta subunits, both of which belong to the superfamily of proteins containing basic helix-loop-helix (bHLH) and PER (see PER1; 602260)-ARNT (126110)-SIM (see SIM1; 603128) (PAS) domains (Hara et al., 2001).
▼ Cloning and Expression
By searching an EST database using mouse Hif3a as query, followed by PCR of a kidney cDNA library, Hara et al. (2001) cloned HIF3A. The deduced 668-amino acid protein contains a bHLH domain and a PAS domain with PAS-A and PAS-B regions in its N-terminal half and an N-terminal transactivation domain (NAD) in its C-terminal half, but it lacks the C-terminal transactivation domain (CAD) found in HIF1A (603348) and HIF1B (ARNT). Immunohistochemical analysis revealed HIF3A expression in distal tubules in human kidney.
By screening databases for proteins containing oxygen-dependent degradation domains (ODDs), Maynard et al. (2003) identified 6 alternatively spliced HIF3A variants that they designated HIF3A1 through HIF3A6. HIF3A1 is the full-length protein reported by Hara et al. (2001). In its N-terminal half, HIF3A1 has a PAS-associated C-terminal (PAC) domain immediately following the PAS-B domain. In its C-terminal half, it has an ODD, which includes the NAD, and a leucine zipper (LZIP) domain that is not present in HIF1A, HIF1B, or any other HIF3A splice variants. HIF3A1 also has 2 LxxLL protein-protein interaction motifs, one upstream of the ODD and the other upstream of the LZIP domain. The deduced 632-amino acid HIF3A2 protein lacks the more C-terminal LxxLL motif and the C-terminal LZIP found in HIF3A1. The other HIF3A variants lack different domains and motifs found in HIF3A1. Northern blot analysis detected tissue-specific expression of 7.5-, 7.0-, 3.0-, and 1.5-kb HIF3A transcripts. Expression was strongest in heart, placenta, and skeletal muscle and weaker in lung, liver, and kidney.
By PCR, Maynard et al. (2005) cloned the HIF3A4 variant from a human cerebellum cDNA library. The deduced 363-amino acid protein contains an N-terminal bHLH domain and PAS-A and PAS-B domains.
Makino et al. (2001) cloned mouse Hif3a, which they called Ipas. The deduced 307-amino acid Ipas protein contains an N-terminal bHLH domain, followed by PAS-A and PAS-B domains, but it lacks transactivation domains. Northern blot analysis detected prominent Ipas expression in eye, with lower levels in cerebellum and cerebrum. In situ hybridization revealed intense Ipas expression in the epithelial cell layer of the cornea and weaker expression in several retinal cell types. In cerebellum, Ipas expression was limited to the Purkinje cell layer. Fluorescence-tagged Ipas showed a predominantly nuclear localization under both normoxic and hypoxic conditions.
▼ Gene Function
Hara et al. (2001) found that hypoxia increased the protein levels of HIF1A and HIF2A in transfected COS-7 cells, whereas the level of HIF3A was unaffected. HIF3A failed to upregulate transcription from a hypoxia-responsive element (HRE), and overexpression of HIF3A in a human renal cell carcinoma cell line reduced HRE-driven transcription. Furthermore, transfection of HIF3A suppressed HIF1A- and HIF2A-mediated gene expression and xenobiotic responsive element-driven gene expression.
Makino et al. (2001) found that expression of mouse Ipas in HeLa cells downregulated expression of a reporter gene driven by an HRE under hypoxic conditions. Hypoxia-dependent activation of the reporter gene by transfected Hif1a was impaired by Ipas coexpression in a dose-dependent manner. Ipas directly interacted with Hif1a, and the Ipas-Hif1a complex did not bind to an HRE probe. Ipas also interfered with the ability of an Hif1a-Arnt heterodimer to bind an HRE. Downregulation of Ipas expression by an Ipas antisense oligonucleotide induced angiogenesis under normal oxygen conditions in mouse cornea and induced Vegf (192240) gene expression in hypoxic corneal cells. Since corneal hypoxia is normally induced during sleep by eye closure, Makino et al. (2001) suggested that Ipas may contribute to the total avascularity and transparency of the cornea by inhibiting hypoxia-induced gene expression.
By immunoprecipitation, Maynard et al. (2005) found that HIF3A4 bound HIF1A following their coexpression in human embryonic kidney cells. HIF3A4 also coprecipitated with ectopically expressed or endogenous ARNT. The HIF1A-ARNT complex bound to an HRE in vitro, but the HIF3A4-ARNT complex did not. Binding of HIF1A-ARNT to an HRE was inhibited by addition of HIF3A4 in a dose-dependent manner. HIF3A4 inhibited HRE-driven transcription in vitro, and it inhibited expression of hypoxia-responsive genes, such as GLUT1 (SLC2A1; 138140) and VEGF, in cultured human renal carcinoma and osteosarcoma cells. Knockdown of HIF3A4 by small interfering RNA increased transcription of hypoxia-inducible genes. Expression of HIF3A4 itself was downregulated by hypoxia.
Maynard et al. (2003) found that HIF3A1, HIF3A2, and HIF3A3 were targeted for ubiquitylation by a VHL (608537)-containing E3 ubiquitin ligase complex in vitro and in vivo. The activity was enhanced in the presence of prolyl hydroxylase (see P4HA1; 176710) and was dependent on oxygen and a proline within the ODD. Ubiquitylation occurred on 2 lysines within the ODD.
▼ Gene Structure
Maynard et al. (2003) determined that the HIF3A gene spans 43 kb and contains 19 exons, including 3 alternatively spliced first exons.
By genomic sequence analysis, Hara et al. (2001) mapped the HIF3A gene to chromosome 19. Maynard et al. (2003) mapped the HIF3A gene to chromosome 19q13.2 by genomic sequence analysis.