Nair Bindu, Johar Kaid, Priya Anusha, Wong-Riley Margaret T T
Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
Biochim Biophys Acta. 2016 Jan;1863(1):1-9. doi: 10.1016/j.bbamcr.2015.10.005. Epub 2015 Oct 18.
Previous studies in our laboratory have shown that the neuron-specific specificity protein 4 (Sp4) transcriptionally regulates many excitatory neurotransmitter receptor subunit genes, such as those for GluN1, GluN2A, and GluN2B of N-methyl-d-aspartate (NMDA) receptors and Gria2 of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. It also regulates Atp1a1 and Atp1b1 subunit genes of Na(+)/K(+)-ATPase, a major energy-consuming enzyme, as well as all 13 subunits of cytochrome c oxidase (COX), an important energy-generating enzyme. Thus, there is a tight coupling between energy consumption, energy production, and excitatory neuronal activity at the transcriptional level in neurons. The question is whether inhibitory neurotransmitter receptors are also regulated by Sp4. In the present study, we tested our hypothesis that Sp4 regulates receptor subunit genes of a major inhibitory neurotransmitter, GABA, specifically GABAA receptors. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, real-time quantitative PCR, chromatin immunoprecipitation, promoter mutational analysis, over-expression and shRNA of Sp4, functional assays, and western blots, we found that Sp4 functionally regulates the transcription of Gabra1 (GABAA α1) and Gabra2 (GABAA α2), but not Gabra3 (GABAA α3) subunit genes. The binding sites of Sp4 are conserved among rats, humans, and mice. Thus, our results substantiate our hypothesis that Sp4 plays a key role in regulating the transcription of GABAA receptor subunit genes. They also indicate that Sp4 is in a position to transcriptionally regulate the balance between excitatory and inhibitory neurochemical expressions in neurons.
我们实验室之前的研究表明，神经元特异性转录因子Sp4可转录调控许多兴奋性神经递质受体亚基基因，如N-甲基-D-天冬氨酸（NMDA）受体的GluN1、GluN2A和GluN2B亚基基因，以及α-氨基-3-羟基-5-甲基-4-异恶唑丙酸（AMPA）受体的Gria2亚基基因。它还调控Na(+)/K(+)-ATP酶（一种主要的耗能酶）的Atp1a1和Atp1b1亚基基因，以及细胞色素c氧化酶（COX，一种重要的产能酶）的所有13个亚基基因。因此，在神经元的转录水平上，能量消耗、能量产生和兴奋性神经元活动之间存在紧密的耦合关系。问题是抑制性神经递质受体是否也受Sp4调控。在本研究中，我们检验了我们的假设，即Sp4调控主要抑制性神经递质γ-氨基丁酸（GABA）的受体亚基基因，特别是GABAA受体。通过多种方法，包括计算机分析、电泳迁移率变动和超迁移分析、实时定量PCR、染色质免疫沉淀、启动子突变分析、Sp4的过表达和短发夹RNA干扰、功能分析以及蛋白质免疫印迹，我们发现Sp4在功能上调控Gabra1（GABAA α1）和Gabra2（GABAA α2）亚基基因的转录，但不调控Gabra3（GABAA α3）亚基基因的转录。Sp4的结合位点在大鼠、人类和小鼠中是保守的。因此，我们的结果证实了我们的假设，即Sp4在调控GABAA受体亚基基因的转录中起关键作用。它们还表明，Sp4能够在转录水平上调控神经元中兴奋性和抑制性神经化学表达之间的平衡。
