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.
- Received March 31, 2016.
- Accepted July 18, 2016.
- © 2016. Published by The Company of Biologists Ltd