Evaluating the role of NRF-1 in 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 analyzed 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 up-regulation 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.