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Effect of hypoxia on gene transcription

Research description

HIF-1 (hypoxia-inducible factor -1) is a transcription factor specifically activated by hypoxic conditions. It is composed of two subunits belonging to the bHLH-PAS family, HIF-1a and ARNT. Only HIF-1a is regulated by hypoxia. HIF-1 upregulates the expression of genes such as erythropoietin, VEGF, glycolytic enzymes, tyrosine hydroxylase, heme oxygenase, all involved in the adaptation of cells and tissues to the hypoxic conditions.

In our Laboratory, several aspects of hypoxia- and HIF-1-mediated regulation of gene transcription are investigated.

  • The activation of HIF-1a by hypoxia involves several steps including stabilization against proteasome-mediated degradation or redox regulation. We are investigating several kinase pathways involved in HIF-1a phosphorylation, such as ERK1/2, casein kinase 2 and glycogen synthase kinase 3.
  • Other proteins are probably involved in the regulation of HIF-1 activity. A new project is being launched aimed to identify new proteins interacting with HIF-1a or HIF-2a. These proteins will be identified by a two-hybrid screen as well as by 2-D gel electrophoresis after co-immunoprecipitation.
  • HIF-1a transcription is enhanced by hypoxia in certain cell types such as HepG2. We have dissected HIF1A promoter and identified a positive hypoxia-responsive cis element in this promoter. The transcription factor which recognizes this DNA sequence is under investigation as well as the pathway leading to its activation during hypoxia.
  • Using differential display RT-PCR, several messengers responsive to hypoxia have been identified. Others are currently being sequenced and their promoter are being cloned.
  • A model for the bHLH domain of HIF-1 has been generated through molecular modeling. This 3D structure has been docked onto the HIF-1 DNA consensus sequence generating a model for the protein/DNA complex.  The amino acids involved in direct contact with DNA have been identified both through computational modeling and site-directed mutagenesis.

Techniques

Reporter gene assay (luciferase, ß-galactosidase, EGFP), gel shift assay (EMSA), northern blot, reverse northern blot, real time RT-PCR, differential display RT-PCR, southern blot, western blot, co-immunoprecipitation, kinase assay, confocal microscopy.