Abstract 989: Transcriptional Analysis of Cardiac Hypoxia Inducible Factor-1
The response to hypoxia in tissues is regulated by the heterodimeric transcription factor hypoxia inducible factor-1 (HIF-1). It coordinates many downstream processes including angiogenesis and glycolytic metabolism. Using a tet-regulated system we can express a mutated form of HIF-1α in cardiac myocytes of transgenic mice that is not degraded in the presence of oxygen. We have used long-oligo microarrays to identify genes that are regulated by transgene expression. We have identified 126 transcripts more than 3 fold upregulated and 36 transcripts down regulated, 24 hours after induction of the transgene. We found that the regulation of 65 percent of a 20 gene sample of these transcripts is confirmed by real-time PCR. We then applied WordSpy and WEEDER software to analyze the promoter regions of these co-regulated genes and identified a motif that is markedly over-represented in a region within 300 base pairs of known HRE consensus sequences, TGYGTCCA. This sequence is listed in the cis-regulatory element database, with an unknown transcription factor, but has been linked to oligonucleotide modulation of protein kinase C. In addition to computational approaches to analyze these HIF-regulated genes, we are applying a chromatin immunoprecipitation-paired-end ditag assay (developed at the Genome Institute of Singapore), in which only 18 base pair ends of immunoprecipitated fragments of the mouse genome are concatamerized together, effectively and rapidly identifying genes to which HIF-1 is bound. The regulated genes identified by these two approaches provide insight into the mechanisms by which HIF-1 modifies cellular and organismal response to hypoxia. In addition to potentially identifying a new oxygen-responsive regulatory sequence, this data has direct pertinence to the effects of hypoxia in the heart and speaks to the utility of hypoxia-stabilized HIF-1α for gene therapy of ischemia.