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- Title
Transcriptional Analysis of Cardiac HIF-1.
- Authors
Takeshita, Shelby; Walton, Chad B.; Shohet, Ralph V.
- Abstract
Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that regulates gene response during hypoxia, including regulating angiogenesis, the formation of new blood vessels, and glycolytic metabolism. This heterodimeric protein is composed of HIF-1α and HIF-1α subunits and consists of a basic helix-loop-helix-PAS domain. HIF-1α is expressed constitutively, whereas HIF-1α is regulated by oxygen concentration and inducible by hypoxia. HIF- 1α possesses an oxygen-dependent degradation domain, which causes it to be targeted for rapid proteosomal degradation during normoxia; hypoxia slows the destruction of HIF-1α. Since HIF-1 plays an important role in angiogenesis, it is a subject of interest in the heart where hypoxia occurs due to coronary artery disease, the leading cause of morbidity and mortality on the Western world. This research project involved the over expression of an oxygenstable form of HIF-1α in the murine heart, to identify the genes regulated by HIF-1. In doing so we are able to determine how HIF-1 directly regulates angiogenesis and glucose metabolism. The study hypothesizes that HIF-1 promotes the expression of genes to stimulate angiogenesis and shifts the citric acid cycle from aerobic to anaerobic glycolysis. To identify the genes, mouse models, chromatin immunoprecipitation (ChIP) assays and other validation techniques, such as electrophoresis mobility shift assay (EMSA) gels and computational analyses were used. Bi-transgenic mice were used as animal models, with a cardiac specific promoter and a mutated form of human HIF-1α, under Tet-off control. The Tet-off system controls gene expression with the use of Doxycycline as an inhibitor to promoter complex binding, preventing the transcription of the transgene. A ChIP assay was used to identify DNA regions that are bound to transcription factors, thereby determining which genes are directly regulated. Since HIF-1α is being over expressed, the DNA sequence identified is composed of the promoter regions regulated by HIF-1. One gene of particular interest that was identified via ChIP analysis was pkc binding protein 1 isoform 2, which may play a role in the regulation of HIF-1. This gene has not been fully characterized, but has been implicated in HIF regulation previously. Genes will be further verified by using additional EMSA and computational analyses. Identifying the genes that HIF-1 directly binds to will give insight to the role that HIF-1 plays in ischemia and coronary artery disease. This will provide important information to assist in the development of HIF-1 gene therapy.
- Subjects
TRANSCRIPTION factors
- Publication
Hawaii Medical Journal, 2008, Vol 67, Issue 6, p160
- ISSN
0017-8594
- Publication type
Abstract