Specificity protein 4 (Sp4) transcriptionally regulates inhibitory GABAergic receptors in neurons

Abstract

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.