Secció de Bioquímica i Biologia Molecular, Departament de Bioquímica i Fisiologia, Facultat de Farmàcia i Ciències de l'Alimentació, Universitat de Barcelona, Barcelona, Spain.
Servei de Bioquímica Clínica, Hospital Sant Joan de Déu, Barcelona, Spain.
J Mol Endocrinol. 2018 Apr;60(3):247-259. doi: 10.1530/JME-17-0142. Epub 2018 Feb 8.
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 , the gene that codes for Gdh, we isolated and characterised the 5' flanking region of from gilthead sea bream (). In addition, tissue distribution, the effect of starvation as well as short- and long-term refeeding on Gdh mRNA levels in the liver of were also addressed. 5'-Deletion analysis of 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 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 , 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 promoter, while refeeding resulted in a slow but gradual restoration of both Gdh and Usf2 mRNA abundance. Herein, we demonstrate that Usf2 transactivates by binding to an E-box located in the proximal region of promoter. In addition, our findings provide evidence for a new regulatory mechanism involving Usf2 as a key factor in the nutritional regulation of transcription in the fish liver.
谷氨酸脱氢酶 (Gdh) 通过催化 l-谷氨酸可逆氧化脱氨生成 α-酮戊二酸，以 NAD 或 NADP 为辅酶，在氨解毒中发挥核心作用。为了深入了解编码 Gdh 的基因的转录调控，我们从金头鲷（）中分离和鉴定了 5'侧翼区。此外，还研究了 组织分布、饥饿以及短期和长期再喂养对 肝脏中 Gdh mRNA 水平的影响。瞬时转染 HepG2 细胞的 启动子 5'缺失分析、电泳迁移率变动分析、染色质免疫沉淀（ChIP）和定点突变分析使我们能够鉴定上游刺激因子 2（Usf2）作为参与 转录调控的新因子。通过逆转录酶耦合定量实时 PCR（RT-qPCR）分析 Gdh 和 Usf2 mRNA 水平的组织分布表明，Gdh 主要在 的肝脏中表达，而 Usf2 则具有广泛的分布。RT-qPCR 和 ChIP 分析表明，长期饥饿将 肝脏中 Gdh 和 Usf2 的表达下调至相似水平，并减少了 Usf2 与 启动子的结合，而再喂养则导致 Gdh 和 Usf2 mRNA 丰度缓慢但逐渐恢复。在此，我们证明 Usf2 通过结合位于 启动子近端区域的 E 盒来反式激活 。此外，我们的研究结果为涉及 Usf2 的新调控机制提供了证据，Usf2 是鱼类肝脏中 转录营养调控的关键因素。
