Affiliation:
1. 1Secció de Bioquímica i Biologia Molecular, Departament de Bioquímica i Fisiologia, Facultat de Farmàcia i Ciències de l’Alimentació, Universitat de Barcelona, Barcelona, Spain
2. 2Servei de Bioquímica Clínica, Hospital Sant Joan de Déu, Barcelona, Spain
Abstract
Glutamate dehydrogenase (Gdh) plays a central role in ammonia detoxification by catalysing reversible oxidative deamination of l-glutamate into α-ketoglutarate using NAD+ or NADP+ as cofactor. To gain insight into transcriptional regulation of glud, the gene that codes for Gdh, we isolated and characterised the 5′ flanking region of glud from gilthead sea bream (Sparus aurata). In addition, tissue distribution, the effect of starvation as well as short- and long-term refeeding on Gdh mRNA levels in the liver of S. aurata were also addressed. 5′-Deletion analysis of glud promoter in transiently transfected HepG2 cells, electrophoretic mobility shift assays, chromatin immunoprecipitation (ChIP) and site-directed mutagenesis allowed us to identify upstream stimulatory factor 2 (Usf2) as a novel factor involved in the transcriptional regulation of glud. Analysis of tissue distribution of Gdh and Usf2 mRNA levels by reverse transcriptase-coupled quantitative real-time PCR (RT-qPCR) showed that Gdh is mainly expressed in the liver of S. aurata, while Usf2 displayed ubiquitous distribution. RT-qPCR and ChIP assays revealed that long-term starvation down-regulated the hepatic expression of Gdh and Usf2 to similar levels and reduced Usf2 binding to glud promoter, while refeeding resulted in a slow but gradual restoration of both Gdh and Usf2 mRNA abundance. Herein, we demonstrate that Usf2 transactivates S. aurata glud by binding to an E-box located in the proximal region of glud promoter. In addition, our findings provide evidence for a new regulatory mechanism involving Usf2 as a key factor in the nutritional regulation of glud transcription in the fish liver.
Subject
Endocrinology,Molecular Biology
Cited by
6 articles.
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