Molecular mechanisms of transcription activation by HLF and HIF1alpha in response to hypoxia: their stabilization and redox signal-induced interaction with CBP/p300

Abstract

Hypoxia-inducible factor 1 α (HIF1α) and its related factor, HLF, activate expression of a group of genes such as erythropoietin in response to low oxygen. Transfection analysis using fusion genes of GAL4DBD with various fragments of the two factors delineated two transcription activation domains which are inducible in response to hypoxia and are localized in the C-terminal half. Their sequences are conserved between HLF and HIF1α. One is designated NAD (N-terminal activation domain), while the other is CAD (C-terminal activation domain). Immunoblot analysis revealed that NADs, which were rarely detectable at normoxia, became stabilized and accumulated at hypoxia, whereas CADs were constitutively expressed. In the mammalian two-hybrid system, CAD and NAD baits enhanced the luciferase expression from a reporter gene by co-transfection with CREB-binding protein (CBP) prey, whereas CAD, but not NAD, enhanced β-galactosidase expression in yeast by CBP co-expression, suggesting that NAD and CAD interact with CBP/p300 by a different mechanism. Co-transfection experiments revealed that expression of Ref-1 and thioredoxin further enhanced the luciferase activity expressed by CAD, but not by NAD. Amino acid replacement in the sequences of CADs revealed a specific cysteine to be essential for their hypoxiainducible interaction with CBP. Nuclear translocation of thioredoxin from cytoplasm was observed upon reducing O 2 concentrations.

The role of oxygen availability in embryonic development and stem cell function

“…HIF-α subunits therefore accumulate in the cytoplasm of O 2 -starved cells, and translocate to the nucleus, where they dimerize with HIF-1β/aryl hydrocarbon receptor nuclear translocator (ARNT) through their PER-ARNT-SIM (PAS) domains (see Box 1), and bind to HIF-response elements (which contain the core recognition sequence 5'-RCGTG-3') located within the promoters, introns, and 3' enhancers of a large number of O 2 -regulated target genes. During this process the C-terminal transcriptional activation domain (TAD) also interacts with coactivators such as p300/CBP (CREB-binding protein; CREB is CRE-response element binding protein) 131 (see figure). This interaction is thought to be required for full HIF activity, but is also regulated by normoxic hydroxylation reactions; in this case at asparagine 803 (see Box 1) 132 .…”

Regulation of HIF-1α Activity by Overexpression of Thioredoxin is Independent of Thioredoxin Reductase Status

“…Initially, the effects of Txn1 overexpression on HIF-1α were reported to be exclusively on its transcriptional activity (Carrero et al, 2000;Ema et al, 1999;Huang et al, 1996), but, more recently, Txn1 overexpres- sion was reported to induce HIF-1α stabilization (Welsh et al, 2002;Zhou et al, 2007). In these studies, no changes in the mRNA levels of HIF-1α were reported.…”

“…These results provide evidence that the effect of Txn1 on HIF-1α is limited to its transcriptional activity. Previously, the mutation of a cysteine in C-TAD of HIF-1α abolished its interaction with the transcriptional co-activator p300, and thus, inhibited HIF-1α transcriptional activity (Ema et al, 1999). A mechanism was proposed, wherein Txn1, through Ref-1, would reduce a cysteine in the transactivation domain of HIF-1α and allow its interaction with p300.…”

“…In addition to the hydroxylation reactions on HIF-1α, other regulatory pathways have been described to be important for the control of HIF-1α levels or activity, such as reactive oxygen species (ROS) (Chandel et al, 1998), diacylglycerol kinase (Aragones et al, 2001), MAP kinase cascades (Conrad et al, 1999a;1999b;Richard et al, 1999), Rac1 (Hirota et al, 2001), the PI3K/Akt pathway (Mazure et al, 1997;Zhong et al, 2000;Zundel et al, 2000) and thioredoxin (Trx) redox activity (Carrero et al, 2000;Ema et al, 1999;Huang et al, 1996;Welsh et al, 2002;Zhou et al, 2007). However, the mechanisms by which these pathways affect HIF-1α stability or function have not been clarified and their implication for HIF-1α signaling remains controversial (Alvarez-Tejado et al, 2002;Chua et al, 2010;Hoffman et al, 2007;Naranjo-Suarez et al, 2012;Srinivas et al, 2001;Vaux et al, 2001).…”

“…Txns have been implicated in many cellular functions, such as cell growth, chemotaxis, antioxidant defense, redox state of cysteine residues in proteins, and regulation of transcription factors. Expression of Txn1 has been described to increase HIF-1α DNA binding and its transcriptional activity (Carrero et al, 2000;Ema et al, 1999;Huang et al, 1996;Welsh et al, 2002;Zhou et al, 2007), and a possible mechanism in which Txn reduces HIF-1α through the nuclear redox regulator, redox factor-1 (Ref-1) has been suggested (Ema et al, 1999). More recent studies have proposed that Txn1 overexpression increases not only HIF-1α activity, but also HIF-1α stability, in various mammalian cell lines (Welsh et al, 2002;Zhou et al, 2007).…”