Transcription Factors

Author: Gianpiero Pescarmona
Date: 09/06/2008



Hypoxia inducible factors are transcription factors, that respond to changes in available oxygen in the cellular environment, increasing their activity in hypoxia.


The highly-conserved transcriptional complex HIF-1, is a heterodimer composed of an alpha and a beta subunit. Both subunits belongs to the basic helix–loop–helix (bHLH) proteins of the PAS FAMILY.

The HIFα/β dimer binds to a core DNA motif (G/ACGTG) in hypoxia-response elements ( HREs ).


HIFα exists in 3 isoforms:

  • HIF1α (5 isoforms reported)
  • HIF2α
  • HIF3α

HIFβ: a constitutively-expressed aryl hydrocarbon receptor nuclear translocator (ARNT) that has been previously identified as a cytosolic dioxin receptor. It forms heterodimers also with AHR (OMIM 600253)and MOP2 (OMIM 603349)


When relevant for the function

  • Primary structure
  • Secondary structure
  • Tertiary structure
  • Quaternary structure

HIF (hypoxia) and VHL (normoxia) play antagonistic roles


mRNA synthesis

protein synthesis

post-translational modifications and degradation

Under normoxic conditions HIF-1 alfa subunit has an exceptionally short half-life, it is subject to rapid turnover and degradation by the ubiquitin proteasome pathway.

Proteolytic regulation of HIF-1α is critically dependent on the von Hippel-Lindau tumour suppressor protein ( VHL). The ODD domain of HIFα subunits could physically interact with VHL and the VHL complex functioned as an ubiquitin ligase capable of ubiquitylating the HIFα subunits at normoxia and targeting them for destruction by the proteasome.
The ODD domain (oxygen-dependent degradation domain) contains N- and C- terminal portions ( N-ODD and C-ODD) that can operate independently.

The enzymatic reactions carried out by the PHD/HPHs and FIH-1 revealed that the hydroxylation reaction requires

  • O2. The hydroxylation of proline and asparagine requires molecular oxygen
  • Iron (Fe2+). PHD/HPHs and FIH-1 are Fe-dependent enzyme. Along with hypoxia, iron chelating agents such as desferoxamine were also able to block the enzymatic reactions and VHL interaction leading to stabilization of HIF-1alpha.
  • 2-oxoglutarate (a citric-acid-cycle intermediate) is required because it undergoes a decarboxylation reaction, consuming the remaining oxygen atom to form succinate and CO2.

ROS (HCV, HBV, drugs )
Local Iron Deficiency

  • Global Iron Deficiency
  • Lactoferrin, ferritin, Asbestos etc
  • Herpesvirus infection (Ribonucleotide Reductase)

Molecular Mechanism of hypoxia and/or iron deficiency

Vitamin C and prolyl hydroxylase

ODD = oxygen - dependent degradation
VHL = von HIPPEL - LINDAU protein, E3 ligase
TAD = trans activation domain
CBP = coactivator binding protein

K = lys
P = pro
N = asn

Hypoxia blocked degradation leading to the accumulation of the HIF-1α protein in the citoplasm and its migration to the nucleus where binds HIFβ and form the HIFα/β dimer.
Oxygen level regulates the degradation via hydroxylation or acetylation-mediated VHL binding and also transcriptional activity of HIF-1:

  1. pVHL interaction is regulated by enzymatic hydroxylation at specific prolyl residues (for human HIF-1 , at Pro 402 in the NODD and at Pro 564 in the CODD). Therefore two proline hydroxylation sites had shown to promote VHL binding in a hydroxylation dependent manner.
  1. FIH (factor inhibiting HIF) hydroxylates N803 under normoxia and inhibits the association of p300/CBP to HIF-1 leading to the downregulation of transcriptional activity of HIF-1.

HIF oxygen-dependent hydroxylation

MDR:Multidrug resistance is the ability of pathologic cells to withstand chemicals that are designed to aid in the eradication of such cells. These pathologic cells include bacterial and neoplastic (tumor) cells

Hypoxia increases cell proliferation rate through reduction of glucose oxidative breakdown and induction of many enzymes (Glutamine synthetase, Glycolytic enzymes, G6PD etc). The high fetal growth rate is at least partially dependent on low fetal pO 2 .

Figure 1 Abreviations. VHL denotes von Hippel-Lindau protein, HIF hypoxia-inducible factor, TGF-{alpha} transforming growth factor {alpha}, VEGF vascular endothelial growth factor A, PDGFbeta platelet-derived growth factor beta, EGFR epidermal growth factor receptor, VEGFR2 VEGF receptor 2, PDGFRbeta PDGF receptor beta, PTEN phosphatase and tensin homologue, TSC1 and TSC2 tuberous sclerosis complex 1 and 2, FKBP12 FK506-binding protein 12 kD, mTOR mammalian target of rapamycin complex 1 kinase, eIF4E eukaryotic translation initiation factor 4E, and S6K S6 kinase.

Used with permission from Brugarolas J. N Engl J Med 2007;356:185-187

HIF-1 alpha and ribosomal entry IRES

HIF Phosphorylation


Generating specificity and diversity in the transcriptional response to hypoxia 2009

Oxygen homeostasis G. Semenza 2009

Genes induced by HIF

that are associated with a broad range of transcriptional targets. These target genes are centrally involved in:

  • systemic responses to hypoxia ( angiogenesis, erythropoiesis)
  • cellular responses (Cell proliferation, alterations in glucose and iron metabolism)

additional genes......

HIF-1 acts as a master regulator of oxygen-regulated gene expression. More than 60 putative HIF-1 target genes have been identified

HIF downstream effects

a deeper insight

cellular localization,
biological function

  • Enzymes
  • Cell signaling and Ligand transport
  • Structural proteins


Cyclic AMP represses the hypoxic induction of hypoxia-inducible factors in PC12 cells.
Torii S, Okamura N, Suzuki Y, Ishizawa T, Yasumoto K, Sogawa K.
J Biochem. 2009 Dec;146(6):839-44. Epub 2009 Aug 11.


HIF and symptoms

Symptoms dependent on VEGF (hyperexpression)

Increased vascular permeability:

  • Edema
  • Proteins efflux from vessels (urine, tissues)
  • Effusions (pleural, pericardial, peritoneal)


2009-05-25T15:59:40 - Gianpiero Pescarmona

HIF and VHL play antagonistic roles

Papers HIF and methylation

Papers VHL and methylation

In regards to HIF it is known that it has a role in epigentic control having some histone demethylases among his targets (JMJD1A human and JMJD1A rat and JMJD family, JMJD28).
The histone demethylases JMJD1A and JMJD2B are transcriptional targets of hypoxia-inducible factor HIF. 2008

Acute inactivation of the VHL gene contributes to protective effects of ischemic preconditioning in the mouse kidney. 2008

A HIF-1alpha-related gene involved in cell protection from hypoxia by suppression of mitochondrial function. 2008

Recently, we reported that acetylcholine-induced hypoxia-inducible factor-1alpha protects cardiomyocytes from hypoxia; however, the downstream factors reducing hypoxic stress are unknown. We identified apoptosis inhibitor (AI) gene as being differentially expressed between von Hippel Lindau (VHL) protein-positive cells with high levels of GRP78 expression and VHL-negative cells with lower GRP levels, using cDNA subtraction. AI decreased GRP78 level, suppressed mitochondrial function, reduced oxygen consumption and, ultimately, suppressed hypoxia-induced apoptosis. By contrast, knockdown of the AI gene increased mitochondrial function. Hypoxic cardiomyocytes and ischemic myocardium showed increased AI mRNA expression. These findings suggest that AI is involved in suppressing mitochondrial function, thereby leading to cellular stress eradication and consequently to protection during hypoxia.

Chronic intermittent fasting improves the survival following large myocardial ischemia by activation of BDNF/VEGF/PI3K signaling pathway.Katare RG et al. J Mol Cell Cardiol. 2009

Relative HIF-1 and HIF-2 contributions to the hypoxic response over time at 1% (A) and 5% (B) oxygen as demonstrated in human neuroblastoma cells. Model based on a summary of data obtained from protein level variations (Western blot), chromatin immunoprocipitations (ChIP) and activation of target genes ± siRNA treatment. Reproduced with permission from Lofstedt et al., Cell Cycle, 2007 Fulltext

  • Poorly oxygenated (hypoxic) tumors are frequently more aggressive compared to corresponding tumors that are better oxygenated. Adaptation to hypoxia is primarily mediated by two closely related hypoxia inducible transcription factor complexes, HIF-1 and HIF-2, which become stabilized and activated at low oxygen levels. Whether HIF-1 and HIF-2 have different roles in tumorigenesis is an open question and an issue we discuss. With focus on HIF-2, we summarize reported phenotypical changes of HIF genetic models and HIF expression patterns during normal development, in adult non-malignant tissues and in tumors. We further address the much-discussed subject of target gene preferences between HIF-1 and HIF-2, given that both transcription factors bind to the same DNA motif. Finally, we also discuss the observations that the oxygen-sensitive HIF-2alpha subunit is accumulated and active under non-hypoxic conditions as exemplified by HIF-2alpha expressing tumor macrophages and neuroblastoma cells located in seemingly well-vascularized tumor regions and how this phenomenon is related to tumor aggressiveness.
2008-11-28T14:35:32 - Gianpiero Pescarmona

Basso peso neonatale e rischio emangioma
L'incidenza degli emangiomi neonatali è aumentata negli ultimi anni, ed il basso peso neonatale è il principale fattore di rischio alla base di questo fenomeno. Ovviamente, la comprensione dei fattori di rischio degli emangiomi neonatali aiuterebbe a comprendere come prevenirli o trattarli in modo più appropriato. Benchè nella maggior parte dei casi si tratti di patologie benigne che tendono a risolversi da sole, una significativa percentuale di essi causa morbidità o cicatrici permanenti. Il ruolo del basso peso neonatale potrebbe riflettere un problema ipossico di base: a questo proposito, si potrebbero ricercare i geni implicati nell'ipossia, oppure metodi per prevenirla nel periodo neonatale. (J Pediatr 2008; 153: 712-5)

2008-10-20T21:07:01 - Gianpiero Pescarmona

Erythropoietin during hypoglycaemia in type 1 diabetes: Relation to basal renin-angiotensin system activity and cognitive function 2009


Both HIF-1beta and presenilin-1 expression were also induced by low glucose. Overexpression of presenilin-1 increased HIF-1beta, suggesting that HIF is downstream of presenilin. Our results provide the first evidence of a presenilin-HIF signaling network in beta-cells. We demonstrate that this pathway is controlled by Ca(2+) flux through intracellular channels, likely via changes in mitochondrial metabolism and ATP.
Glucose and endoplasmic reticulum calcium channels regulate HIF-1beta via presenilin in pancreatic beta-cells. 2008

Loss of ARNT/HIF1beta mediates altered gene expression and pancreatic-islet dysfunction in human type 2 diabetes. 2005

Involvement of hypoxia-inducible factor 1 in human cancer. 2002

Abnormal angiogenesis and responses to glucose and oxygen deprivation in mice lacking the protein ARNT. 1997

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