Transactivation and Inhibitory Domains of Hypoxia-inducible Factor 1α

Abstract

Hypoxia-inducible factor 1 (HIF-1) binds to cis-acting hypoxia-response elements within the erythropoietin, vascular endothelial growth factor, and other genes to activate transcription in hypoxic cells. HIF-1 is a basic helix-loop-helix transcription factor composed of HIF-1␣ and HIF-1␤ subunits. Here, we demonstrate that HIF-1␣ contains two transactivation domains located between amino acids 531 and 826. When expressed as GAL4 fusion proteins, the transcriptional activity of these domains increased in response to hypoxia. Fusion protein levels were unaffected by changes in cellular O 2 tension. Two minimal transactivation domains were localized to amino acid residues 531-575 and 786 -826. The transcriptional activation domains were separated by amino acid sequences that inhibited transactivation. Deletion analysis demonstrated that the gradual removal of inhibitory domain sequences (amino acids 576 -785) was associated with progressively increased transcriptional activity of the fusion proteins, especially in cells cultured at 20% O 2 . Transcriptional activity of GAL4/HIF-1␣ fusion proteins was increased in cells exposed to 1% O 2 , cobalt chloride, or desferrioxamine, each of which also increased levels of endogenous HIF-1␣ protein but did not affect fusion protein levels. These results indicate that increased transcriptional activity mediated by HIF-1 in hypoxic cells results from both increased HIF-1␣ protein levels and increased activity of HIF-1␣ transactivation domains.Human cells require O 2 for essential metabolic processes, most notably oxidative phosphorylation. Hypoxia is a significant pathophysiologic component of many cardiovascular, hematologic, and pulmonary disorders (reviewed in Ref.

PI4KIIα is a novel regulator of tumor growth by its action on angiogenesis and HIF-1α regulation

“…The levels of HIF-1a protein were then detected by western blotting with cells transfected with GFP acting as a negative control, whereas cells treated with 1% O 2 for 24 h acted as positive controls (Jiang et al, 1997). Results showed that overexpression of PI4KIIa, a novel player in tumor growth J Li et al PI4KIIa leads to a large accumulation of HIF-1a, while HIF-1a expression was also significantly induced by hypoxia (1% O 2 treated for 24 h).…”

Non-hypoxic pathway mediates the induction of hypoxia inducible factor 1 alpha (HIF-1 alpha ) in vascular smooth muscle cells

“…C-terminal transactivation domains can be found on both HIF-1α and HIF-1β (6)(7)(8)(9). The mouse HIF-1α gene the ubiquitous inducer of HIF-1α in all cells tested, other stimuli, such as insulin, IGF-1,2 and EGF, have also been shown to increase HIF-1α protein levels in certain cell types (18)(19)(20).…”

Structural basis for recruitment of CBP/p300 by hypoxia-inducible factor-1α

“…Two mechanisms for this additional level of hypoxic regulation have been proposed. First, a region between NTAD and CTAD that inhibits transactivation was recently shown to form part of a binding site for the novel inhibitory factor, FIH-1 (18,21). According to this model, FIH-1 may dissociate from HIF-1␣ during hypoxia and thereby permit transactivation through CTAD.…”

“…1). HIF-1␣ also contains an N-terminal transactivation domain (NTAD) that transactivates less effectively than CTAD alone; however, NTAD and CTAD together function synergistically (13,14,17,18). CH1 and CH3 are homologous Zn 2ϩ -binding domains of CBP͞p300 containing numerous cysteine and histidine residues (Fig.…”