Characterization of the Rat GRIK5 Kainate Receptor Subunit Gene Promoter and Its Intragenic Regions Involved in Neural Cell Specificity
2001; Elsevier BV; Volume: 276; Issue: 45 Linguagem: Inglês
10.1074/jbc.m101895200
ISSN1083-351X
AutoresLi‐Jin Chew, Xiaoqing Yuan, Steven E. Scherer, Lixin Qie, Fei Huang, William Hayes, Vittorio Gallo,
Tópico(s)Photochromic and Fluorescence Chemistry
ResumoThe GRIK5 (glutamatereceptor ionotropickainate-5) gene encodes the kainate-preferring glutamate receptor subunit KA2. The GRIK5 promoter is TATA-less and GC-rich, with multiple consensus initiator sequences. Transgenic mouse lines carrying 4 kilobases of the GRIK5 5′-flanking sequence showed lacZreporter expression predominantly in the nervous system. Reporter assays in central glial (CG-4) and non-neural cells indicated that a 1200-base pair (bp) 5′-flanking region could sustain neural cell-specific promoter activity. Transcriptional activity was associated with the formation of a transcription factor IID-containing complex on an initiator sequence located 1100 bp upstream of the first intron. In transfection studies, deletion of exonic sequences downstream of the promoter resulted in reporter gene activity that was no longer neural cell-specific. When placed downstream of the GRIK5 promoter, a 77-bp sequence from the deleted fragment completely silenced reporter expression in NIH3T3 fibroblasts while attenuating activity in CG-4 cells. Analysis of the 77-bp sequence revealed a functional SP1-binding site and a sequence resembling a neuron-restrictive silencer element. The latter sequence, however, did not display cell-specific binding of REST-like proteins. Our studies thus provide evidence for intragenic control of GRIK5promoter activity and suggest that elements contributing to tissue-specific expression are contained within the first exon. The GRIK5 (glutamatereceptor ionotropickainate-5) gene encodes the kainate-preferring glutamate receptor subunit KA2. The GRIK5 promoter is TATA-less and GC-rich, with multiple consensus initiator sequences. Transgenic mouse lines carrying 4 kilobases of the GRIK5 5′-flanking sequence showed lacZreporter expression predominantly in the nervous system. Reporter assays in central glial (CG-4) and non-neural cells indicated that a 1200-base pair (bp) 5′-flanking region could sustain neural cell-specific promoter activity. Transcriptional activity was associated with the formation of a transcription factor IID-containing complex on an initiator sequence located 1100 bp upstream of the first intron. In transfection studies, deletion of exonic sequences downstream of the promoter resulted in reporter gene activity that was no longer neural cell-specific. When placed downstream of the GRIK5 promoter, a 77-bp sequence from the deleted fragment completely silenced reporter expression in NIH3T3 fibroblasts while attenuating activity in CG-4 cells. Analysis of the 77-bp sequence revealed a functional SP1-binding site and a sequence resembling a neuron-restrictive silencer element. The latter sequence, however, did not display cell-specific binding of REST-like proteins. Our studies thus provide evidence for intragenic control of GRIK5promoter activity and suggest that elements contributing to tissue-specific expression are contained within the first exon. glutamate receptor embryonic day initiator kilobase(s) chloramphenicol acetyltransferase base pair(s) kainate cell-specific enhancer transcription factor 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside neuron-restrictive silencer element analysis of variance protected least significant difference Three structurally related mammalian glutamate receptor (GluR)1 gene families encode N-methyl-d-aspartate, α-amino-3-hydroxymethylisoxazole-4-propionic acid, and kainate receptor subunits (1Nakanishi S. Science. 1992; 258: 597-603Crossref PubMed Scopus (2303) Google Scholar, 2Hollman M. Heinemann S. Annu. Rev. Neurosci. 1994; 17: 31-108Crossref PubMed Scopus (3668) Google Scholar, 3Dingledine R. Borges K. Bowie D. Traynelis S.F. Pharmacol. Rev. 1999; 51: 7-61PubMed Google Scholar). 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The kainate receptor subunit KA2 is encoded by the GRIK5gene (30Szpirer C. Molne M. Antonacci R. Jenkins N.A. Finelli P. Sziperer J. Riviere M. Rocchi M. Gilbert D.J. Copeland N.G. Gallo V. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11849-11853Crossref PubMed Scopus (20) Google Scholar, 31Huang F. Gallo V. J. Biol. Chem. 1997; 272: 8618-8627Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). KA2 transcripts can be detected in the rat as early as embryonic day 10 (E10) in the neural tube (32Scherer S. Gallo V. J. Neurosci. Res. 1998; 52: 356-368Crossref PubMed Scopus (26) Google Scholar) and at E12 in the cortical plate (9Herb A. Burnashev N. Werner P. Sakmann B. Wisden W. Seeburg P.H. Neuron. 1992; 8: 775-785Abstract Full Text PDF PubMed Scopus (469) Google Scholar). These findings suggest that KA2 may play a developmental role prior to synapse formation (33Gallo V. Pende M. Scherer S. Molne M. Wright P. Neurochem. Res. 1995; 20: 549-560Crossref PubMed Scopus (25) Google Scholar). At birth, the GRIK5 gene is expressed throughout the central nervous system, although its level of expression varies considerably between different brain areas and distinct cell types (11Wisden W. Seeburg P.H. J. Neurosci. 1993; 13: 3582-3596Crossref PubMed Google Scholar, 12Bahn S. Volk B. Wisden W. J. Neurosci. 1994; 14: 5525-5547Crossref PubMed Google Scholar). Since KA2 associates with other kainate receptor subunits, it follows that different functional receptor subtypes can be formed as a result of a stringent qualitative and quantitative control of GRIK5expression (34Sahara Y. Noro N. Iida Y. Soma K. Nakamura Y. J. Neurosci. 1997; 17: 6611-6620Crossref PubMed Google Scholar). To gain an understanding of how kainate receptor subunit expression is regulated, we have previously cloned the GRIK5 gene and characterized its structure (31Huang F. Gallo V. J. Biol. Chem. 1997; 272: 8618-8627Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). We have also identified an intronic element of this gene, which displays functional features of a silencer (31Huang F. Gallo V. J. Biol. Chem. 1997; 272: 8618-8627Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). More recently, we described the binding of nuclear orphan receptor proteins to this sequence to down-regulate GRIK5transcription (35Chew L.-J. Huang F. Boutin J.-M. Gallo V. J. Biol. Chem. 1999; 274: 29366-29375Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). In this study, we analyzed the GRIK5 promoter and regulatory regions sufficient for tissue-specific transcription of the gene in both cultured cells and transgenic mice. We also found that GRIK5, like other glutamate receptor genes of the α-amino-3-hydroxymethylisoxazole-4-propionic acid and N-methyl-d-aspartate classes (36Myers S.J. Dingledine R. Borges K. Annu. Rev. Phrmacol. Toxicol. 1999; 39: 221-241Crossref PubMed Scopus (94) Google Scholar), possesses a TATA-less and initiator (Inr)-containing promoter region that is GC-rich. This promoter is regulated by elements located within its first exon, and its selective expression in neural cells may involve a mechanism of preferential repression in non-neural cells. Restriction enzymes and DNA-modifying enzymes were from New England Biolabs Inc. (Beverly, MA). Polyacrylamide gel electrophoresis-purified oligonucleotides were purchased from Life Technologies, Inc. Radionuclides were from PerkinElmer Life Sciences. Large-scale plasmid DNA preparations were carried out using a QIAGEN Plasmid Maxi kit. All animal procedures were in accordance with the National Institutes of Health Animal Welfare Guidelines. CG-4, HeLa, and NIH3T3 cells were cultured as previously described (31Huang F. Gallo V. J. Biol. Chem. 1997; 272: 8618-8627Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 37Louis J.C. Magal E. Muir D. Manthrope M. Varon S. J. Neurosci. Res. 1992; 31: 193-204Crossref PubMed Scopus (362) Google Scholar). Cortical astrocytes were prepared as previously described (38McCarthy K.D. De Vellis J. J. Cell Biol. 1980; 85: 890-902Crossref PubMed Scopus (3398) Google Scholar, 39Gallo V. Armstrong R.C. J. Neurosci. 1995; 15: 394-406Crossref PubMed Google Scholar). The isolation and characterization of GRIK5 genomic clones from a rat Sprague-Dawley genomic library (λDASH II; Stratagene, La Jolla, CA) have been described previously (31Huang F. Gallo V. J. Biol. Chem. 1997; 272: 8618-8627Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). The nucleotide sequence reported in this paper has been previously submitted to the GenBank™/EBI Data Bank with accession number U81010. The BamHI site in the 2-kb EcoRI-BamHI fragment defines the exon 1/intron 1 boundary (see Fig. 3 A). The GRIK5 2Kb-CAT construct was generated as previously described (31Huang F. Gallo V. J. Biol. Chem. 1997; 272: 8618-8627Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). GRIK5-CAT deletion constructs were generated by restriction enzyme digestion and religation of GRIK52Kb-CAT (see Fig. 2). Apa5-CAT was generated by subcloning an 800-bp ApaI fragment from 2Kb-CAT into pLITMUS39 (New England Biolabs Inc.) to form pLITApa5 and subsequently inserting into pCATBasic (Promega, Madison WI) which will be subsequently referred to as Basic-CAT. Apa5A-, Apa5B-, Eag-, Sph-Apa-, and 2KbΔEag-CAT constructs were derived by digestion and religation of Apa5-CAT. Deletion constructs of Apa5A-CAT such as Apa5AΔSP1(a)Inr(a), Apa5AΔInr(a)(b), and Apa5AΔInr(b) were made by site-directed mutagenesis to generate appropriate restriction enzyme sites at the positions of Inr(a) (+1) and/or Inr(b) (+130) (see Fig. 3 Aand Table I). Apa5A-KCSE-CAT was formed by ligating a double-stranded oligonucleotide between the XbaI and XbaI sites of Apa5-CAT from which the XbaI-XbaI fragment had previously been removed.Table IOligonucleotides used for gel mobility shift assays and mutagenesisNameUseNucleotide sequenceInr(a)Gel shiftTCCCTGCCTCCCTCCCTTCATCTCTCCCCACCAInr(b)Gel shiftCCACAACCACCACCCTACCCACTCCCCInr(c)Gel shiftATCCTCTCCTCCCACCTCATCTCTCCACCTCGTmAMutagenesisGCCCGCCCTCCTCCCTGCCTGgagccCgTcgACTCTCCCCACCACCACCmBMutagenesisCACAACCACCACCCTAgatAtcaaCCCACCCCCCCGCGC600InrGel shiftGCCCTCCCCCACTTCCCCCGATGSP1(a)Gel shiftTCCTCCTGCCCGCCCTCCTCCCTGCCTConsensus SP1Gel shiftATTCGATCGGGGCGGGGCGAGCAP2Gel shiftGATCGAACTGACCGCCCGCGGCCCGCK(u)Gel shiftCTTTTCTCCACCCAGTCCTCACCCCCAGTGCCCCCCCCConsensus NRSEGel shiftCGCGCTGTCCGCGGTGCTGAAK(d)Gel shiftGGCCGGCCCGGCTAGGGGAGGGGGCGGGGGCCCmK(d)Gel shiftGGCCGGCCCGGCTAGGGGAaaaaataaGGGCCC62Gel shiftTCCTTTCTGTGGCCTCTGACCTTTCCTOne strand of each complementary pair is shown. Mutated nucleotides are in lowercase boldface lettering. mA has been mutated to insert the restriction sites XhoI and SalI, and mB contains a mutation of Inr(b) to introduce an EcoRV site. Open table in a new tab One strand of each complementary pair is shown. Mutated nucleotides are in lowercase boldface lettering. mA has been mutated to insert the restriction sites XhoI and SalI, and mB contains a mutation of Inr(b) to introduce an EcoRV site. Transient transfections were performed in all cells in serum-free medium by lipofection (2 μl of LipofectAMINE/μg of DNA; Life Technologies, Inc.) as previously described (31Huang F. Gallo V. J. Biol. Chem. 1997; 272: 8618-8627Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Cells were plated in 100-mm dishes, transfected with 10 μg of plasmid DNA for each GRIK5 construct, and cotransfected with 2 μg of pPolIIplacF.gal (Grant MacGregor, Emory University) to normalize for transfection efficiency. Cells were harvested 40 h after transfection, and cell extracts were prepared in 0.25 mTris buffer (pH 8.0) and assayed for CAT activity after heat inactivation of endogenous deacetylase for 10 min at 60 °C. CAT assays were carried out using n-butyryl-CoA as specified by the manufacturer (Promega). CAT activity results were obtained by liquid scintillation counting of xylene extracts. β-Galactosidase assays were performed as described by Nielsen et al. (40Nielsen D.A. Chou J. Mackrell A.J. Casadaban M.J. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 5198-5202Crossref PubMed Scopus (145) Google Scholar). For all cell types, at least three independent transfection experiments were performed for each CAT construct. Statistical analysis was performed using Statview Version 5.0. Total RNA was prepared from tissues, CG-4 cells and astrocytes. Poly(A)+ RNAs were purified by an Oligotex kit (QIAGEN Inc.). RNA samples (2 μg/lane) were resolved by electrophoresis through a formaldehyde-containing 1% denaturing agarose gel, electrotransferred to Nytran membranes (Schleicher & Schüll), cross-linked to the membranes by UV irradiation (Stratalinker, Stratagene), and hybridized with a random-primed [α-32P]dCTP-labeled EcoRI-StuI KA2 cDNA fragment (595 bp). The specific activity of the probe was 108 cpm/μg of DNA. The blot was hybridized in 50% formamide at 42 °C and washed at high stringency with 0.1× SSC at 60 °C. To generate the pSP72Apa5A template plasmid, a HindIII-XbaI fragment from the subclone pLITApa5 was inserted into the corresponding sites of pSP72 (Promega). The insert was subsequently shortened at the 3′-end by digestion with EagI and EcoRI, filled in with Klenow enzyme, and religated. The template pSP72Apa5B was constructed by inserting an XbaI-StuI fragment from pLITApa5 into the SmaI and EcoRV sites of pSP72. Both templates were linearized with HindIII, and antisense [α-32P]UTP-labeled RNA probes were generated by transcription with T7 RNA polymerase (Maxiscript kit, Ambion Inc., Austin, TX). Total RNA was isolated by a single-step method using RNAzol (41Chomzynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Google Scholar). Following gel purification of probes, RNase protection assays were performed with 30 μg of total RNA using the RPAIII kit from Ambion Inc. Hybridization was performed overnight at 55 °C. Reaction products were digested with a 1:100 dilution of RNase A/T1 mixture at 37 °C for 30 min, precipitated, and resolved on a 6% denaturing polyacrylamide gel alongside an M13 sequencing ladder. The ladder was generated with 35S-ATP using the −40 sequencing primer supplied with the U. S. Biochemical Corp. sequencing kit. Nuclear extracts from CG-4 cells were made and DNase I footprinting was performed as described by Huang and Gallo (31Huang F. Gallo V. J. Biol. Chem. 1997; 272: 8618-8627Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar) and Chew et al. (35Chew L.-J. Huang F. Boutin J.-M. Gallo V. J. Biol. Chem. 1999; 274: 29366-29375Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Footprinting probes were generated by polymerase chain reaction and are shown in Fig. 8. The sequences of gel-shift probes, competitors, and mutagenic primers are given in TableI. For gel-shift assays, the probes were end-labeled by T4 polynucleotide kinase with [γ-32P]ATP (PerkinElmer Life Sciences), and purified on Sephadex G-50 columns. The reactions were carried out in a total volume of 20 μl of binding buffer containing 25 mm HEPES (pH 7.5), 60 mm KCl, 10% glycerol, 5 mm MgCl2, 1 mm dithiothreitol, 1 mm EDTA, and 50 ng/μl poly(dI-dC) or poly(dG-dC) for Inr competitions. The binding reactions were incubated on ice for 30 min with 6–15 μg of nuclear proteins. For experiments with recombinant TFIID (Santa Cruz Biotechnology, Santa Cruz, CA), binding reactions containing up to 160 ng of protein were incubated at room temperature for 15 min in the absence of poly(dI-dC). Unlabeled competitors were mixed with probe prior to addition of binding protein(s). For antibody supershift assays, reactions were preincubated with specific antisera against SP1, SP3, SP4, AP2, TFIIB, and TFIID (Santa Cruz Biotechnology Inc.) and YY1 (Geneka, Montreal, Canada) at room temperature for 30 min prior to addition of the probe. After adding 2 μl of 0.1% bromphenol blue loading dye, samples were loaded onto a 5% TGE (1× TGE: 0.05 m Tris, 0.4m glycine, and 0.002 m EDTA)- or 5% Tris borate/EDTA-polyacrylamide gel and electrophoresed at 100 V at 4 °C. The gels were then dried and autoradiographed. The 4.3-kb BamHI GRIK5 fragment was cloned upstream of the bacterial lacZ coding region (31Huang F. Gallo V. J. Biol. Chem. 1997; 272: 8618-8627Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Following restriction digestion and purification from agarose gel, the DNA fragment containing 4.3-β-gal was purified by three consecutive ethanol precipitations, a chloroform extraction, and a final ethanol precipitation. The DNA was finally filtered through a Millex GV4 0.22-μm filter (Millipore Corp., Marlborough, MA) before microinjection into fertilized eggs and implantation into pseudo-pregnant foster mothers, after injected eggs developed into blastocysts (42Brinster R.L. Chen H.Y. Trumbauer M.E. Yagle M.K. Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 471-487Crossref Scopus (784) Google Scholar). Putative founders were screened for transgene integration by both polymerase chain reaction and Southern blotting of genomic tail DNA. Two stable transgenic lines were obtained for 4.3-β-gal. Expression of the bacterial lacZ gene in transgenic mice was detected using the luminescence assay (CLONTECH, Palo Alto, CA). Tissue homogenate was prepared by sonication in lysis solution containing 100 mmKHPO4, 0.2% Triton X-100, and 1 mmdithiothreitol. After centrifugation for 5 min, 25 μl of the supernatant was assayed by incubation with 200 μl of reaction buffer at room temperature for 60 min, and luminescence was measured in a luminometer according to the manufacturer's instructions (CLONTECH). Luminescence was normalized to protein concentration in each sample. Histochemical staining was performed as follows. Mice were anesthetized and perfused transcardially with 4% paraformaldehyde in 0.1m phosphate buffer (pH 7.4). Tissues were removed, immersed in the same fixative for 2 h at 4 °C, and stored in phosphate-buffered saline + 0.05% sodium azide. Tissue sections (100 μm) were cut using a Vibratome (Technical Products International Inc., St. Louis, MO). β-Galactosidase expression was analyzed by overnight incubation at 37 °C in X-gal staining solution (5 mm K3Fe(CN)6, 5 mmK4Fe(CN)6, 2 mm MgCl2, 0.02% Nonidet P-40, 0.01% sodium deoxycholate and 1 mg/ml X-gal). For whole embryo tissue sections, pregnant mice were perfused with phosphate-buffered saline, and the embryos (E15) were removed, immersion-fixed in 4% paraformaldehyde in 0.1 m phosphate buffer (pH 7.4), and stained as described above for brain tissue sections. After staining, frozen sections (100 μm) were then obtained on a freezing microtome (Microm International GmbH, Walldorf, Germany). In situhybridization protocols using 20- and 50-μm fresh frozen cryostat tissue sections were performed as described by Hayes and Loh (43Hayes W.P. Loh Y.P. Development. 1990; 110: 747-757PubMed Google Scholar) and Gallo et al. (44Gallo V. Upson L.M. Hayes W.P. Vyklicky L. Winters C.A. Buonanno A. J. Neurosci. 1992; 12: 1010-1023Crossref PubMed Google Scholar). The probe used was derived from the BamHI-SacI fragment (bp +452 to +1463) of KA2 cDNA (9Herb A. Burnashev N. Werner P. Sakmann B. Wisden W. Seeburg P.H. Neuron. 1992; 8: 775-785Abstract Full Text PDF PubMed Scopus (469) Google Scholar) inserted between the BamHI and SacI sites of pSP72. Antisense riboprobes were generated by in vitro transcription with T7 RNA polymerase following linearization of the template with HindIII. To delineate the genomic sequences responsible for tissue-specific GRIK5 expression in vivo, transgenic mouse lines were generated using 4.3 kb of 5′-flanking region. According to the numbering system used in this study (see Figs.2 and 3), this flanking region spans −3231 to +1100. Fusion genes were constructed that contained the promoter fragment and the bacterial lacZ coding region. Of 14 4.3-kb construct founders analyzed, two were found to carry the transgene by both polymerase chain reaction and Southern blotting of tail DNA (data not shown). These animals were bred to produce two independent lines, which displayed germ-line transmission of the transgene. In both lines, the pattern of β-galactosidase activity was clearly restricted to the central nervous system (Fig. 1 C) (data not shown), demonstrating that the 4.3-kb GRIK5 fragment containing the promoter is sufficient to direct expression to the rodent central nervous system. In addition, transgenic mice were also generated with the 2Kb-CAT and 4Kb-CAT constructs (31Huang F. Gallo V. J. Biol. Chem. 1997; 272: 8618-8627Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar) with very similar results (data not shown). Expression of the 4.3-β-gal transgene was analyzed in different areas of the mouse brain (Fig. 1, D–I) and compared with expression of endogenous KA2 transcripts (Fig. 1, A and B). The distribution of β-galactosidase activity detected in the embryonic brain at E16 was similar to that of the endogenous GRIK5 mRNAs (Fig. 1, A and D). Both transgenic lines showed similar transgene expression patterns. In the adult brain, high levels of β-galactosidase activity were found in areas with abundant GRIK5 mRNA (Fig. 1, compare B and E). The hippocampus (Fig. 1 H) and pyriform cortex (Fig. 1 I) were among the areas that displayed the highest levels of GRIK5 mRNA and β-galactosidase activity. A series of GRIK5 promoter constructs (31Huang F. Gallo V. J. Biol. Chem. 1997; 272: 8618-8627Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar) was assayed in cells of neural origin (central glia and CG-4 cells) and non-neural origin (NIH3T3 and HeLa cells). CG-4 cells are central glia with properties of oligodendrocyte progenitors and were shown to support high levels of GRIK5 promoter activity (31Huang F. Gallo V. J. Biol. Chem. 1997; 272: 8618-8627Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 35Chew L.-J. Huang F. Boutin J.-M. Gallo V. J. Biol. Chem. 1999; 274: 29366-29375Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). As reported previously (31Huang F. Gallo V. J. Biol. Chem. 1997; 272: 8618-8627Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar), CAT activity driven by 2 kb of the GRIK5promoter was significant in CG-4 cells and undetectable in NIH3T3 and HeLa cells (Fig. 2) (data not shown). Inclusion of an additional 2.3 kb of 5′-flanking sequence to generate a 4.3-kb construct did not significantly modify GRIK5 promoter activity (31Huang F. Gallo V. J. Biol. Chem. 1997; 272: 8618-8627Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar), indicating that 2 kb is sufficient for robust neural cell-specific GRIK5 expression. Deletion analysis of 2Kb-CAT indicated that there is a negative regulatory element(s) between the EcoRI (−931) and ApaI (−112) sites and that the core promoter(s) lies within the 1200-bp fragment (Fig. 2). (Reference numbers assigned to restriction enzyme sites are based on physical mapping of the transcription initiation cluster shown in Fig. 3, with +1 indicating the start of the first exon.) A large ApaI deletion in the 2Kb-CAT construct (2KbΔApa5-CAT) completely abolished transcriptional activity (Fig. 2), thus providing evidence that minimal promoter sequences lie between −112 and +723. Indeed, this ApaI fragment (Apa5-CAT) was found to maintain cell type-specific transcriptional activity in CG-4 cells (Fig. 2). Interestingly, removal of sequences 3′ of XbaI from Apa5-CAT (Apa5A-CAT) resulted in a significant 2–3-fold increase in CAT activity compared with 1200-CAT, whereas the 3′-segment (Apa5B-CAT) was found to be inactive, demonstrating a core promoter element within Apa5A-CAT (−112 to +331) and repressor activity of Apa5B-CAT. Further analysis of these upstream sequences showed that sequences between −112 and +223 (Eag-CAT) could function as a minimal promoter (Fig. 2). In support of this observation, further 5′-deletion to the EagI site (2KbΔEag-CAT) dramatically attenuated activity. Importantly, the emergence of significant CAT activity in non-neural cells, NIH3T3 (Fig. 2) and HeLa (data not shown), with Apa5A-CAT, Apa-Sph-CAT, and Eag-CAT supports the notion that transcription could initiate within the EagI fragment, and neural cell specificity was determined by sequences located downstream of SphI (+503) (Fig. 2). Since the GRIK5 promoter region contained neither TATAA nor CCAAT consens
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