Does the goldfish COX4-1 gene promoter possess cold-responsive elements?
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Mitochondrial biogenesis permits tissues to increase mitochondrial content in response to environmental and physiological stressors. In many species of fish mitochondrial biogenesis is triggered by cold exposure. The greater capacity to produce ATP may compensate for energy shortages when body temperature decreases. In goldfish, cold acclimation increases COX activity (and content), paralleled by increases in COX4-1 transcript levels. These transcriptional changes are accompanied by increases in nuclear respiratory factor-1 (NRF-1) mRNA and nuclear protein levels. Since NRF-1 regulates mitochondrial biogenesis in mammals, my thesis explored whether the increase in NRF-1 caused the change in COX4-1 mRNA that is associated with mitochondrial biogenesis. I used reporter genes under the control of the goldfish COX4-1 proximal promoter to characterize the COX4-1 gene in an effort to identify cold responsive elements. Preliminary analysis of the promoter sequence suggested that there was a putative NRF-1 site at -444/-414 on the proximal promoter, and a second putative NRF-1 element in the 5’UTR. I created a series of reporters that differed in length and analyzed these promoters in several cell types. In mouse myoblasts (C2C12), a decrement in promoter activity was seen when a critical region from 230 bp to 159 bp was deleted. In C2C12 myotubes, deletion of a region of the promoter from -452 to -312 led to a 30% loss in activity. The 1592bp reporter gene transfected into rainbow trout gonad (RTG-2) cells was unaffected by deletions until shortened to 114 bp, and promoter activity was not affected by temperature. When plasmids were transfected into goldfish white muscle, no increase in luciferase expression was seen in cold-acclimated fish that showed a 6.5-fold increase in COX4-1 mRNA. This goldfish in vivo model also created an opportunity to determine if species that differ in thermal tolerance possessed promoters that differed in thermal sensitivity. COX4-1 promoters from the eurythermal goldfish and the stenothermal zebrafish showed the same magnitude of change in promoter activity. Chromatin immunoprecipitation analysis was conducted on goldfish white muscle to assess if acclimation affected the amount of NRF-1 bound to the COX4-1 promoter. I saw no significant change in binding of NRF-1 to the promoter. While I found no evidence of NRF-1 binding to the proximal promoter of COX4-1, it remains possible that the up-regulation of COX4-1 transcript is a result of NRF-1 binding to an upstream distal promoter region or in an indirect manner through regulating other transcription factors.