Abstract
To identify new dimerization partners for the aromatic hydrocarbon receptor nuclear translocator (Arnt), we used its N-terminal region (amino acids 1-470) as a target in a two-hybrid screening procedure, and we cloned the murine form of hypoxia-inducible factor 1alpha (HIF1alpha). Sequence comparisons reveal substantial identity between mouse and human HIF1alpha. Hypoxia induces a 10-fold accumulation of phosphoglycerate kinase 1 mRNA in wild type mouse hepatoma (Hepa 1c1c7) cells; the induction mechanism is Arnt dependent because induction does not occur in Arnt-defective cells. Furthermore, induction of phosphoglycerate kinase 1 mRNA requires Arnt's N-terminal region, which mediates DNA binding and heterodimerization; in contrast, induction does not require Arnt's C-terminal region, which mediates transactivation. We also show that a GAL4-HIF1alpha fusion protein transactivates a GAL4-dependent gene in the absence of Arnt, that HIF1alpha's transactivation capability is inducible by hypoxia, and that both hypoxia responsiveness and transactivation capability reside within the C-terminal 83 amino acids of HIF1alpha. Our findings generate new insights into the mechanism by which Arnt and HIF1alpha induce transcription in response to hypoxia.
Highlights
To identify new dimerization partners for the aromatic hydrocarbon receptor nuclear translocator (Arnt), we used its N-terminal region as a target in a two-hybrid screening procedure, and we cloned the murine form of hypoxia-inducible factor 1␣ (HIF1␣)
We demonstrate the functional importance of this observation by showing that Arnt is required for induction of the glycolytic enzyme phosphoglycerate kinase 1 (PGK1) in response to hypoxia
Cloning of a Dimerization Partner for Arnt—Previous observations indicate that Arnt forms heterodimers with aromatic hydrocarbon receptor (AhR) and Sim, both of which contain basic helix-loop-helix (bHLH) and PAS domains [37]
Summary
AhR nuclear translocator; AhR, aromatic hydrocarbon receptor; HIF1␣, hypoxia-inducible factor 1␣; CAT, chloramphenicol acetyltransferase; PGK1, phosphoglycerate kinase 1; PCR, polymerase chain reaction; kb, kilobase; bHLH, basic helix-loop-helix. As is typical of many transcription factors, AhR and Arnt have modular organizations Both AhR and Arnt contain basic helix-loop-helix (bHLH) motifs; the HLH domains mediate heterodimerization between AhR and Arnt, while the basic regions are responsible for the binding of the AhR/Arnt heterodimer to DNA (16 –18). Others [1] have suggested that Arnt might play a more general role in mediating cellular responses to environmental stimuli and, that cells might contain additional proteins that serve as heterodimerization partners for Arnt In support of this idea, studies of human hypoxia-inducible factor 1, which regulates cellular responses to low oxygen tension, reveal that it contains two bHLH/PAS proteins, hypoxia-inducible factor 1␣ (HIF1␣) and Arnt [27]. Our studies provide new insights into the mechanism by which cells adapt to low oxygen tension, and they emphasize the importance of Arnt as a regulator of cellular responses to environmental stimuli
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