Trans-activators Regulating Neuronal Glucose Transporter Isoform-3 Gene Expression in Mammalian Neurons
2004; Elsevier BV; Volume: 279; Issue: 25 Linguagem: Inglês
10.1074/jbc.m402735200
ISSN1083-351X
AutoresAugustine Rajakumar, Shanthie Thamotharan, Nupur Raychaudhuri, Ram K. Menon, Sherin U. Devaskar,
Tópico(s)RNA Research and Splicing
ResumoThe murine facilitative glucose transporter isoform 3 is developmentally regulated and is predominantly expressed in neurons. By employing the primer extension assay, the transcription start site of the murine Glut 3 gene in the brain was localized to -305 bp 5′ to the ATG translation start codon. Transient transfection assays in N2A neuroblasts using murine GLUT3-luciferase reporter constructs mapped enhancer activities to two regions located at -203 to -177 and -104 to -29 bp flanking a previously described repressor element (-137 to -130 bp). Dephosphorylated Sp1 and Sp3 proteins from the 1- and 21-day-old mouse brain nuclear extracts bound the repressor elements, whereas both dephosphorylated and phosphorylated cAMP-response element-binding protein (CREB) in N2A, 1- and 21-day-old mouse brain nuclear extracts bound the 5′-enhancer cis-elements (-187 to -180 bp) of the Glut 3 gene, and the Y box protein MSY-1 bound the sense strand of the -83- to -69-bp region. Sp3, CREB, and MSY-1 binding to the GLUT 3 DNA was confirmed by the chromatin immunoprecipitation assay, whereas CREB and MSY-1 interaction was detected by the co-immunoprecipitation assay. Furthermore, small interference RNA targeted at CREB in N2A cells decreased endogenous CREB concentrations, and CREB mediated GLUT 3 transcription. Thus, in the murine brain similar to the N2A cells, phosphorylated CREB and MSY-1 bound the Glut 3 gene trans-activating the expression in neurons, whereas Sp1/Sp3 bound the repressor elements. We speculate that phosphorylated CREB and Sp3 also interacted to bring about GLUT 3 expression in response to development/cell differentiation and neurotransmission. The murine facilitative glucose transporter isoform 3 is developmentally regulated and is predominantly expressed in neurons. By employing the primer extension assay, the transcription start site of the murine Glut 3 gene in the brain was localized to -305 bp 5′ to the ATG translation start codon. Transient transfection assays in N2A neuroblasts using murine GLUT3-luciferase reporter constructs mapped enhancer activities to two regions located at -203 to -177 and -104 to -29 bp flanking a previously described repressor element (-137 to -130 bp). Dephosphorylated Sp1 and Sp3 proteins from the 1- and 21-day-old mouse brain nuclear extracts bound the repressor elements, whereas both dephosphorylated and phosphorylated cAMP-response element-binding protein (CREB) in N2A, 1- and 21-day-old mouse brain nuclear extracts bound the 5′-enhancer cis-elements (-187 to -180 bp) of the Glut 3 gene, and the Y box protein MSY-1 bound the sense strand of the -83- to -69-bp region. Sp3, CREB, and MSY-1 binding to the GLUT 3 DNA was confirmed by the chromatin immunoprecipitation assay, whereas CREB and MSY-1 interaction was detected by the co-immunoprecipitation assay. Furthermore, small interference RNA targeted at CREB in N2A cells decreased endogenous CREB concentrations, and CREB mediated GLUT 3 transcription. Thus, in the murine brain similar to the N2A cells, phosphorylated CREB and MSY-1 bound the Glut 3 gene trans-activating the expression in neurons, whereas Sp1/Sp3 bound the repressor elements. We speculate that phosphorylated CREB and Sp3 also interacted to bring about GLUT 3 expression in response to development/cell differentiation and neurotransmission. Glucose, an essential substrate for brain oxidative metabolism, is transported across the blood-brain barrier and into neurons and glia by a family of structurally related membrane-spanning glycoproteins termed the facilitative glucose transporters (1Bell G.I. Burant C.F. Takeda J. Gould G.W. J. Biol. Chem. 1993; 268: 19161-19184Abstract Full Text PDF PubMed Google Scholar, 2Devaskar S.U. Mueckler M.M. Pediatr. Res. 1992; 31: 1-13Crossref PubMed Scopus (128) Google Scholar). Of the 14 major isoforms cloned to date (1Bell G.I. Burant C.F. Takeda J. Gould G.W. J. Biol. Chem. 1993; 268: 19161-19184Abstract Full Text PDF PubMed Google Scholar, 2Devaskar S.U. Mueckler M.M. Pediatr. Res. 1992; 31: 1-13Crossref PubMed Scopus (128) Google Scholar, 3Joost H.G. Bell G.I. Best J.D. Birnbaum M.J. Charron M.J. Chen Y.T. Doege H. James D.E. Lodish H.F. Moley K.H. Moley J.F. Mueckler M. Rogers S. Schurmann A. Seino S. Thorens B. Am. J. Physiol. 2002; 282: E974-E976Crossref PubMed Scopus (346) Google Scholar, 4Carayannopoulos M.O. Chi M.M. Cui Y. Pingsterhaus J.M. McKnight R.A. Mueckler M. Devaskar S.U. Moley K.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7313-7318Crossref PubMed Scopus (271) Google Scholar, 5Phay J.E. Hussain H.B. Moley J.F. Genomics. 2000; 66: 217-220Crossref PubMed Scopus (177) Google Scholar, 6McVie-Wylie A.J. Lamson D.R. Chen Y.T. Genomics. 2001; 72: 113-117Crossref PubMed Scopus (128) Google Scholar, 7Wu X. Li W. Sharma V. Godzik A. Freeze H.H. 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Hoshima T. Biochem. J. 1994; 300: 125-131Crossref PubMed Scopus (60) Google Scholar). We and others have reported previously that although the spatial distribution of GLUT 3 in brain is not age-dependent (12Fields H.M. Rinaman L. Devaskar S.U. Brain Res. 1999; 846: 260-264Crossref PubMed Scopus (20) Google Scholar), a temporal distribution exists with low amounts noted during the embryonic/fetal and early postnatal stages and peak amounts at day 14-21 (13Khan J.Y. Rajakumar R.A. McKnight R.A. Devaskar U.P. Devaskar S.U. Am. J. Physiol. 1999; 45: R892-R900Google Scholar), which coincides with the timing of synaptogenesis (14Sachs G.M. Jacobson M. Caviness Jr., V.S. J. Comp. Neurol. 1986; 246: 395-408Crossref PubMed Scopus (82) Google Scholar, 15Minelli A. Alonso-Nanclares L. Edwards R.H. DeFelipe J. Conti F. Neuroscience. 2003; 117: 337-346Crossref PubMed Scopus (76) Google Scholar, 16Stern E.A. Maravall M. Svoboda K. Neuron. 2001; 31: 305-315Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). In addition, GLUT 3 localization to the synaptic region and its vesicular trafficking, which involves SNAP-25 and syntaxin-1, proteins of the SNARE complex present in synaptic vesicles, supports a role for GLUT 3 in neurotransmission (17Greenlee M.H.W. Uemura E. Carpenter S.L. Doyle R.T. Buss J.E. J. Neurosci. Res. 2003; 73: 518-525Crossref PubMed Scopus (10) Google Scholar). Brain 2-deoxyglucose uptake serves as a surrogate marker for neuronal activity (18Chugani H.T. Phelps M.E. Science. 1986; 231: 840-843Crossref PubMed Scopus (540) Google Scholar); thus GLUT 3, which mediates this glucose uptake, must play a major role in fueling neurotransmission (19Maher F. Davies-Hill T.M. Simpson I.A. Biochem. J. 1996; 315: 827-831Crossref PubMed Scopus (112) Google Scholar). Depolarization of neurons in vitro by the presence of extracellular K+ ions or N-methyl-D-aspartate led to an increase in GLUT 3 concentrations, providing credence to this concept (20Maher F. Simpson I.A. Mol. Cell Neurosci. 1994; 5: 369-375Crossref PubMed Scopus (35) Google Scholar). Both the processes of neuro-development and depolarization of cultured neurons cause a pre-translational increase in neuronal GLUT 3 expression (13Khan J.Y. Rajakumar R.A. McKnight R.A. Devaskar U.P. Devaskar S.U. Am. J. Physiol. 1999; 45: R892-R900Google Scholar, 20Maher F. Simpson I.A. Mol. Cell Neurosci. 1994; 5: 369-375Crossref PubMed Scopus (35) Google Scholar). Furthermore, conditions of substrate deficiency such as chronic hypoglycemia or hypoxic ischemia, which depolarize neurons, also pre-translationally increase neuronal GLUT 3 concentrations (21Lee D.E. Chung M.Y. Lee J.U. Kang D.G. Paek Y.W. Diabetes Res. Clin. Pract. 2000; 47: 15-23Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 22Vannucci S.J. Reinhart R. Maher F. Bondy C.A. Lee W.H. Vannucci R.C. Simpson I.A. Brain Res. Dev. Brain Res. 1998; 107: 255-264Crossref PubMed Scopus (108) Google Scholar). By determining transcriptional mechanism(s) underlying the pre-translational increase in neuronal GLUT 3 expression, we had previously characterized the murine Glut 3 promoter. We demonstrated that the murine Glut 3 promoter activity resides in the -203- to +237-bp region of the gene, with reference to the transcriptional start site (23Rajakumar R.A. Thamotharan S. Menon R.K. Devaskar S.U. J. Biol. Chem. 1998; 273: 27474-27483Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Additionally, we demonstrated nuclear factors Sp1 to repress and Sp3 to activate Glut 3 gene transcription in cultured murine neuroblasts (23Rajakumar R.A. Thamotharan S. Menon R.K. Devaskar S.U. J. Biol. Chem. 1998; 273: 27474-27483Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). In the present study, we extended these observations by determining whether Sp1 and Sp3 present in postnatal murine brain nuclear extracts could bind to the identified Glut 3 promoter cis-elements. We have also confirmed that by in vivo Sp3, and we established that in vitro and in vivo phosphorylated cyclic AMP-regulatory element-binding (pCREB) 1The abbreviations used are: pCREB, phosphorylated cAMP-regulatory element-binding protein; ChiP, chromatin immunoprecipitation; Si, small interference RNA; CREB, cAMP-response element-binding protein; PBS, phosphate-buffered saline; EMSA, electromobility shift assay; oligo, oligonucleotide; PIPES, 1,4-piperazinediethanesulfonic acid; CBP, CREB-binding protein; AP-1, activator protein-1. protein and the mouse Y box-binding protein-1 (MSY-1) bind the Glut 3 promoter region and activate GLUT 3 expression in neurons. We speculate that although Sp1/Sp3 along with MSY-1 may regulate the transcriptional activation of GLUT 3 during neuro-development, trans-activation of GLUT 3 expression by pCREB may mediate the processes of neuronal synaptic activity and neuro-protection under conditions of substrate deficiency. Oligonucleotides—Synthetic oligonucleotides (Invitrogen) were used in these experiments (nucleotides altered in mutant oligonucleotides are indicated by boldface type). Double-stranded oligonucleotides were generated by annealing the synthetic oligonucleotides with respective complementary sequences. Animals—Balb/c mice were purchased from The Jackson Laboratories (Bar Harbor, ME) and housed in cages. The protocol for the care and use of animals was approved by the Animal Care and Use Committee of the Magee Womens Research Institute in accordance with the guidelines set by the National Institutes of Health. Mice were allowed access to laboratory chow and water ad libitum and were maintained in 12-h light-dark cycles. Cells—N2A murine neuroblastoma cells (American Tissue Culture Collection, Manassas, VA) were grown at 37 °C with 95% air, 5% CO2 in poly-l-lysine-coated culture flasks and maintained in Dulbecco's modified Eagle's medium supplemented with 2 mm glutamine, penicillin (100 units/ml), streptomycin (100 units/ml), and 10% fetal bovine serum (23Rajakumar R.A. Thamotharan S. Menon R.K. Devaskar S.U. J. Biol. Chem. 1998; 273: 27474-27483Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). RNA Studies—Poly(A+)-enriched RNA was extracted as per the manufacturer's instructions using the miniribosep extraction kit (Collaborative Biomedical Products, Bedford, MA) from confluent cultured N2A cells (∼1 × 108 cells) or whole brains from the 1- and 21-day-old mice. The extracted RNA was subjected to Northern blot analysis as described previously (23Rajakumar R.A. Thamotharan S. Menon R.K. Devaskar S.U. J. Biol. Chem. 1998; 273: 27474-27483Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). A 32P-labeled 1.5-kb XhoI-XbaI fragment of the murine GLUT 3 cDNA served as the probe (24Nagamatsu S. Kornhauser J.M. Burant C.F. Seino S. Mayo K.E. Bell G.I. J. Biol. Chem. 1992; 267: 467-472Abstract Full Text PDF PubMed Google Scholar). Inter-lane loading variability was standardized by re-hybridization of the stripped filters with a 32P-labeled rat 18 S rRNA probe (25Chan Y.L. Gutell R. Noller H.F. Wool I.G. J. Biol. Chem. 1984; 259: 224-230Abstract Full Text PDF PubMed Google Scholar). Protein Studies—Thirty to fifty μg of either cellular or brain homogenate or extracted nuclear protein (26Wildeman A.G. Sassone-Corsi P. Grundstrom T. Zenke M. Chambon P. EMBO J. 1984; 3: 3129-3133Crossref PubMed Scopus (99) Google Scholar) were solubilized in 50 mm Tris, pH 6.8, containing 2% SDS and the protein concentration determined by the Bio-Rad dye-binding assay (27Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (222135) Google Scholar). Western blot analysis was carried out as described previously (23Rajakumar R.A. Thamotharan S. Menon R.K. Devaskar S.U. J. Biol. Chem. 1998; 273: 27474-27483Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The primary antibody consisted of an affinity-purified rabbit anti-mouse GLUT 3 IgG that was generated against a keyhole limpet-linked terminal 17 amino acids of the mouse GLUT 3 protein and the isoform specificity previously characterized by us (23Rajakumar R.A. Thamotharan S. Menon R.K. Devaskar S.U. J. Biol. Chem. 1998; 273: 27474-27483Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The primary anti-mouse GLUT 3 antibody was used at a 1:500 dilution, and the incubation with filters containing the transferred proteins was carried out at room temperature for 16 h. To detect nuclear proteins, the rabbit anti-synthetic human Sp1 peptide (436-454 amino acid region), anti-synthetic human Sp3 peptide (676-695 amino acids at the C-terminal region) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-rat CREB (Upstate Biotechnology, Inc., Lake Placid, NY), and anti-mouse c-Jun (247-263 amino acids) (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies were employed. Antibodies raised in rabbit against the MSY-1 peptide sequence NH2-DPPAENSSAPEAEQGGAECOOH (28Schwartzbauer G. Yu J.H. Cheng H. Menon R.K. J. Biol. Chem. 1998; 273: 24760-24769Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) were affinity-purified by using an Amino-link Immobilization kit (Pierce). In certain instances 125I-labeled goat anti-rabbit secondary antibody (50,000 cpm/sample, PerkinElmer Life Sciences) was used to detect the primary antigen-antibody complex. Autoradiography of the filters was carried out for optimal lengths of time to maintain linearity of the signal. Detection of protein bands for MSY-1 was carried out by subjecting the immunoblots to the chemiluminescent method (Amersham Biosciences) of detection. Primer Extension Assay—Primer extension was carried out as described previously (23Rajakumar R.A. Thamotharan S. Menon R.K. Devaskar S.U. J. Biol. Chem. 1998; 273: 27474-27483Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Briefly, an antisense oligonucleotide (Cruachem Inc., Dulles, VA) complementary to the +299 to +320 bp of the murine GLUT 3 mRNA (CTTCGTTGTCCCCATGGTCCCA) was end-labeled with [γ-32P]ATP (DuPont). ∼5 × 106 cpm (50 fmol) of the labeled oligonucleotide and 5 μg of poly(A+) mRNA were mixed in 1 μl of the hybridization buffer (80% formamide, 0.4 m NaCl, 40 mm PIPES, pH 6.8, and 1 mm EDTA) and incubated for 10 min at 95 °C. Hybridization between the mRNA and labeled oligonucleotide was accomplished for 16 h at 42 °C. Reverse transcription was initiated by adding 30 units of avian myeloblastosis virus-reverse transcriptase (Promega, Madison, WI) to the mRNA/oligonucleotide mixture in 20 μl of a mixture consisting of 50 mm Tris, pH 8.3, 6 mm MgCl2, 40 mm KCl, 10 units of RNasin, 0.625 mm dNTPs, and the reaction was carried out at 42 °C for 1 h. The primer-extended products were purified using Jetsorb (Genomed Inc., Research Triangle Park, NC) and separated on 8% polyacrylamide gels. RNase Protection Assay—A 341-bp fragment spanning -203 to +237 bp of the mouse Glut 3 gene was amplified by PCR and cloned into pGEM3z(f). 32P-Labeled antisense RNA probe was synthesized by the Sp6 polymerase using a riboprobe kit (Promega, Madison, WI). About 50,000 cpm (∼0.5 fmol) of gel-purified riboprobe was hybridized overnight at 45 °C with 5 μg of mRNA from the 21-day-old mouse brain in a hybridization buffer containing 80% formamide, 100 mm sodium citrate, pH 6.4, 300 mm sodium acetate, pH 6.4, and 1 mm EDTA. The hybrid was digested with 200 μl of a 1:50 dilution of RNase A (250 units/ml) and RNase T1 (10,000 units/ml) for 30 min at 37 °C prior to analysis on a 5% polyacrylamide gel. Transient Transfection and Reporter Expression Assays—A ∼1.8-kb fragment of the mouse GLUT 3 spanning the -1553- to +237-bp region was amplified by PCR and cloned into an enhancerless and promoterless firefly luciferase reporter gene containing vector (pGL2-basic: Promega, Madison, WI). Subsequently, serial 5′-deletional mouse GLUT 3-Luc fusion gene constructs were created by using a PCR-based strategy employing primers listed in Table I. The sequence and orientation of the individual clones were confirmed by direct DNA sequencing. The sequence information was managed using the MacVector5.0 sequence analysis program (Oxford Molecular Group, Campbell, CA).Table IPrimers used to make the 5′-deletional constructsDNA constructSequence informationNucleotide orientationbp−15535′-CCT ACG TGT ATG TCT GSense−8575′-CCT AGG CCT CAG TGT CACSense−5735′-CCT AGA GCC GCG GCA AGT GSense−3725′-CTG TGT ACT TCA CTT GAT CCTSense−2035′-CAA CTA AAA GCA GCA CTG ASense−1775′-CTG CGT TTA GAC TGA TTCSense−1045′-TCT GTG AAA CAA AAA GGC TGSense−295′-CAC GAG GAG GAT GTG GTA AAA AGSense+585′-GAA GAT CAG ATA AGA GGT CACSense+1585′-AGG CTG GGC TTC GGG CTGSense+3055′-GGT CCC AAC CTA GTT CTC AAG TCSense Open table in a new tab Transient transfection of cultured cells was carried out using Lipofectin (Invitrogen) according to the manufacturer's instructions. Briefly, 5 μg of the pGL2 fusion constructs were incubated at room temperature for 30 min with Lipofectin (25 μl) and 200 μl of serum-free Dulbecco's modified Eagle's medium. After thorough washing with Dulbecco's modified Eagle's medium, the cells were exposed to this preincubated DNA-Lipofectin complex. pRL-Tk plasmid DNA (thymidine kinase promoter-driven Renilla luciferase, 0.5 μg (Promega, Madison, WI)) was cotransfected with each individual transfectant to standardize the results for transfection efficiency. The luciferase reporter activity was assessed by the dual luciferase assay (Promega, Madison, WI). Briefly, 36-48 h of post-transfection the cells were washed with PBS and lysed using 0.5 ml of passive lysis buffer (Promega, Madison, WI). The supernatant on centrifugation at 10,000 rpm for 10 min was stored at -70 °C until analysis. Twenty μl of this cellular extract was mixed with 100 μl of the luciferase assay buffer, and the firefly luciferase activity was measured as light output (15 s) in a Monolight 2010 luminometer (Analytical Luminescence, San Diego, CA) (23Rajakumar R.A. Thamotharan S. Menon R.K. Devaskar S.U. J. Biol. Chem. 1998; 273: 27474-27483Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Subsequently the Renilla luciferase activity was estimated after the addition of 100 μl of the Stop and Glo reagent, and the light output (15 s) was measured separately. The Renilla-driven luciferase activity was used to standardize the Glut 3 promoter-driven firefly luciferase activity for transfection efficiency. The corrected Glut 3 promoter-driven luciferase activity was expressed as a percentage of the SV40 promoter-driven luciferase activity that served as the positive control in every transfection experiment. Small Interference (Si) RNA Transfection Experiments—SiRNA was constructed to target the mouse CREB (Dharmacom Inc., Dallas, TX) sequence between the +810- to +830-bp coding region by using the following oligonucleotides: sense, 5′-GAGAGAGGUCCGUCUAAUGUU-3′, and antisense, 5′-CAUUAGACGGACCUCUCUCUU-3′ (29Yang E.J. Yoon J.H. Chung K.C. J. Biol. Chem. 2004; 279: 1827-1837Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Complementary oligonucleotides were converted to a 2′-hydroxyl annealed and desalted duplex strands with a 9-base spacer, thereby creating a short hairpin RNA that was driven by the RNA polymerase III promoter followed by a (T)5 RNA poly III transcriptional stop signal. Co-transfections into N2A cells were performed by using the -203-bp GLUT3-luciferase DNA construct (2 μg) and the constructed SiRNA targeted at CREB (100 nm) in 6-well plates using Trans-It-LTI and Trans-IT-TKO (Mirus Corp., Madison, WI) as transfection reagents, respectively (29Yang E.J. Yoon J.H. Chung K.C. J. Biol. Chem. 2004; 279: 1827-1837Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 30Cheng J.C. Moore T.B. Sakamoto K.M. Mol. Genet. Metab. 2003; 80: 121-128Crossref PubMed Scopus (66) Google Scholar). PGL2 basic consisting of the firefly luciferase as the reporter in the absence of a promoter region was used as a negative control. To assess the transfection efficiency, 0.25 μg of pRL-TK plasmid DNA (thymidine kinase promoter-driven Renilla luciferase; Promega, Madison, WI) was co-transfected as well. Luciferase activity was measured in 10 μl of the N2A cellular extract (Zylux FB 15 tube luminometer, Fisher) at 48 h post-transfection by the dual luciferase assay system (Promega, Madison, WI). In addition, 30 μg of protein from SiRNA-transfected N2A cell lysates was subjected to SDS-PAGE, and the separated proteins were transferred onto nitrocellulose membranes. Each nitrocellulose membrane was washed twice for 10 min in distilled water. The membranes were then blocked by incubating with freshly prepared 5% bovine milk solution in PBS for 1 h at room temperature under constant agitation. The membranes were then washed with distilled H2O and incubated with 1:1000 dilution of the anti-CREB (Upstate Biotechnology, Inc.) antibody that was diluted in PBS containing 1% bovine milk solution. The incubation with primary antibody was conducted overnight with constant agitation at 4 °C, washed twice with distilled H2O, and incubated with the secondary antibody that was the anti-rabbit horseradish peroxidase-conjugated IgG (Sigma) diluted in a PBS, 1% milk solution for 1.5 h at room temperature with agitation. The nitrocellulose membranes were washed twice with diluted H2O, followed by a wash in PBS, 0.05% Tween 20 for 5 min, and rinsed twice in distilled H2O. Detection of protein bands for CREB was carried out by subjecting the immunoblots to the chemiluminescent method (Amersham Biosciences) of detection and then exposing them to x-ray film. Autoradiography of the filters was carried out for optimal lengths of time to maintain linearity of the signal (31Zovein A. Flowers-Ziegler J. Thamotharan S. Shin D. Sankar R. Nguyen K. Gambhir S. Devaskar S.U. Am. J. Physiol. 2004; 286: R273-R282Crossref PubMed Scopus (63) Google Scholar). Electromobility Shift Assay (EMSA)—Nuclear extracts from the N2A cells, 1- and 21-day-old mouse brains were prepared as described by Wildeman et al. (26Wildeman A.G. Sassone-Corsi P. Grundstrom T. Zenke M. Chambon P. EMBO J. 1984; 3: 3129-3133Crossref PubMed Scopus (99) Google Scholar). Briefly, brain tissue was obtained, or 5 × 108 cells were retrieved by a rubber policeman and suspended in 10 mm Hepes, pH 7.8, 10 mm KCl, 1.5 mm MgCl2, 0.5 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, and 25% glycerol to extract the nuclear proteins. The extracted nuclear proteins were collected as the supernatant after centrifugation at 10,000 rpm for 30 min and precipitated with solid (NH4)2SO4 by centrifugation at 15,000 rpm for 15 min, and the supernatant was stored in aliquots at -70 °C. The concentration of the solubilized nuclear protein was measured by the method of Bradford (27Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (222135) Google Scholar), and the concentration was adjusted to 1 mg/ml. Synthesized double-stranded oligonucleotides using the primers listed in Table II were end-labeled with [γ-32P]ATP and T4 polynucleotide kinase. Approximately 6 fmol of the labeled DNA oligoprobe was added to 5 μg of nuclear extract in a final volume of 20 μl containing 1 μg of poly(dl-dC), 10 mm Tris-HCl, pH 7.5, 50 mm NaCl, 0.5 mm EDTA, 1 mm MgCl2, 4% glycerol, 1 mm dithiothreitol and incubated for 15 min at room temperature. Subsequently, the DNA-protein complexes were separated from the unbound DNA by electrophoresis through a 5% non-denaturing polyacrylamide gel in a 90 mm Tris borate, 2 mm EDTA buffer (23Rajakumar R.A. Thamotharan S. Menon R.K. Devaskar S.U. J. Biol. Chem. 1998; 273: 27474-27483Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The gels were dried and subjected to autoradiography in the presence of intensifying screens (PerkinElmer Life Sciences) at -80 °C. Competition experiments included the addition of 10-1000-fold excess of unlabeled DNA oligonucleotides, whereas supershift analysis included the addition of 2 μg of the respective antibody to the reaction mix for 15 min.Table IIPrimers for gel-shift analysisPrimers used for gel-shift analysis− 195 to − 1735′ -AGCAGCACTGACTCTACTCTGCG-sense− 173 to − 1955′ -CGCAGAGTAGAGTCAGTGCTGCT-antisense− 144 to − 1195′ -CAAATTTGCCCGCCCTCCAAACTTG-sense− 119 to − 1445′ -CAAGTTTGGAGGGCGGGCAAATTTG-antisenseMutated primers used for gel-shift analysis− 195 to − 1735′ -AGCAGCACTGAAAAAACTCTGCG-sense− 173 to − 1955′ -CGCAGAGTTTTTTCAGTGCTGCT-antisense− 149 to − 1235′ -GCCTTCAAATTTGCTTGCTTTCCAAAC-sense− 123 to − 1495′ -GTTTGGAAAGCAAGCAAATTTGAAGGC-antisense Open table in a new tab Oligo-affinity Purification of MSY-1—In order to determine more precisely where MSY-1 binds within the -89 to -66 region on the Glut 3 promoter, MSY-1 was isolated from N2A nuclear extracts by using biotinylated oligonucleotides linked to streptavidin paramagnetic beads (32Kelm Jr., R.J. Cogan J.G. Elder P.K. Strauch A.R. Getz M.J. J. Biol. Chem. 1999; 274: 14238-14245Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Oligonucleotides corresponding to portions of the mouse GLUT 3 sense and antisense sequence between -90 and -61 bp were biotinylated at either the 5′ or 3′ ends. Oligo 177, 5′-GAAAAGGGGAAGGAA-biotin-3′, corresponded to the sense strand from -75 to -61 bp. Oligo 178, 5′-biotin-TTCCTTCCCCTTTTC-3′ corresponded to the antisense strand between -61 and -75 bp. Oligo 179, 5′-biotin-AGGCTGTCGGCTCTT-3′, corresponded to the sense strand between -90 and -76 bp, and oligo 180, 5′-AAGAGCCGACAGCCT-biotin-3′, corresponded to the antisense strand between -76 and -90 bp. In separate 500-μl reactions, 100 μg of N2A nuclear protein was mixed with 100 pmol of biotinylated oligonucleotide plus 50 μg of poly(dA) in EMSA binding buffer and incubated for 20 min at room temperature. The protein bound to the biotinylated oligo was then purified by using streptavidin-conjugated paramagnetic beads according to the manufacturer's instructions (Dynal Inc., Lake Success, NY). The bound protein was eluted by resuspending the beads in 40 μl of Laemmli buffer, incubating for 3 min at 65 °C, then adding 2 μl of 14.3 m β-mercaptoethanol, and heating for 5 min at 90 °C. The eluted protein was then analyzed by Western blot analysis by probing with antibodies against MSY-1. Identification of MSY-1 as a Glut 3 Promoter Binding Protein by Shift-Western Analysis—Because the MSY-1 antibody available for the current studies did not supershift MSY-1 protein when complexed with single-stranded DNA (data not shown), the presence of MSY-1 in the protein-DNA complex was ascertained by the shift-Western technique (28Schwartzbauer G. Yu J.H. Cheng H. Menon R.K. J. Biol. Chem. 1998; 273: 24760-24769Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Briefly, following a preparative electromobility shift analysis, the location of the DNA-protein complex was determined by using a PhosphorImager. The portion of the gel containing the shifted band was excised and transferred to nitrocellulose membrane (Bio-Rad) and subjected to Western blot analysis using the antibody raised against MSY-1 (28Schwartzbauer G. Yu J.H. Cheng H. Menon R.K. J. Biol. Chem. 1998; 273: 24760-24769Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Confirmation of the Sp3, CREB, and MSY-1 DNA Bindability by the Chromatin Immunoprecipitation Assay—Chromatin immunoprecipitation (ChIP) assay was performed as described by Boyd and Farnham (33Boyd K.E. Farnham P.J. Mol. Cell. Biol. 1997; 17: 2529-2537Crossref PubMed Scopus (141) Google Scholar). N2A murine neuroblastoma cells (∼2 × 107 in a 150-mm culture dish) were fixed with 1% formaldehyde for 15 min at room temperature. The cell pellet was resuspended in cell lysis buffer (5 mm Pipes (KOH), pH 8.0, 8.5 mm KCl, 0.5% Nonidet P-40) containing protease inhibitors and homogenized with a type B Dounce homogenizer. The nuclei were lysed in the nuclear lysis buffer (50 mm Tr
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