Differentiating the functional role of hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha (EPAS-1) by the use of RNA interference: erythropoietin is a HIF-2alpha target gene in Hep3B and Kelly cells

FASEB J. 2004 Sep;18(12):1462-4. doi: 10.1096/fj.04-1640fje. Epub 2004 Jul 1.


Activation of the hypoxia-inducible factor alpha-subunits, HIF-1alpha and HIF-2alpha, seems to be subject to similar regulatory mechanisms, and transgene approaches suggested partial functional redundancy. Here, we used RNA interference to determine the contribution of HIF-1alpha vs. HIF-2alpha to the hypoxic gene induction. Surprisingly, most genes tested were responsive only to the HIF-1alpha siRNA, showing no effect by HIF-2alpha knock-down. The same was found for the activation of reporter genes driven by hypoxia-responsive elements (HREs) from the erythropoietin (EPO), vascular endothelial growth factor, or phosphoglycerate kinase gene. Interestingly, EPO was the only gene investigated that showed responsiveness only to HIF-2alpha knock-down, as observed in Hep3B and Kelly cells. In contrast to the EPO-HRE reporter, the complete EPO enhancer displayed dependency on HIF-2alpha regulation, indicating that additional cis-acting elements confer HIF-2alpha specificity within this region. In 786-0 cells lacking HIF-1alpha protein, the identified HIF-1alpha target genes were regulated by HIF-2alpha. Overexpression of the HIFalpha subunits in different cell lines also led to a loss of target gene specificity. In conclusion, we found a remarkably restricted target gene specificity of the HIFalpha subunits, which can be overcome in cells with perturbations in the pVHL/HIF system and under forced expression.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Basic Helix-Loop-Helix Transcription Factors
  • Cell Line
  • Cell Line, Tumor
  • Enhancer Elements, Genetic / genetics
  • Erythropoietin / genetics*
  • Genes, Reporter / genetics
  • Humans
  • Hypoxia
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • Protein Subunits / genetics
  • Protein Subunits / metabolism
  • RNA Interference*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism
  • Response Elements / genetics
  • Sensitivity and Specificity
  • Trans-Activators / genetics
  • Trans-Activators / metabolism*
  • Transcription Factors / chemistry
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*
  • Transcriptional Activation
  • Transfection


  • Basic Helix-Loop-Helix Transcription Factors
  • HIF1A protein, human
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • Protein Subunits
  • RNA, Messenger
  • RNA, Small Interfering
  • Trans-Activators
  • Transcription Factors
  • Erythropoietin
  • endothelial PAS domain-containing protein 1