Activation of Hypoxia-inducible Factor-1; Definition of Regulatory Domains within the α Subunit

Abstract

Hypoxia-inducible factor-1 (HIF-1), a heterodimeric DNA binding complex composed of two basic-helix-loophelix Per-AHR-ARNT-Sim proteins (HIF-1␣ and -1␤), is a key component of a widely operative transcriptional response activated by hypoxia, cobaltous ions, and iron chelation. To identify regions of HIF-1 subunits responsible for oxygen-regulated activity, we constructed chimeric genes in which portions of coding sequence from HIF-1 genes were either linked to a heterologous DNA binding domain or encoded between such a DNA binding domain and a constitutive activation domain. Sequences from HIF-1␣ but not HIF-1␤ conferred oxygenregulated activity. Two minimal domains within HIF-1␣ (amino acids 549 -582 and amino acids 775-826) were defined by deletional analysis, each of which could act independently to convey inducible responses. Both these regions confer transcriptional activation, and in both cases adjacent sequences appeared functionally repressive in transactivation assays. The inducible operation of the first domain, but not the second, involved major changes in the level of the activator fusion protein in transfected cells, inclusion of this sequence being associated with a marked reduction of expressed protein level in normoxic cells, which was relieved by stimulation with hypoxia, cobaltous ions, or iron chelation. These results lead us to propose a dual mechanism of activation in which the operation of an inducible activation domain is amplified by regulation of transcription factor abundance, most likely occurring through changes in protein stability.Hypoxia-inducible factor-1, a DNA binding complex first identified as a factor critical for the inducible activity of the erythropoietin 3Ј enhancer (1), is now recognized to be a key component of a widely operative transcriptional control system responding to physiological levels of cellular hypoxia (2-5). Deletional and mutational analysis of cis-acting sequences has demonstrated functionally critical HIF-1 1 binding sites in many oxygen-regulated promoters and enhancers (6 -12). The importance of HIF-1 in the regulation of such genes has been confirmed by the reduction or abrogation of hypoxia-inducible expression in mutant cells (13,14) that are unable to form a functional HIF complex (15)(16)(17)(18)(19). Together these studies have provided strong evidence for a critical role for HIF-1 in the regulation of genes involved in a variety of important biological processes that include glucose transport and metabolism, vascular growth, vasomotor regulation, erythropoiesis, iron metabolism, and catecholamine synthesis (reviewed in Ref. 5).As is observed for HIF-1-responsive genes (20 -22), the HIF-1 complex is inducible by particular transition elements such as cobaltous ions and by iron chelating agents such as desferrioxamine (DFO) but not by inhibitors of mitochondrial respiration such as cyanide or azide (3,8,23). These distinctive features have led to the proposal of a specific oxygen sensing mechanism underlying these responses, most probably involvin...

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