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J. Biol. Chem., Vol. 279, Issue 39, 40451-40461, September 24, 2004

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Transcriptional Regulation of the Mouse Gene Encoding the -4 Subunit of the GABA_A Receptor^*

Limei Ma, Lihua Song, Gina E. Radoi, and Neil L. Harrison

From the Departments of Anesthesiology and Pharmacology, Weill Medical College, Cornell University, New York, New York 10021

Received for publication, June 18, 2004

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The -aminobutyric acid type A receptors (GABA_A-Rs) mediate fast inhibitory synaptic transmission in the brain. The 4 subunit of the GABA_A-R confers distinct pharmacological properties on the receptor and its expression pattern exhibits plasticity in response to physiological and pharmacological stimuli, including withdrawal from progesterone and alcohol. We have analyzed the promoter region of the mouse GABRA4 gene that encodes the 4 subunit and found that the promoter has multiple transcriptional initiation sites and lacks a TATA box. The minimal promoter for GABRA4 spans the region between –444 to –19 bp relative to the coding ATG and shows high activity in cultured mouse cortical neurons. Both Sp3 and Sp4 transcription factors can interact with the two Sp1 binding sites within the minimal promoter and are critical for maximal activity of the promoter in neurons.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The -aminobutyric acid type A receptors (GABA_A-Rs)¹ are ligand-gated Cl^– channels that mediate fast inhibitory synaptic transmission in the mammalian brain (1–3). The pentameric receptor complexes are assembled from five subunits that are encoded by a family of 19 genes, including 1–6, 1–3, 1–3, , , , 1–3, and (4, 5). Along with several splicing variants, these subunits can form a large number of GABA_A-R subtypes, which differ not only in subunit composition and stoichiometry, but also exhibit distinct physiological and pharmacological properties (3–8). A number of clinically important drugs, such as benzodiazepines, inhaled anesthetics and barbiturates, as well as alcohols and neurosteroids, facilitate GABA_A-R function, and thereby enhance inhibitory synaptic transmission (4, 5, 7–12).

In the adult brain, the expression of the GABA_A-R subunits varies among brain regions and neuron types, and this regional specificity determines the pharmacological and functional properties of local GABAergic transmission (12–14). The expression pattern of the GABA_A-Rs is not static, and alterations of subunit expression are observed in a number of neurological, psychiatric, and physiological conditions (reviewed in Ref. 12) including temporal lobe epilepsy (15), Huntington's disease (16), focal ischemia (17), progesterone withdrawal (11), and alcohol withdrawal/dependence (18–22).

GABRA4, the gene that encodes the 4 subunit, is normally expressed at low levels in the brain, except in the ventrobasal thalamus, striatum, nucleus accumbens, olfactory tubercle, dentate gyrus, CA1 region of the hippocampus, and the outer layers of the cortex (14, 23). GABA_A-Rs containing the 4 subunit have unusual pharmacological properties that are distinct from those of receptors containing the more common 1or 2 subunits (24–26). These include insensitivity to benzodiazepines (26), high affinity for GABA (25), slower desensitization kinetics (24), and high sensitivity to facilitation by alcohol (27, 28). An increase in levels of GABRA4 mRNA and membrane protein is observed in an animal model of premenstrual syndrome and postpartum depression, in which rats undergo withdrawal from high levels of progesterone treatment (11, 29, 30). Withdrawal of progesterone is accompanied by a series of behavioral changes, including reduced seizure threshold, increased anxiety, and locomotor activity, all of which occur in parallel with the increase of GABRA4 expression. An elevation of GABRA4 expression is also observed in the rat following electroshock (31), in an animal model of temporal lobe epilepsy (15), and during withdrawal from alcohol (21, 22, 32). These findings have led to the speculation that the 4 subunit of the GABA_A-R might be involved in neuronal hyperexcitability.

Despite accumulating reports on the plasticity of the 4 subunit, the molecular mechanisms controlling this process are little understood at present. In this report we have analyzed the promoter region of GABRA4 in cultured cortical neurons by a luciferase reporter assay and identified two Sp1 binding sites within the proximal promoter region that are critical for promoter activity. The Sp1 binding site, also known as the GC box, contains an asymmetric hexanucleotide core GGGCGG sequence that is recognized by the members of Sp/KLF family of transcription factors with zinc finger motifs (33–35). The closely related Sp1, Sp3, and Sp4 have a similar binding affinity to the classic Sp1 site, as do other subfamily members, such as BTEB1, TIEG1, and TIEG2 (33–35). Our results indicate that both Sp3 and Sp4 can interact with the Sp1 sites in the GABRA4 promoter and are critical for promoter activity in neurons.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Construction of GABRA4 Reporter Plasmids and DNA Sequence Analysis—For constructing plasmids pLuc-P1 through pLuc-P8, pLuc-P11, and pLuc-P12, various promoter fragments of GABRA4 were generated by direct PCR amplification of genomic DNA isolated from 129 x 1/Svj mice (Jackson Laboratory, Bar Harbor, ME) using the Expand Long Template System (Roche Applied Science, Indianapolis, IN). The PCR primers (Table I) were designed based on the published mouse genome sequence (36) using Amplify 1.0 software (University of Wisconsin) at default settings. Restriction sites for MluI and NheI were included at the 5'-end of the PCR primers to facilitate subsequent cloning steps. PCR products were gel purified using the QIAquick gel extraction kit (Qiagen, Valencia, CA) and ligated into pGEM-T Easy vector (Promega, Madison, WI). Mini-prep DNA was isolated from white colonies and screened by MluI and NheI digestion. Fragments from the MluI and NheI digestion were gel purified and ligated into the MluI and NheI sites of pGL3-basic vector (pGL-BV) (Promega). Mutant vectors pLuc-P71 through pLuc-P78 were generated from the wild-type plasmid pLuc-P7 using the ExSite PCR-based system (Stratagene, La Jolla, CA) with primers listed in Table I. Constructs pLuc-P7A, pLuc-P7B, and pLuc-P7AB were generated from the pLuc-P7 vector using the QuikChange site-directed mutagenesis kit (Stratagene) with primers listed in Table I. All promoter constructs were confirmed by DNA sequencing.

5'-Rapid Amplification of cDNA Ends (RACE) and Primer Extension Assays—Total RNA was isolated from the brain tissue of adult C57/BL6 mice (Jackson Laboratory and Charles River Laboratories, Wilmington, MA) using the TRIzol reagent (Invitrogen). 5'-RACE analysis of transcription initiation sites was carried out by synthesizing RACE-ready cDNA from total brain RNA using the SMART RACE cDNA amplification kit (Clontech, Palo Alto, CA) and by using the FirstChoice RLMRACE kit (Ambion, Austin, TX). The RACE-ready cDNA was then subjected to PCR amplification using the universal anchor primer and the GABRA4 specific primer anti-4 (5'-CGCAATCGCGGGTACCTTCTGGACAG-3'), which anneals to the region +8 to +33 relative to the coding ATG. PCR amplification was performed by denaturing the cDNA for 2 min at 94 °C followed by 35 cycles of 94 °C for 10 s, 55 °C for 30 s, and 72 °C for 2 min. PCR products were subcloned into pGEM-T Easy vector (Promega) and clones containing the PCR inserts were sequenced to locate the 5'-end of the transcript for GABRA4.

The primer extension assay was carried out by using the Primer Extension System (Promega). 3 x 10⁶ cpm of the [-³²P]ATP (Perkin-Elmer Life Sciences, Boston, MA) labeled anti-4 primer was annealed to 30–40 µg of total brain RNA at 55 °C for 30 min following denaturation at 95 °C for 5 min. Annealed samples were slowly cooled to room temperature prior to addition of reverse transcriptase. After incubating for 1 h at 42 °C, the reverse transcription (RT) reaction mixture was purified by phenol-chloroform extraction followed by ethanol precipitation. The primer extension products were analyzed on a 6% denaturing TBE gel containing 8 M urea using a DNA sequencing size marker prepared with the AmpliCycle sequencing kit (Applied Biosystems, Foster City, CA).

RT-PCR and Real-time PCR Analyses—For RT-PCR analysis, cDNA was reverse transcribed from 0.5 to 1 µg of total RNA in a 30-µl reaction containing 0.25 µg of random hexamer (Qiagen), 1 mM dNTP, 2.5 units of RNaseOut (Invitrogen), and 5 units of reverse transcriptase (New England Biolabs, Beverly, MA) with 1x RT reaction buffer. The RT reaction mixture was purified by one step of phenol-chloroform extraction. The primers for GAPDH, GB450 (5'-ACCACAGTCCATGCCATCAC-3') and GB449 (5'-TCCACCACCCTGTTGCTGTA-3'), which amplify both human and mouse genes, were described previously (37). The primers for GABRA4 mRNA were individually designed and the resulting PCR products were confirmed by DNA sequencing. GABRA4 RT-PCR primers for human (HEK293 cells) were: sense (5'-CTGGACAAAAGGTCCTGAG-3') and antisense (5'-CTCTCTGCACTGGAGCAGC-3'), and for mouse (neurons and astrocytes) were: sense (5'-TACACCTGGACCAAAGGCCC-3') and antisense (5'-TCTGTGTGTTTCTCCTTCAGCACA-3'). The 50-µl PCR reaction mixture contained 2 µl of cDNA, 0.2 µM of each primer, 0.2 mM of each dNTP, 2.5 units of Taq polymerase, and 1x PCR buffer with Mg²⁺ (Roche Applied Science). PCR amplification was carried out by denaturing the cDNA for 2 min at 94 °C followed by 28 cycles (GAPDH) or 35 cycles (GABRA4) of 94 °C for 10 s, 58 °C for 30 s, 72 °C for 2 min, and a final step of elongation for 7 min at 72 °C.

For real-time PCR analysis, 0.5–1 µg of total RNA was isolated by TRIzol reagent (Invitrogen) and was treated with RNase-free DNase I (Promega) for 15 min followed by heat inactivation. cDNA was prepared using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) with random hexamer followed by RNase H digestion. Real-time PCR primers were designed by OligoPerfect Designer (Invitrogen) using default settings, which generates T_m matched primer pairs that amplify 80–250 bp of target sequence. The GAPDH primers were forward (5'-AACTTTGGCATTGTGGAAGG-3') and reverse (5'-ACACATTGGGGGTAGGAACA-3') and amplified a product of 223 bp. The GABRA4 primers were forward (5'-TAAACGAATCCCCAGGACAG-3') and reverse (5'-GACGCAGCCTGTTGTCATAA-3') and amplified a product of 105 bp. The amplified PCR products were confirmed by DNA sequencing. Real-time PCR were carried out in a DNA Engine Thermocycler (MJ Research, Reno, NV) using SYBR green I dye. The 50-µl reaction mixtures contained 0.5 µM of each primer, 1 to 2 µl of cDNA, and 25 µl of Platinum Quantitative PCR SuperMix-UDG (Invitrogen). The conditions for realtime PCR were 2 min at 95 °C, and 45 (GAPDH) or 50 (GABRA4) cycles for 15 s at 95 °C, 30 s at 60 °C, 30 s at 72 °C followed by a gradient step from 55 to 95 °C to obtain a melting curve for each sample. Plasmids containing GAPDH and GABRA4 cDNA were linearized by EcoRI digestion and were used as PCR standards in 5-fold serial dilutions. The cycle threshold (C_t) line was positioned manually at the point where the fluorescent signals surpassed background noise and started to increase linearly. Standard curves were determined for each reaction by plotting C_t as a function of log [DNA]. C_t for each sample was obtained from the fluorescence trace where it intersected with the C_t line. DNA concentration was obtained by converting the C_t to an initial quantity by interpolation from the standard curve run on the same plate. The homogeneity of PCR products was analyzed by the observation of a single peak of the fluorescence versus temperature trace at the melting temperature and by agarose gel electrophoresis. In each experiment, the average values of triplicate samples were used for each data point, and a minimum of three independent neuronal cultures were included for each analysis to obtain the final results. A control sample with no reverse transcriptase in the RT reaction was included in each experiment to monitor for genomic DNA contamination.

Cell Culture, Transfection, and Dual Luciferase Assay—Mouse cortical neurons were prepared as previously described with modifications (38). Briefly, cerebral cortices were isolated from embryonic day 17 or 18 C57/BL6 mice (Jackson Laboratory and Charles River Laboratories). The dissected cortices were minced in 1x Dulbecco's phosphate-buffered saline (Mediatech, Herndon, VA) and treated for 30 min at 37 °C with 20 units/ml papain (Calbiochem, San Diego, CA) in 1x Earle's balanced salts solution (Sigma) containing 1 mM L-cysteine (Calbiochem) and 0.5 mM EDTA (Sigma). Digested cortical cells were further dissociated in Earle's balanced salts solution containing 2 mg/ml albumin (Sigma) and 100 units/ml DNase I (Worthington, Lakewood, NJ) by gentle trituration with a fire-polished Pasteur pipette. The resulting single cell suspension was layered on top of a 20 mg/ml albumin solution in Earle's balanced salt solution for a single step of discontinuous gradient centrifugation at 800 rpm for 10 min at room temperature. The cell pellet was then resuspended in phenol red-free minimum essential medium (PRF-MEM) (Invitrogen) supplemented with 5% horse serum (Sigma) and 0.5 mM L-glutamine (Sigma). Cells were seeded at a concentration of 1 x 10⁶ cells/ml in 12-well plates (Corning, Corning, NY) pre-coated overnight with 0.05 mg/ml poly-D-lysine (Sigma). On day 3 in vitro, half of the media was replaced with phenol red-free neurobasal medium (Invitrogen) containing 0.5 mM glutamine, 1x B27 supplement (Invitrogen), and the anti-mitotic agents 10^–4 M uridine and 10^–5 M 5-fluoro-2-deoxyuridine (Sigma). Fresh PRF neurobasal medium was added to replace half of the media every other day thereafter. Astrocytes were prepared by culturing the cortical cells at a density of 2 x 10⁵ cells/ml in 5% equine serum (HyClone, Logan, UT) PRF-MEM. Astrocytes continued to propagate and constituted more than 90% of the cells in the mixed culture population after 2 weeks of expansion. Transfection of neurons was generally performed on days 7–9 in vitro for luciferase analysis, whereas transfection of astrocytes was performed after 2 weeks in culture. HEK293 cells were cultured as described previously (39) and transfected when the cells reached 70–80% confluence. All cells were maintained at 37 °C in a humidified environment of 5% CO₂.

Plasmid DNA used for transfection was obtained using the HiSpeed maxi-prep kit (Qiagen) and re-purified by sodium acetate and ethanol precipitation. For each construct, at least three independent plasmid DNA preparations were used for transfection experiments. For each transfection, 1 µg of plasmid DNA was added to 5 ng of CMV promoter driven Renilla luciferase construct (pRL-CMV) (Promega) for co-transfection. The pRL-CMV construct served as the internal control for the dual luciferase assay (Promega). Transfection was performed using a combination of the Nupherin (Biomol%20Research%20Laboratories">Biomol Research Laboratories, Plymouth Meeting, PA) and LipofectAMINE 2000 (Invitrogen) reagents. 1 µg of plasmid DNA was first mixed with 6 µg of Nupherin in 150 µl of PRF-MEM for 15 min and then combined with 150 µl of PRF-MEM containing 3 µl of LipofectAMINE 2000 for another 15 min at room temperature. The culture media was replaced with 300 µl of transfection media containing the LipofectAMINE-Nupherin-DNA complex. After incubating for 1 h, transfection media was replaced with 1 ml of culture media. Luciferase activity was analyzed 20–24 h post-transfection with a Turner Design 20/20 luminometer (Turner Designs, Sunnyvale, CA) using the dual-luciferase reporter assay system (Promega). Triplicate samples were measured for each construct, and the average values of luciferase light units were used for data analysis. The negative control pGL-BV plasmid was the pGL3-basic vector (Promega), which contained no eukaryotic promoter or enhancer sequence. The luciferase activity was defined as the ratio of the light units of GABRA4 promoter driven firefly luciferase versus CMV promoter driven Renilla luciferase. The relative luciferase activity for each sample was normalized to that of the SV40 minimal promoter (P_SV40) driven luciferase construct for comparison among different experiments. The expression vectors pEVR2/Sp1, pRC/CMV-Sp3, and pRC/CMV-Sp4 were obtained from Professor G. Suske (40, 41).

Gel Shift Assay—Mouse brain nuclear extract was prepared as described previously with modifications (42). Briefly, the brain tissue was frozen in liquid nitrogen and shattered into tissue powder. About 500 mg of tissue was lysed in buffer A containing 10 mM HEPES, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.5 mM dithiothreitol and protease inhibitor mixture (Sigma) and homogenized for 10 strokes using a glass homogenizer with loose type pestle. Tissue debris was discarded by centrifuging the homogenate at 2,000 rpm for 30 s at 4 °C, and nuclei were collected by centrifuging the supernatant at 5,000 rpm for 5 min at 4 °C. The nuclei were re-suspended in 200–500 µl of buffer B containing 20 mM HEPES, pH 7.9, 25% glycerol, 420 mM NaCl, 1.2 mM MgCl₂, 0.2 mM EDTA, 0.5 mM dithiothreitol, and protease inhibitor mixture and incubated for 30 min at 4 °C by gentle shaking. Nuclear extract was collected by centrifuging the lysate at 14,000 rpm at 4 °C for 15 s.

Oligos Sp1-A (5'-GAGCGGCGAGGGAGGGGGCGGGCGCGCAGGTC-3') and Sp1-B (5'-GCGCGCAGGTCCCGCCTCCCCTGGCCGCGT-3') were synthesized (Qiagen) and double-stranded oligos were prepared by annealing the complementary strands in 10 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA buffer, pH 8.0. The consensus Sp1 binding sites were underlined in the oligo sequences. The sequences of mutant oligos MutA and MutB were listed in Table I. The Sp1 core oligo was obtained from Promega. The in vitro binding reactions were carried out using a gel shift kit (Roche Applied Science). The oligos were either end-labeled with [-³²P]ATP or digoxigenin. About 10 x 10³ cpm or 0.4 ng of labeled oligo was used for each binding reaction. Human rSp1 and HeLa nuclear extract were obtained from Promega. 0.8 µg of antibodies against c-Myc (C-19), Sp1 (PEP 2), Sp3 (D-20), and Sp4 (V-20) (Santa Cruz Biotechnology) were used in supershift analysis. For the digoxigenin-labeled probes, the DNA-protein complex was blotted to a Zeta probe membrane (Bio-Rad) by capillary contact transfer for 0.5–2 h. The DNA was cross-linked to the membrane with a UV cross-linker (Stratagene). Immunological detection of digoxigenin-labeled DNA was then carried out according to the manufacturer's instructions (Roche Applied Science).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Identification of the Transcriptional Initiation Site for GABRA4 —Previously we isolated a GABRA4 cDNA clone from a mouse library (28); the 5'-end of this clone is located at –144 bp relative to the coding ATG. As a first step in characterizing the mouse GABRA4 promoter, we conducted RACE analysis of total RNA isolated from the adult mouse brain to map the 5'-end of the GABRA4 mRNA. We designated the nucleotide residue A of the coding ATG as the +1 position in GABRA4. Sequence analysis of the RACE PCR products revealed multiple potential start sites for the GABRA4 mRNA clustered in the first exon within a region from –150 to –113 bp (Figs. 1 and 2B), which included the beginning of our cDNA clone at position –144. The sequences of the RACE PCR products corresponded exactly with the published mouse genomic sequence (36) without interruption, indicating that no intron was present at the 5'-end of GABRA4 coding exon 1. In a search of the GenBank^TM data base, we found that the 5'-end of a mouse neocortex GABRA4 cDNA transcript AK043806 [GenBank] was located at position –114, which was within the initiation region that we had identified. A second GABRA4 transcript AK013727 [GenBank] , which was isolated from the hippocampus, was reported to originate at position –499, about 300–400 bp further upstream. We wondered whether multiple transcripts for GABRA4 might exist, or whether the short transcripts we detected might have arisen as the result of preferential amplification of short fragments. We therefore designed three additional PCR primers that annealed to the sequence upstream of position –150. These primers should have enabled the amplification of the long transcript of GABRA4 mRNA. However, we were not able to detect any long PCR products, even after two rounds of nested PCR with an additional 30 cycles. This observation suggested that the short transcripts we identified did in fact represent the major products of GABRA4 transcription in the mouse.

View larger version (36K): [in this window] [in a new window]   FIG. 1. Sequence alignment of the 5'-flanking region of mouse and human GABRA4. Mouse and human GABRA4 were aligned using the ClustalW program. Nucleotide residue A in the coding ATG was designated as +1 for both genes. Transcription start sites identified by 5'-RACE and primer extension analyses are indicated by open circles and arrowheads, respectively. The 5'-end of the cDNA AK013727 [GenBank] and AK043806 [GenBank] are marked by flag symbols and the 5'-end of our own GABRA4 cDNA clone is marked by a star symbol. The two 16-bp repeats, putative Inr, E-Box, Sp1, AP1, and c-Myb elements are boxed and labeled.

View larger version (25K): [in this window] [in a new window]   FIG. 2. Mapping of the transcription initiation sites for GABRA4. A, primer extension analysis of total brain RNA isolated from adult mouse. Lanes 1–4, a DNA marker generated by a sequencing reaction. Lane 5, a representative primer extension reaction using the 32P-labeled anti-4 primer. The sizes of the primer extension products are as indicated. B, a schematic map of the putative transcription start sites based on the results obtained from primer extension and 5'-RACE analyses. The 5'-end of the AK013727 [GenBank] and AK043806 [GenBank] cDNA clones are also indicated. The positions of the start sites are indicated by the numbers in parentheses.

Consensus Sequence Elements in the 5'-Flanking Region of GABRA4 —We searched the 5'-proximal region of GABRA4 for sequence motifs that were commonly found in the promoters of genes expressed in a neuron-specific manner. Sequence analysis of a 3.7-kb fragment of the 5'-flanking region revealed no TATA box, but the presence of two tandem direct repeats, between –1267 and –1214 bp, and between –143 and –112 bp, and one short poly-pyrimidine track at –1213 bp. The repeats located at –1267 bp contained 10 CTCTGT motifs, whereas the repeats at –143 bp contained two direct repeats of 16 nucleotides long. In many TATA-less promoters, an initiator (Inr) element with the consensus sequence YYANWYY had been identified and found to direct transcription (43). Several elements bearing the Inr consensus sequence were found within the region of –1.0 kb to +1 bp but none of these putative Inr elements was located near the transcription start sites that we identified (Fig. 1). Neural-specific promoters very often contain a 21-bp neuron-restrictive silencer element (NRSE) in the proximal promoter region (44–46). In an attempt to search for NRSE-like elements in the GABRA4 promoter, we examined a 10-kb region of the mouse gene upstream and downstream of the coding ATG. No sequence motif that shared >70% homology with the consensus NRSE was found within this region. This observation indicates that elements other than the NRSE are likely present in the GABRA4 promoter to control neuronal-specific expression.

Identification of the Minimal Promoter Region for Mouse GABRA4—To identify the minimal promoter region, we employed the luciferase reporter assay to test the ability of promoter fragments to direct the expression of the reporter gene in cultured cortical neurons and cells derived from non-neuronal origins. GABRA4 is expressed in a limited number of brain regions including the outer layers of the cortex (14, 23). We reasoned that the promoter constructs should be active in an environment in which endogenous GABRA4 was expressed. We first used RT-PCR analysis to determine whether the endogenous GABRA4 was expressed in cultured cortical neurons. With similar levels of GAPDH expression, GABRA4 mRNA was minimally detectable in cortical cells after 2 days in vitro, and then increased from 4 days in vitro, reaching its peak after 10 days in vitro (Fig. 3A, lanes 1–5). As negative controls for RT-PCR, we synthesized cDNA from 10 day in vitro cells either without the addition of reverse transcriptase or without RNA in the RT reactions. No RT-PCR signals were detected from these two control samples for either GAPDH or GABRA4 message (Fig. 3, lanes 7 and 8).

View larger version (22K): [in this window] [in a new window]   FIG. 3. The expression of endogenous GABRA4 mRNA and the activity of promoter construct pLuc-P1 in cultured mouse cortical neurons. A, lanes 1–5, RT-PCR analysis of RNA isolated from mouse cortical neuron cultures at 2, 4, 6, 8 and 10 days in vitro (DIV). GAPDH expression level was also analyzed for each sample and was used to normalize cDNA template input for RT-PCR. Lanes 6–8, RT-PCR control reactions using day-10 in vitro samples. Lane 6, day-10 in vitro RNA in a standard RT-PCR reaction. Lane 7, no reverse transcriptase added in the RT reaction. Lane 8, no RNA added in the RT reaction. B, real-time PCR analysis of GABRA4 expression in cortical neurons at days 2, 4, 6, 8, and 10 in vitro. The concentrations of GABRA4 and GAPDH cDNA were measured for each sample based on the standard curves generated using linearized plasmid DNA of known concentrations. The relative concentration of GABRA4 mRNA was then expressed in terms of the mean ratio of GABRA4 versus GAPDH± S.E. (n = 4). C, the pLuc-P1 promoter construct and the promoter-less control vector pGL-BV were transfected into neurons cultured for 2, 4, 6, 8, and 10 days. Luciferase activity was assayed 20–24 h after transfection to measure the promoter activity in these cultures. DIV, days in vitro.

We then employed the dual luciferase assay to test whether a 3.7-kb GABRA4 promoter fragment P1 could recapitulate the time course of endogenous GABRA4 expression. The P1 fragment was directly cloned by PCR amplification of a region from –3716 to –19 bp of GABRA4. The firefly luciferase construct that carried the P1 promoter fragment was designated as pLuc-P1. A CMV promoter driven Renilla luciferase construct pRL-CMV was co-transfected with the promoter construct as internal control for normalization in dual luciferase assay. We transfected the construct pLuc-P1 into neurons after various times in culture and assayed for luciferase activity. The luciferase activity was defined as the ratio of the light units of GABRA4 promoter driven firefly luciferase versus CMV promoter driven Renilla luciferase. The relative luciferase activity for each sample was normalized to that of the SV40 minimal promoter-driven luciferase construct P_SV40 for comparison among different experiments. The pLuc-P1 construct did not elicit significant luciferase activity on 2 days in vitro, but the promoter activity of this construct increased with time in culture and reached activity of 104% relative to the control P_SV40 in neurons after 10 days in vitro (Fig. 3C). The negative control pGL-BV vector, which contained the luciferase coding sequence but no eukaryotic promoter or enhancer sequence, only gave rise to basal levels of expression (Fig. 3C). This result demonstrates that a 3.7-kb fragment of the 5' region of GABRA4 is able to drive the expression of the luciferase reporter gene in a neuronal environment and that the time course of promoter activity in neurons is consistent with the profile of endogenous GABRA4 expression. The promoter activity of pLuc-P1 was consistently reproducible between days 7 and 9 in vitro, so neuronal cultures at this age were used for all subsequent experiments. No endogenous GABRA4 transcript was detected in primary mouse astrocytes or human embryonic kidney cells (HEK293) (data not shown). The activity of the pLuc-P1 construct was low in both astrocytes and HEK293 cells (Fig. 4, A and B).

View larger version (28K): [in this window] [in a new window]   FIG. 4. GABRA4 promoter construct activity in cultured neurons, astrocytes, and HEK293 cells. A and C, schematic illustrations of the promoter constructs used for the transfection experiments. The 5' and 3' boundaries of the promoter for each construct are identified by numbers. The positions of the two direct repeats, transcription initiation sites, and 5'-end of AK013727 [GenBank] cDNA for GABRA4 are marked on each construct. B and D, luciferase reporter analysis of promoter constructs in cultured cortical neurons (n = 6–36), astrocytes (n = 6–9), and HEK293 cells (n = 6–12). The relative luciferase activity for each construct is calculated as the ratio of the luciferase activities of GABRA4 promoter driven construct versus the Psv40 driven construct. The results are expressed as mean relative luciferase activity ± S.E.

Promoter constructs pLuc-P1 through pLuc-P7, which carried 3.7-, 2.6-, 2.3-, 2.2-, 1.3-, 0.9-, and 0.4-kb fragments of the 5' region of GABRA4, respectively, gave rise to similar levels of luciferase activity in cultured cortical neurons, but little to no activity in astrocytes or HEK293 cells (Fig. 4, A and B). When the construct was truncated to 166 bp, as in pLuc-P8, the promoter activity in neurons decreased sharply relative to all the other constructs analyzed (Fig. 4, A and B). We reasoned that the sudden change of activity between pLuc-P7 and pLuc-P8 might be because of the loss of one or more positive regulatory element(s).

We also assessed the functional significance of the putative Inr element at position –488, because it was located close to the 5'-end of the published long GABRA4 transcript AK013727 [GenBank] . Two deletion constructs, pLuc-P11 and pLuc-P12, were generated by PCR, in which the start sites for the short transcripts were eliminated. These two deletion constructs showed drastically reduced promoter activity (Fig. 4, C and D), indicating the loss of critical transcriptional elements in these constructs and the inability of the putative upstream Inr element to support GABRA4 expression. Taken together, these results support the idea that the minimal promoter region for driving neuron-specific expression of GABRA4 is located within the region –444 to –19.

cis-Regulatory Elements Critical for GABRA4 Minimal Promoter Activity—To delineate the elements that were critical for GABRA4 promoter activity, we generated internal deletion mutants of the pLuc-P7 construct and assayed their activity for directing transcription in neurons. Deletion constructs pLuc-P71, -2, -3, -7, and -8 showed similar promoter activity compared with the parental pLuc-P7 construct (Fig. 5). These deletions eliminated the two repeats at –143 bp and a series of putative binding sites for transcription factors, specifically an Sp1 site located at the 5'-end of the promoter fragment, Ap1, c-Myb, and E-box. These findings suggest that the potential binding sites within these regions are not required for GABRA4 promoter activity in neurons in a transient transfection assay. The pLuc-P74, -5, and -6 constructs harbored deletions that eliminated the two Sp1 sites close to the 3'-end of the promoter and other elements (Fig. 5A). We designated the Sp1 site at position –214 as Sp1-A, and the one at position –198 as Sp1-B. In construct pLuc-P75, deletion of the c-Myb, E-box, and the Sp1-A site resulted in about 60% reduction of promoter activity compared with the pLuc-P7 construct (Fig. 5B). In construct pLuc-P74, deletion of the Sp1-A site resulted in about 50% reduction of promoter activity, suggesting that the Sp1-A site and its surrounding sequences were critical for promoter activity (Fig. 5B). Deletion of the two Sp1 sites and the two direct repeats at –143 bp (as in pLuc-P76) drastically reduced the promoter activity by >80%, whereas deletion of the repeats alone, as in pLuc-P77, did not affect the promoter activity significantly (Fig. 5B). These results suggest that the two Sp1 sites within the region –257 to –179 are critical for maximal GABRA4 promoter activity.

View larger version (23K): [in this window] [in a new window]   FIG. 5. Internal deletion analysis of the pLuc-P7 promoter construct in cortical neurons. A, schematic illustration of the deletion constructs used. The deletion boundaries for each construct are identified by numbers. The positions of the direct repeats and potential transcription factor binding sites are indicated on each construct. B, luciferase reporter analysis of mutant promoter constructs in cultured cortical neurons (n = 7–36). The asterisks represent significant differences at the level of p < 0.01 when different constructs are compared with pLuc-P7 using one-way analysis of variance and post-hoc Dunnett's test.

View larger version (39K): [in this window] [in a new window]   FIG. 6. Two Sp1 elements in the GABRA4 promoter are critical for maximal promoter activity. A and B, gel shift assay of rSp1 binding to Sp1-A and Sp1-B oligos. 10 ng of rSp1 were incubated with 0.02 nM digoxigenin-labeled oligos. Protein-DNA complex was resolved on a non-denaturing 0.5 x TBE gel. In A and B, lanes 1, free probes with no rSp1 added to the binding reaction. Lanes 2–11, rSp1 was incubated with labeled Sp1-A (A) or Sp1-B (B) oligo and unlabeled competitors as indicated. The concentrations of competitors were: 0.02 nM (lanes 3, 6, and 9), 0.2 nM (lanes 4, 7, and 10), and 2 nM (lanes 5, 8, and 11). C, luciferase reporter analysis of promoter constructs with mutations at Sp1 binding sites in cultured cortical neurons (n = 7–36). The asterisks represent significant differences at the level of p < 0.01 (**) and p < 0.05 (*) when different constructs are compared with pLuc-P7 using one-way analysis of variance and post-hoc Dunnett's test.

The Sp/KLF family of transcription factors comprises at least 16 mammalian gene products that share the signature zinc finger motifs for DNA binding (33–35). Among them, Sp1, Sp3, and Sp4 are closely related transcriptional activators that recognize identical DNA sequences. To investigate which Sp/KLF transcription factors interact with the GABRA4 promoter, we conducted gel shift analysis using nuclear extract isolated from adult mouse brain.

Three DNA-protein complexes were formed when Sp1-A double-stranded oligo was incubated with the brain nuclear extract (Fig. 7A, lane 2). Addition of unlabeled Sp1-A oligo was able to abolish the formation of all three complexes, whereas addition of MutA oligo did not inhibit the complex formation (Fig. 7A, lanes 3 and 4). The Sp1 core oligo was able to abolish the formation of complex I and weakly inhibit complex III, but did not affect the formation of complex II (Fig. 7A, lane 5). Addition of 0.8 µg of rabbit antibodies against c-Myc (control) and Sp1 did not alter the binding pattern of the brain nuclear extract to the Sp1-A oligo (Fig. 7A, lanes 6 and 7). However, addition of rabbit antibodies against Sp3 and Sp4 abolished the formation of complex I without affecting complexes II and III significantly (Fig. 7A, lanes 8 and 9). We noticed that no supershifted band was observed when anti-Sp3 and -Sp4 antibodies were added in the binding reactions although the formation of complex I was abolished (Fig. 7A, lanes 8 and 9). These results implied that the addition of antibodies in the reactions might prevent the formation of DNA-protein complex at the Sp1-A site. Taken together, these results indicate that multiple factors can interact with the Sp1-A oligo and that both Sp3 and Sp4 are present in the bound complexes at the Sp1-A site.

View larger version (29K): [in this window] [in a new window]   FIG. 7. Identification of the Sp/KLF factors bound to the Sp1-A and Sp1-B sites using nuclear extracts derived from brain. Gel shift assay of brain nuclear extract binding to the Sp1-A and Sp1-B sites. 8–10 µg of brain nuclear extract were incubated with 10 x 103 cpm of 32P-labeled Sp1-A (A) or Sp1-B (B) oligo. Protein-DNA complex was resolved on a non-denaturing 0.5 x TBE gel. A, lane 1, free Sp1-A probe with no nuclear extract added. Extract was incubated without competitors or antibodies (lane 2) and with competitors at 100x excess: unlabeled Sp1-A (lane 3), MutA (lane 4), or Sp1-core oligo (lane 5). Extract was incubated with 0.8 µg of antibodies against c-Myc (lane 6), Sp1 (lane 7), Sp3 (lane 8), or Sp4 (lane 9) in the binding reactions. Three DNA-protein complexes were formed at the Sp1-A site as indicated. B, lane 1, free Sp1-B probe with no nuclear extract added to the binding reaction. Extract was incubated without competitors or antibodies (lane 2) and with competitors at 100x excess: unlabeled Sp1-B (lane 3), MutB (lane 4), or Sp1-core oligo (lane 5). Extract was incubated with 0.8 µgof antibodies against c-Myc (lane 6), Sp1 (lane 7), Sp3 (lane 8), or Sp4 (lane 9) in the binding reactions. The DNA-protein complex is indicated as complex II and the supershifted DNA-protein-antibody complex is identified as complex I.

View larger version (30K): [in this window] [in a new window]   FIG. 8. Identification of the Sp/KLF factors bound to the Sp1-A and Sp1-B sites using nuclear extract derived from HeLa cells. Gel shift assay of HeLa nuclear extract binding to the Sp1-A and Sp1-B sites. 8–10 µg of HeLa nuclear extract were incubated with 10 x 103 cpm of 32P-labeled Sp1-A (A) or Sp1-B (B) oligo. Protein-DNA complex was resolved on a non-denaturing 0.5x TBE gel. A, lane 1, free Sp1-A probe with no nuclear extract added to the binding reaction. Extract was incubated without competitors or antibodies (lane 2) and with competitors at 100x excess: unlabeled Sp1-A (lane 3), MutA (lane 4), or Sp1-core oligo (lane 5). Extract was incubated with 0.8 µg of antibodies against c-Myc (lane 6), Sp1 (lane 7), Sp2 (lane 8), Sp3 (lane 9), or Sp4 (lane 10) in the binding reactions. Two DNA-protein complexes formed at the Sp1-A site are indicated as complexes II and III. The supershifted complex is identified as complex I. B, lane 1, free Sp1-B probe with no nuclear extract added. Extract was incubated without competitors or antibodies (lane 2) and with competitors of 100x excess: unlabeled Sp1-B (lane 3), MutB (lane 4), or Sp1-core oligo (lane 5). Extract was incubated with 0.8 µg of antibodies against c-Myc (lane 6), Sp1 (lane 7), Sp2 (lane 8), Sp3 (lane 9), or Sp4 (lane 10) in the binding reactions. DNA-protein complexes are indicated as complexes II and III and the supershifted DNA-protein-antibody complex is labeled as complex I.

The Roles of Sp3 and Sp4 in Regulating the GABRA4 Promoter—To further investigate whether Sp3 and Sp4 were functionally involved in the expression of GABRA4, we analyzed the influence of Sp3 and Sp4 on the endogenous GABRA4 expression by real-time PCR. Expression vectors carrying Sp1, Sp3, and Sp4 cDNA were transfected into day 7 in vitro neurons and the expression of endogenous GABRA4 was analyzed 2 days after transfection. Transfection of pcDNA3.1 vector that did not contain any Sp/KLF factor was used as a negative control. Overexpression of Sp1 slightly increased the GABRA4 level to about 1.3-fold over the control. In contrast, overexpression of Sp3 and Sp4 augmented GABRA4 expression to about 2.3-fold (Fig. 9A). We also examined whether expression of the Sp/KLF factors could affect the promoter activity on the plasmid construct. Co-transfection of the pLuc-P7 vector with pcDNA3.1, Sp1, Sp3, or Sp4 expression vectors was carried out in day 7 in vitro neurons and the luciferase activity was assayed 2 days after transfection. We observed that overexpression of Sp1 did not affect the pLuc-P7 promoter activity, whereas expression of Sp3 and Sp4 increased the promoter activity to 1.3- and 1.7-fold (Fig. 9B). These results indicate that both Sp3 and Sp4 are able to augment GABRA4 expression and are likely to be involved in the regulation of this gene in vivo.

View larger version (11K): [in this window] [in a new window]   FIG. 9. The effects of the Sp/KLF family of transcription factors on endogenous GABRA4 mRNA expression and the activity of promoter construct pLuc-P7 in cultured mouse cortical neurons. A, real-time PCR analysis of GABRA4 expression in cortical neurons transfected with expression vectors of Sp1, Sp3, and Sp4. The vectors pEVR2/Sp1, pRC/CMV-Sp3, and pRC/CMV-Sp4, which carried Sp1, Sp3, and Sp4, respectively, were transfected into day-7 in vitro neurons, and RNA was collected 48 h later. The pcDNA3.1 vector was used as a negative control. The concentrations of GABRA4 and GAPDH were measured for each sample based on the standard curves generated using linearized plasmid DNA of known concentrations. The relative concentration of GABRA4 mRNA was then expressed in terms of the mean ratio of GABRA4 versus GAPDH. The results are reported as the mean increase (relative to the negative control) ± S.E. (n = 4). The asterisks represent significant differences at the level of p < 0.01 (*) and p < 0.05 (**) by one-way analysis of variance and post-hoc Dunnett's test. B, the vectors pEVR2/Sp1, pRC/CMV-Sp3, and pRC/CMV-Sp4, which carried Sp1, Sp3, and Sp4, respectively, were co-transfected with pLuc-P7 construct into day 7 in vitro neurons and luciferase activity was measured 48 h later. The pcDNA3.1 vector was used as a negative control. The results are reported as the mean increase (relative to the negative control) ± S.E. (n = 4). The asterisks represent significant differences at the level of p < 0.01 (*) by one-way analysis of variance and post-hoc Dunnett's test.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Sequence alignment has revealed that two conserved Sp1 binding sites, Sp1-A and Sp1-B, are critical for GABRA4 expression (Fig. 1). The Sp1 binding site is recognized by the Sp/KLF family members of transcription factors (33–35). Similar binding affinity to the classic Sp1 site has been observed in the closely related Sp1, Sp3, and Sp4 factors, as well as other subfamily members such as BTEB1, TIEG1, and TIEG2 (33–35, 40, 56). The ubiquitously expressed Sp1 and Sp3 and the relatively brain-specific Sp4 and BTEB1 are transcriptional activators that are able to stimulate transcription, possibly by recruiting TAFs, TFIID, and RNA polymerase to the initiation sites (40, 57).

Gel shift analyses of brain-derived nuclear extract suggest that specific protein-DNA complexes are formed at both Sp1 sites. The Sp1-A oligo forms three distinct complexes, one of which includes both Sp3 and Sp4, whereas the identity and function of the other complexes are yet to be identified. A single DNA-protein complex is formed at the Sp1-B site that contains both Sp3 and Sp4, with Sp3 as the dominant factor present in the complex. In contrast, in nuclear extract derived from the non-neuronal HeLa cell line, the bound complexes contained Sp1 and Sp3, with Sp1 as the dominant factor in the complex.

These observations suggest that the GABRA4 promoter may be differentially regulated in neurons and non-neuronal cells, contributing to the neuronal specificity of the promoter. Sp3 has previously been shown to act as a bi-functional transcriptional activator and repressor, depending on promoter context and acetylation state of the protein (59, 60). One possible explanation of the neural specificity of the GABRA4 promoter is that, in non-neuronal cells, the presence of Sp3 at the promoter acts to suppress transcription, whereas in neurons Sp3 interacts with Sp4 to activate transcription. How the Sp/KLF factors regulate neural-specific gene expression is an interesting question and needs to be investigated further.

Using 5'-RACE and primer extension assays, we have mapped the transcription initiation sites of GABRA4 to the region between –184 to –113 bp relative to the coding ATG (Figs. 1 and 2). Transcripts with different start sites for GABRA4 are reported in the GenBank^TM data base, including AK043806 [GenBank] that originated from –114 bp and AK013727 [GenBank] that originated from –499 bp. Our deletion analyses suggest that the initiation region we have identified is able to support high levels of luciferase reporter expression in cultured cortical neurons, whereas the putative initiation site at –499 bp alone is not able to give rise to reporter gene expression to a comparable level (Fig. 4, C and D). These results suggest that the initiation region reported here includes the major start sites for the GABRA4 gene and is functionally important for high levels of promoter activity.

As has been described for many neuron-specific genes, including genes encoding members of the GABA_A-R family and other ligand-gated ion channels, the GABRA4 promoter lacks a TATA box or other classic elements associated with the start of transcription and contain multiple start sites (37, 48, 49, 52, 61). Multiple start sites can give rise to mRNAs that differ in their 5'-untranslated region, which may potentially influence their post-transcriptional regulation (37, 58, 61). For example, GluR2 is transcribed from a region –481 to –286 bp 5' to the coding ATG (52). Because of the presence of a 32–34-bp imperfect GU repeat in the long GluR2 transcripts, the translation efficiency of these mRNA species is greatly inhibited possibly as the result of formation of a highly stable secondary structure within the repeats (58). In the case of GABRA4, several groups have shown that the GABRA4 transcript is present in all regions of the thalamus (23) but the 4 protein is only expressed in the dorsal lateral geniculate region (14). These observations suggest that post-transcriptional regulation of GABRA4 may indeed contribute to the diversity of GABRA4 protein expression. Although there are two 16-nucleotide repeats spanning the initiation region of GABRA4, we failed to find a stable stem-loop structures formed around this region. In addition, deletion of these repeats had no effect on promoter activity in vitro. However, we have not assessed the effects of different GABRA4 transcripts on translation efficiency of the gene. Whether the diversity of GABRA4 transcripts is critical for post-transcriptional regulation of GABRA4 expression remains to be further investigated.

In this study, we have analyzed the proximal promoter region of GABRA4 as the first step in unraveling the molecular mechanisms involved in the plasticity of GABRA4 in a number of physiological and pathophysiological conditions. The major conclusions of this report are that GABRA4 is transcribed from different locations within a region –184 to –113 bp 5' to the coding ATG, and that the transcription factors Sp3 and Sp4 are critical in regulating the activity of the GABRA4 promoter in neurons.

	FOOTNOTES

 
^* This work was supported in part by National Institute on Alcohol Abuse and Alcoholism Grant AA13646 (to N. L. H.) and a Charles Dana Foundation Postdoctoral Fellowship (to L. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by the Dept. of Anesthesiology at the Weill Cornell Medical College to the CV Starr laboratory. To whom correspondence should be addressed: 525 East 68th St., A-1040, New York, NY 10021. Tel.: 212-746-1150; Fax: 212-746-4879; E-mail: neh2001{at}med.cornell.edu.

¹ The abbreviations used are: GABA_A-R, -aminobutyric acid type A receptor; RACE, 5'-rapid amplification of cDNA ends; RT, reverse transcriptase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PRF-MEM, phenol red-free minimum essential medium; CMV, cytomegalovirus; NRSE, neuron-restrictive silencer element.

	ACKNOWLEDGMENTS

 
We thank Zhigao Li, Adam Light, Jazmine Liu, and Dingyi Fu for their early efforts in the project and Johanna Dizon for careful reading of the manuscript. We are grateful to Professor G. Suske for providing the Sp1, Sp3, and Sp4 expression vectors.

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