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An Initiator Element Mediates Autologous Down- Regulation of the Human Type a ␥-Aminobutyric Acid Receptor ␤1 Subunit Gene

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An Initiator Element Mediates Autologous Down- Regulation of the Human Type a ␥-Aminobutyric Acid Receptor ␤1 Subunit Gene An initiator element mediates autologous down- regulation of the human type A ␥-aminobutyric acid receptor ␤1 subunit gene Shelley J. Russek, Sabita Bandyopadhyay, and David H. Farb* Laboratory of Molecular Neurobiology, Department of Pharmacology, Boston University School of Medicine, 80 East Concord Street, Boston, MA 02118 Edited by Erminio Costa, University of Illinois, Chicago, IL, and approved May 15, 2000 (received for review November 19, 1999) The regulated expression of type A ␥-aminobutyric acid receptor The ␤1 subunit gene is located in the ␤1-␣4-␣2-␥1 gene cluster (GABAAR) subunit genes is postulated to play a role in neuronal on chromosome 4 (12) and is most highly expressed in the adult maturation, synaptogenesis, and predisposition to neurological rat hippocampus. Seizure activity decreases hippocampal ␤1 disease. Increases in GABA levels and changes in GABAAR subunit mRNA levels by about 50% while increasing ␤3 levels (11). gene expression, including decreased ␤1 mRNA levels, have been Because the subtype of ␤ subunit influences the sensitivity of the observed in animal models of epilepsy. Persistent exposure to GABAAR to GABA and to allosteric modulators such as GABA down-regulates GABAAR number in primary cultures of etomidate, loreclezole, barbiturates, and mefenamic acid (13– neocortical neurons, but the regulatory mechanisms remain un- 17), a change in ␤ subunit composition may alter receptor known. Here, we report the identification of a TATA-less minimal function and pharmacology in vivo. Modulation of receptor ␤ promoter of 296 bp for the human GABAAR ␤1 subunit gene that function by phosphorylation is also influenced by subunit is neuron specific and autologously down-regulated by GABA. ␤1 composition and contributes to increased inhibition during states promoter activity, mRNA levels, and subunit protein are decreased of neuronal excitability (18). by persistent GABAAR activation. The core promoter, 270 bp, To determine whether a transcriptional mechanism controls contains an initiator element (Inr) at the major transcriptional start autologous regulation of GABAAR subunit gene expression, we site. Three concatenated copies of the 10-bp Inr and its immediate have begun by identifying the minimal ␤1 promoter and the 3؅ flanking sequence produce full neural specific activity that is genomic mechanism that controls inhibition of promoter activity down-regulated by GABA in transiently transfected neocortical in primary neocortical cultures exposed to persistent GABAAR neurons. Taking these results together with those of DNase I activation. footprinting, electrophoretic mobility shift analysis, and 2-bp mu- tagenesis, we conclude that GABA-induced down-regulation of ␤1 Materials and Methods subunit mRNAs involves the differential binding of a sequence- Genomic Walking by Using PCR. Inverse PCR (19) was carried out specific basal transcription factor(s) to the Inr. The results support using MspI and NcoIwith(5Ј-CCATGGTCTGTTGTGCACACA Ј Ј Ј a transcriptional mechanism for the down-regulation of ␤1 subunit G-3 ) and (5 -ACTGTCCACATTACTAACTCTGAT3- ) prim- GABAAR gene expression and raises the possibility that altered ers. Additional upstream sequence was obtained by performing levels of sequence-specific basal transcription factors may contrib- primer extension with Taq polymerase at 55°C for 3 min, yielding ute to neurological disorders such as epilepsy. a single-stranded cDNA that was recovered by nick column chro- matography (Pharmacia). The cDNA (15.4 ␮l) was tagged at the 3Ј ␮ transcription ͉ neural specific ͉ epilepsy end with a cytosine homopolymeric tail, using 2.0 l of terminal transferase (Pharmacia) in 2.2 ␮lof10ϫ phosphate buffer (Phar- ␮ ␥ macia) and 0.4 l of 100 mM dGTP for 15 min at 37°C, and then ABA ( -aminobutyric acid) is the major transmitter at inactivated at 75°C for 10 min. After ethanol precipitation, cDNAs Ginhibitory chemical synapses; yet little is known about the were amplified by PCR using a sense primer that was complemen- mechanisms underlying the genetic regulation of type A GABA tary to the tail (5Ј-ATAATGGTACCGGGGGGGGGGGGG-3Ј) receptor (GABAAR) number and composition. GABAAR num- and a gene-specific antisense primer directed upstream of the ber and subunit mRNA levels are down-regulated in primary original primer used for the first extension reaction, yielding 112 bp brain cultures as a response to persistent activation of the of additional upstream sequence. GABAAR (1–3). Moreover, down-regulation of subunit mRNAs precedes down-regulation of receptor number, indicating that Mapping of Transcriptional Start Sites. The locations of transcrip- GABA-induced GABAAR down-regulation is a consequence of tional initiation sites were determined by primer extension, S1 decreased mRNA levels (4). nuclease protection (20), and single-stranded ligation of com- Whereas the effects of GABA are inhibitory in the adult brain, plementary ends (SLIC) (21) using human brain total RNA they are excitatory during embryogenesis and early postnatal life (CLONTECH). We modified the SLIC procedure by synthesiz- (5). A variety of observations indicate that GABAAR subunit ing a ligation primer (5Ј-CTCATACATAGTACGAATTCGG- gene expression is regulated by receptor activation (6, 7) regard- less of age. GABAARs may play a critical role in the development of the central nervous system and so determine its future This paper was submitted directly (Track II) to the PNAS office. susceptibility to seizures (8). Extracellular GABA is increased Abbreviations: GABA, ␥-aminobutyric acid; GABAAR, type A GABA receptor; Inr, initiator during paroxysmal hippocampal activity in epileptic patients (9), element; ␤-gal, ␤-galactosidase; EMSA, electrophoretic mobility-shift analysis; ␤1-P, ␤1 promoter; Sv40-P, Sv40 promoter; Sv40-E, Sv40 enhancer; Mt, mutation; SLIC, single- and GABA uptake is inhibited in genetic absence epilepsy rats stranded ligation of complementary ends. (10). Both of these changes may lead to increases in the activity Data deposition: The sequence reported in this paper has been deposited in the GenBank of cell surface GABAARs and subsequent changes in GABAAR database (accession no. AF285168). function through down-regulation of subunit specific gene ex- ␣ ␤ *To whom reprint requests should be addressed. E-mail: [email protected]. pression. A decrease in the levels of 1 and 1GABAAR subunit The publication costs of this article were defrayed in part by page charge payment. This mRNAs precedes the onset of spontaneous seizures in pilo- article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. carpine-induced status epilepticus (11). §1734 solely to indicate this fact. 8600–8605 ͉ PNAS ͉ July 18, 2000 ͉ vol. 97 ͉ no. 15 Downloaded by guest on October 1, 2021 TACTGATTAC-3Ј) that contained an amino group at its 3Ј end. This amino group prevents excess ligation primer from initiating a polymerase extension reaction. An antisense primer (5Ј- TCACTTCGGAGACCATGTCTATGC-3Ј) that recognizes a sequence 300 bp downstream from the end of the ␤1 cDNA was used with avian myeloblastosis virus reverse transcriptase to produce a DNA copy of the 5Ј end of ␤1 subunit mRNAs. A second round of modified SLIC was performed by using an antisense primer that contained sequence immediately upstream of the most 5Ј end of the first SLIC products. Transfection Assays. Primary hippocampal, neocortical, muscle, and fibroblast cells were derived from 18-day-old rat embryos and grown in defined media (22). Neuronal cultures were exposed to 1 ␮M cytosine arabinoside on days 2 and 3 in vitro. Cells were transfected 1 wk after dissociation. PCR products were cloned into Promega GeneLight plasmids (pGL) and into PNASS␤ (Clonetech), for fluorescence imaging of ␤-galactosidase (␤-gal) activity in intact neurons (Imagene Green; Molecular Probes). For fluorescence imaging of ␤-gal activity in hippocampal neurons, on 35-mm dishes, cells were incubated with fresh media (1 ml) containing 2.0–2.5 ␮loffresh lipophilic fluorescein di-␤-galactopyranoside (FDG) (Imagene Green; Molecular Probes). Internal deletions and site-directed mutagenesis constructs were generated by using the Kunkel Fig. 1. Nucleotide sequence for the 5Ј end of the human ␤1 subunit gene. method (23). Mt(Ϫ1,ϩ2) contains mutation of ‘‘C’’ at Ϫ1 to ‘‘T’’ Transcriptional start sites mapped by S1 nuclease protection are indicated by ϩ, and ‘‘G’’ at ϩ2 to ‘‘C.’’ Inserts for luciferase constructs contain- primer extension by *, and modified SLIC by ‚ for round one and { for round two. ing one or three copies of the initiator (Inr) and flanking The first nucleotide, A, of the most 5Ј transcriptional initiation site is designated ϩ sequence, 5Ј-TGCGCAGGTCCATTCGGGAATTAC-3Ј, were as 1. Positions of Inr elements are indicated by overlining. Half brackets mark the 5Ј and 3Ј ends of ␤1-P (Ϫ436͞ϩ105), T5.1–T5.6, and T3.1–T3.5 constructs. The produced by using oligonucleotide synthesis. Cells were trans- 5Ј end of the human cDNA sequence (25) is indicated by a bracket. fected by using the calcium phosphate technique (24), or by bombardment using the Bio-Rad Biolistic Particle Delivery System, both yielding 5%–8% efficiency. Chemiluminescence Electrophoretic Mobility-Shift Analysis (EMSA) and DNase 1 Footprint- (Tropix, Bedford, MA) was used to monitor both luciferase ing. DNase 1 footprinting (20) was performed by using a 5Ј activity driven by ␤1-P, and ␤-gal activity driven by the phos- end-labeled HincII͞MseI fragment of the ␤1 gene. Double- phoglucokinase (PGK) promoter in cotransfected
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