An initiator element mediates autologous downregulation of the human type A γ-aminobutyric acid receptor β1 subunit gene

Abstract

The regulated expression of type A ␥-aminobutyric acid receptor (GABAAR) subunit genes is postulated to play a role in neuronal maturation, synaptogenesis, and predisposition to neurological disease. Increases in GABA levels and changes in GABA AR subunit gene expression, including decreased ␤1 mRNA levels, have been observed in animal models of epilepsy. Persistent exposure to GABA down-regulates GABAAR number in primary cultures of neocortical neurons, but the regulatory mechanisms remain unknown. Here, we report the identification of a TATA-less minimal promoter of 296 bp for the human GABAAR ␤1 subunit gene that is neuron specific and autologously down-regulated by GABA. ␤1 promoter activity, mRNA levels, and subunit protein are decreased by persistent GABAAR activation. The core promoter, 270 bp, contains an initiator element (Inr) at the major transcriptional start site. Three concatenated copies of the 10-bp Inr and its immediate 3 flanking sequence produce full neural specific activity that is down-regulated by GABA in transiently transfected neocortical neurons. Taking these results together with those of DNase I footprinting, electrophoretic mobility shift analysis, and 2-bp mutagenesis, we conclude that GABA-induced down-regulation of ␤1 subunit mRNAs involves the differential binding of a sequencespecific basal transcription factor(s) to the Inr. The results support a transcriptional mechanism for the down-regulation of ␤1 subunit GABA AR gene expression and raises the possibility that altered levels of sequence-specific basal transcription factors may contribute to neurological disorders such as epilepsy.transcription ͉ neural specific ͉ epilepsy G ABA (␥-aminobutyric acid) is the major transmitter at inhibitory chemical synapses; yet little is known about the mechanisms underlying the genetic regulation of type A GABA receptor (GABA A R) number and composition. GABA A R number and subunit mRNA levels are down-regulated in primary brain cultures as a response to persistent activation of the GABA A R (1-3). Moreover, down-regulation of subunit mRNAs precedes down-regulation of receptor number, indicating that GABA-induced GABA A R down-regulation is a consequence of decreased mRNA levels (4).Whereas the effects of GABA are inhibitory in the adult brain, they are excitatory during embryogenesis and early postnatal life (5). A variety of observations indicate that GABA A R subunit gene expression is regulated by receptor activation (6, 7) regardless of age. GABA A Rs may play a critical role in the development of the central nervous system and so determine its future susceptibility to seizures (8). Extracellular GABA is increased during paroxysmal hippocampal activity in epileptic patients (9), and GABA uptake is inhibited in genetic absence epilepsy rats (10). Both of these changes may lead to increases in the activity of cell surface GABA A Rs and subsequent changes in GABA A R function through down-regulation of subunit specific gene expression. A decrease in the levels of ␣1 and ␤1 GABA A R subunit...

Egr3 stimulation of GABRA4 promoter activity as a mechanism for seizure-induced up-regulation of GABA A receptor α4 subunit expression

“…Primary neurons were transfected as described in ref. 22. Expression constructs for Egr3 were generously provided by J. Milbrandt (Washington University, St. Louis), and ZnEgr was provided by J. Baraban (The Johns Hopkins University School of Medicine, Baltimore).…”

“…A variety of evidence suggests that GABRs vary their rates of transcription in response to extracellular signals (19)(20)(21)(22). In this study, we used stimulation of PKC in primary cultured hippocampal neurons (HNs) to increase levels of GABRA4 transcripts, mirroring changes shown in several epilepsy models (6)(7)(8).…”

Brain-derived Neurotrophic Factor (BDNF)-induced Synthesis of Early Growth Response Factor 3 (Egr3) Controls the Levels of Type A GABA Receptorα4 Subunits in Hippocampal Neurons

“…Primary Neuronal Cell Culture-Primary hippocampal neurons were cultured from prenatal day 18 Sprague-Dawley rats (E18) (Charles River Laboratories) as described previously (22,23).…”

Regionally selective effects of GABA on hypothalamic GABAA receptor mRNA in vitro

“…Notably, this effect is autologous because it occurred in synaptically isolated neurons. Similarly, stimulation of GABA A Rs altered transcription in cells of the mammalian circadian clock in the absence of cellular activity [35], and downregulated a promoter of the human β 1 subunit of GABA A R [36].…”