Nair Bindu, Wong-Riley Margaret T T
From the Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin 53226.
From the Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
J Biol Chem. 2016 Oct 21;291(43):22583-22593. doi: 10.1074/jbc.M116.742304. Epub 2016 Sep 13.
Brain-derived neurotrophic factor (BDNF) is an active neurotrophin abundantly expressed throughout the nervous system. It plays an important role in synaptic transmission, plasticity, neuronal proliferation, differentiation, survival, and death. The Bdnf gene in rodents has eight non-coding exons and only a single coding exon (IX). Despite its recognized regulation by neuronal activity, relatively little is known about its transcriptional regulation, and even less about the transcription factor candidates that may play such a role. The goal of the present study was to probe for such a candidate that may regulate exon IX in the rat Bdnf gene. Our in silico analysis revealed tandem binding sites for nuclear respiratory factor 2 (NRF-2) on the promoter of exon IX. NRF-2 is of special significance because it co-regulates the expressions of mediators of energy metabolism (cytochrome c oxidase) and mediators of neuronal activity (glutamatergic receptors). To test our hypothesis that NRF-2 also regulates the Bdnf gene, we performed electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), promoter cloning, and site-directed mutagenesis, real-time quantitative PCR (RT-qPCR), and Western blotting analysis. Results indicate that NRF-2 functionally regulates exon IX of the rat Bdnf gene. The binding sites of NRF-2 are conserved between rats and mice. Overexpressing NRF-2 up-regulated the expression of Bdnf exon IX, whereas knocking down NRF-2 down-regulated such expression. These findings are consistent with our hypothesis that NRF-2, in addition to regulating the coupling between neuronal activity and energy metabolism, also regulates the expression of BDNF, which is intimately associated with energy-demanding neuronal activity.
脑源性神经营养因子（BDNF）是一种在整个神经系统中大量表达的活性神经营养因子。它在突触传递、可塑性、神经元增殖、分化、存活和死亡中发挥重要作用。啮齿动物的Bdnf基因有八个非编码外显子，只有一个编码外显子（IX）。尽管其受神经元活动调控已得到公认，但对其转录调控了解相对较少，对于可能发挥这种作用的转录因子候选物了解更少。本研究的目的是探寻可能调控大鼠Bdnf基因外显子IX的候选物。我们的计算机分析揭示了外显子IX启动子上核呼吸因子2（NRF-2）的串联结合位点。NRF-2具有特殊意义，因为它共同调节能量代谢介质（细胞色素c氧化酶）和神经元活动介质（谷氨酸能受体）的表达。为了验证我们的假设，即NRF-2也调控Bdnf基因，我们进行了电泳迁移率变动分析（EMSA）、染色质免疫沉淀（ChIP）、启动子克隆、定点诱变、实时定量PCR（RT-qPCR）和蛋白质免疫印迹分析。结果表明，NRF-2在功能上调控大鼠Bdnf基因的外显子IX。NRF-2的结合位点在大鼠和小鼠之间是保守的。过表达NRF-2上调了Bdnf外显子IX的表达，而敲低NRF-2则下调了这种表达。这些发现与我们的假设一致，即NRF-2除了调节神经元活动与能量代谢之间的耦合外，还调节与能量需求性神经元活动密切相关的BDNF的表达。
