BioProject

The response of cells to hypoxia is characterised by co-ordinated regulation of many genes. More...

The response of cells to hypoxia is characterised by co-ordinated regulation of many genes. Studies of the regulation of the expression of many of these genes by oxygen has implicated a role for the heterodimeric transcription factor hypoxia inducible factor (HIF). The mechanism of oxygen sensing which controls this heterodimeric factor is via oxygen dependent prolyl and asparaginyl hydroxylation by specific 2-oxoglutarate dependent dioxygenases (PHD1, PHD2, PHD3 and FIH-1). Whilst HIF appears to have a major role in hypoxic regulation of gene expression, it is unclear to what extent other transcriptional mechanisms are also involved in the response to hypoxia. The extent to which 2-oxoglutarate dependent dioxygenases are responsible for the oxygen sensing mechanism in HIF-independent hypoxic gene regulation is also unclear. Both the prolyl and asparaginyl hydroxylases can be inhibited by dimethyloxalylglycine (DMOG). Such inhibition can produce activation of the HIF system with enhanced transcription of target genes and might have a role in the therapy of ischaemic disease. We have examined the extent to which the HIF system contributes to the regulation of gene expression by hypoxia, to what extent 2-oxoglutarate dependent dioxygenase inhibitor can mimic the hypoxic response and the nature of the global transcriptional response to hypoxia. We have utilised microarray assays of mRNA abundance to examine the gene expression changes in response to hypoxia and to DMOG. We demonstrate a large number of hypoxically regulated genes, both known and novel, and find a surprisingly high level of mimicry of the hypoxic response by use of the 2-oxoglutarate dependent dioxygenase inhibitor, dimethyloxalylglycine. We have also used microarray analysis of cells treated with small interfering RNA (siRNA) targeting HIF-1alpha and HIF-2alpha to demonstrate the differing contributions of each transcription factor to the transcriptional response to hypoxia. Candidate transcripts were confirmed using an independent microarray platform and real-time PCR. The results emphasise the critical role of the HIF system in the hypoxic response, whilst indicating the dominance of HIF-1alpha and defining genes that only respond to HIF-2alpha. Keywords: Hypoxia response, gene knockdown, chemical treatment Overall design: MCF7 breast cancer cell lines were grown under conditions of either normoxia (21% oxygen) or hypoxia (1% oxygen) for 16 hours in an Invivo2 Hypoxia Workstation (Ruskin Technologies, UK). All culture media comprised DMEM, 2mM L-Glutamine and 10% Fetal Bovine Serum. Total RNA was extracted from each sample using the Absolutely RNA RT-PCR Miniprep kit (Stratagene). In total, 7 different types of sample were analysed by microarray technology. These were: ‘normoxia’ - cells grown in normoxic (21% oxygen) conditions; ‘hypoxia’ - cells grown in hypoxic conditions (1% oxygen) for 16 hours; ‘DMOG’ - cells grown in normoxic conditions and exposed to the 2-oxoglutarate dependent dioxygenase inhibitor dimethyloxalylglycine, DMOG (2 mM) for 16 hours; ‘OF’ - cells grown in hypoxic conditions as above and exposed to oligofectamine transfection reagent (Invitrogen) alone; ‘HIF1’ - cells grown in hypoxic conditions as above and exposed to HIF-1alpha siRNA; ‘HIF2’ - cells grown in the same hypoxic conditions with HIF-2alpha siRNA and ‘HIF12’ - cells grown in the same hypoxic conditions with both HIF-1alpha and HIF-2alpha siRNA. Three independent samples were analysed for each experimental condition. All of the analyses for the hypoxia and DMOG samples were compared to the normoxia samples whilst the siRNA transfected samples were compared against the OF samples. The normoxia, hypoxia and DMOG samples (replicates 1-3) were arrayed to HG-U133A Genechips and sample types OF, HIF1, HIF2 and HIF12 (replicates 1-3) were arrayed to HG-U133 plus 2 Genechips (Affymetrix). The normoxia, hypoxia and DMOG samples (replicates 2-3) along with the samples OF, HIF1, HIF2 and HIF12 (replicates 4-6) were arrayed to the ‘whole genome’ Sentrix Human-6 Expression BeadChips (Illumina). All labelling, hybridisation and scanning steps were performed according to the manufacturers’ instructions. Less...