title: Evaluating the role of NRF-1 in the regulation of the goldfish COX4-1 gene in response to temperature.
abstract: Cold acclimation in fish typically increases muscle mitochondrial enzymes. In mammals, stressors that increase mitochondrial content are mediated though transcriptional regulators, including nuclear respiratory factor-1 (NRF-1). Focusing on the goldfish gene for cytochrome c oxidase (COX) subunit 4-1, we analysed the regulatory regions in various contexts to identify a mechanistic link between NRF-1 and cold-induced mitochondrial proliferation. Promoter analysis implicated two putative NRF-1 sites: one in the proximal promoter and a second in exon 1, which encodes the 5' untranslated region (5'-UTR). Transfection into mouse myoblasts showed that deletion of a region that included the proximal NRF-1 site reduced promoter activity by 30%; however, mutagenesis of the specific sequence had no effect. Thermal sensitivity analyses performed in rainbow trout gonadal fibroblasts (RTG-2) showed no effect of temperature (4 vs 19°C) on reporter gene expression. Likewise, reporters injected into muscle of thermally acclimated goldfish (4 vs 26°C) showed no elevation in expression. There was no difference in thermal responses of COX4-1 promoter reporters constructed from homologous regions of eurythermal goldfish and stenothermal zebrafish genes. NRF-1 chromatin immunoprecipitation of thermally acclimated goldfish muscle showed no temperature effect on NRF-1 binding to either the proximal promoter or 5'-UTR. It remains possible that the cold-induced upregulation of COX4-1 expression is a result of NRF-1 binding to distal regulatory regions or through indirect effects on other transcription factors. However, the proximal promoter does not appear to play a role in mediating the thermal response of the COX4-1 gene in fish.
authors.affiliation: Department of Biology, Queen's University, Kingston, Canada K7L 3N6.
authors.forename: Ge
authors.initials: G
authors.lastname: Gao
authors.affiliation: Department of Biology, Queen's University, Kingston, Canada K7L 3N6 chris.moyes@queensu.ca.
authors.forename: Christopher D
authors.identifier: 0000-0003-3506-2837
authors.initials: CD
authors.lastname: Moyes
mesh_terms: D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D003576:Electron Transport Complex IV / Q000235:genetics* / Q000378:metabolism; D006054:Goldfish / Q000235:genetics*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D051266:Nuclear Respiratory Factor 1 / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D013696:Temperature; D014162:Transfection
chemical_list: D007527:Isoenzymes; D051266:Nuclear Respiratory Factor 1; D003576:Electron Transport Complex IV
keywords: Bioenergetics; Goldfish; Mitochondria; Nuclear respiratory factor 1; Oxidative phosphorylation; Thermal acclimation