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• W2085104568 abstract "Spinal muscular atrophy, an autosomal recessive disorder, is caused by loss of the SMN1 (survival motor neuron) gene while retaining the SMN2 gene. SMN1 produces a majority of full-length SMN transcript, whereas SMN2 generates mostly an isoform lacking exon 7. Here, we demonstrate a novel cAMP-response element, CRE-II, in the SMN promoter that interacts with the cAMP-response element-binding (CREB) family of proteins. In vitro DNase I protection analysis and in vivo genomic footprinting of the SMN promoter using the brain and liver nuclei from SMN2 transgenic mice revealed footprinting at the CRE-II site. Site-directed mutation of the CRE-II element caused a marked reduction in the SMN promoter activity revealed by transient transfection assay. Activation of the cAMP pathway by dibutyryl cAMP (0.5 mm) alone or in combination with forskolin (20 μm) caused a 2–5-fold increase in the SMN promoter activity but had no effect on the CRE-II mutated promoter. Electrophoretic mobility shift assay and a UV-induced DNA-protein cross-linking experiment confirmed that CREB1 binds specifically to the CRE-II site. Transient overexpression of CREB1 protein resulted in a 4-fold increase of the SMN promoter activity. Intraperitoneal injection of epinephrine in mice expressing two copies of the human SMN2 gene resulted in a 2-fold increase in full-length SMN transcript in the liver. Combined treatment with dibutyryl cAMP and forskolin significantly increased the level of both the full-length and exon 7-deleted SMN (exonΔ7SMN) transcript in primary hepatocytes from mice expressing two copies of human SMN2 gene. Similar treatments of type I spinal muscular atrophy mouse and human fibroblasts as well as HeLa cells resulted in an augmented level of SMN transcript. These findings suggest that the CRE-II site in SMN promoter positively regulates the expression of the SMN gene, and treatment with cAMP-elevating agents increases expression of both the full-length and exonΔ7SMN transcript. Spinal muscular atrophy, an autosomal recessive disorder, is caused by loss of the SMN1 (survival motor neuron) gene while retaining the SMN2 gene. SMN1 produces a majority of full-length SMN transcript, whereas SMN2 generates mostly an isoform lacking exon 7. Here, we demonstrate a novel cAMP-response element, CRE-II, in the SMN promoter that interacts with the cAMP-response element-binding (CREB) family of proteins. In vitro DNase I protection analysis and in vivo genomic footprinting of the SMN promoter using the brain and liver nuclei from SMN2 transgenic mice revealed footprinting at the CRE-II site. Site-directed mutation of the CRE-II element caused a marked reduction in the SMN promoter activity revealed by transient transfection assay. Activation of the cAMP pathway by dibutyryl cAMP (0.5 mm) alone or in combination with forskolin (20 μm) caused a 2–5-fold increase in the SMN promoter activity but had no effect on the CRE-II mutated promoter. Electrophoretic mobility shift assay and a UV-induced DNA-protein cross-linking experiment confirmed that CREB1 binds specifically to the CRE-II site. Transient overexpression of CREB1 protein resulted in a 4-fold increase of the SMN promoter activity. Intraperitoneal injection of epinephrine in mice expressing two copies of the human SMN2 gene resulted in a 2-fold increase in full-length SMN transcript in the liver. Combined treatment with dibutyryl cAMP and forskolin significantly increased the level of both the full-length and exon 7-deleted SMN (exonΔ7SMN) transcript in primary hepatocytes from mice expressing two copies of human SMN2 gene. Similar treatments of type I spinal muscular atrophy mouse and human fibroblasts as well as HeLa cells resulted in an augmented level of SMN transcript. These findings suggest that the CRE-II site in SMN promoter positively regulates the expression of the SMN gene, and treatment with cAMP-elevating agents increases expression of both the full-length and exonΔ7SMN transcript. Proximal spinal muscular atrophy (SMA) 1The abbreviations used are: SMA, spinal muscular atrophy; CRE, cAMP-response element; mCRE, mutant CRE; CREB, CRE-binding protein; CREM, CRE modulator; STAT1, signal transducer and activator of transcription 1; EHMN, embryonic hybrid motor neuron; Bt2cAMP, dibutyryl cyclic AMP; LM-PCR, ligation-mediated PCR; RT, reverse transcriptase; EMSA, electrophoretic mobility shift assay; NMDA, N-methyl-d-aspartate; ATF, activating transcription factor. 1The abbreviations used are: SMA, spinal muscular atrophy; CRE, cAMP-response element; mCRE, mutant CRE; CREB, CRE-binding protein; CREM, CRE modulator; STAT1, signal transducer and activator of transcription 1; EHMN, embryonic hybrid motor neuron; Bt2cAMP, dibutyryl cyclic AMP; LM-PCR, ligation-mediated PCR; RT, reverse transcriptase; EMSA, electrophoretic mobility shift assay; NMDA, N-methyl-d-aspartate; ATF, activating transcription factor. is a common autosomal recessive disorder characterized by loss of motor neurons in the spinal cord (1Crawford T.O. Pardo C.A. Neurobiol. Dis. 1996; 3: 97-110Crossref PubMed Scopus (398) Google Scholar). SMA occurs with a frequency of 1 in 10,000 live births with a carrier frequency of 1 in 50 (2McAndrew P.E. Parsons D.W. Simard L.R. Rochette C. Ray P.N. Mendell J.R. Prior T.W. Burghes A.H. Am. J. Hum. Genet. 1997; 60: 1411-1422Abstract Full Text PDF PubMed Scopus (448) Google Scholar, 3Pearn J. J. Med. Genet. 1978; 15: 414-417Crossref PubMed Scopus (24) Google Scholar) and is the leading genetic cause of infant mortality (4Roberts D.F. Chavez J. Court S.D. Arch. Dis. Child. 1970; 45: 33-38Crossref PubMed Scopus (137) Google Scholar). Based on age of onset and severity of the disease, SMA patients are often classified as type I, II, or III (5Munsat T.L. Davies K.E. Neuromuscul. Disord. 1992; 2: 423-428Abstract Full Text PDF PubMed Scopus (592) Google Scholar). All three forms of SMA are caused by loss or mutation of the telomeric survival motor neuron gene (SMN1), but the centromeric survival motor neuron gene (SMN2) is retained (6Bussaglia E. Clermont O. Tizzano E. Lefebvre S. Burglen L. Cruaud C. Urtizberea J.A. Colomer J. Munnich A. Baiget M. Melki J. Nat. Genet. 1995; 11: 335-337Crossref PubMed Scopus (206) Google Scholar, 7Lefebvre S. Burglen L. Reboullet S. Clermont O. Burlet P. Viollet L. Benichou B. Cruaud C. Millasseau P. Zeviani M. Cell. 1995; 80: 155-165Abstract Full Text PDF PubMed Scopus (2932) Google Scholar, 8Parsons D.W. McAndrew P.E. Monani U.R. Mendell J.R. Burghes A.H. Prior T.W. Hum. Mol. Genet. 1996; 5: 1727-1732Crossref PubMed Scopus (127) Google Scholar, 9Hahnen E. Forkert R. Marke C. Rudnik-Schoneborn S. Schonling J. Zerres K. Wirth B. Hum. Mol. Genet. 1995; 4: 1927-1933Crossref PubMed Scopus (271) Google Scholar, 10Talbot K. Ponting C.P. Theodosiou A.M. Rodrigues N.R. Surtees R. Mountford R. Davies K.E. Hum. Mol. Genet. 1997; 6: 497-500Crossref PubMed Scopus (146) Google Scholar). The SMN1 and SMN2 gene differ functionally by a single nucleotide change in exon 7 that does not alter an encoded amino acid but does alter the activity of an exon splice enhancer (11Lorson C.L. Hahnen E. Androphy E.J. Wirth B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6307-6311Crossref PubMed Scopus (1133) Google Scholar, 12Monani U.R. Lorson C.L. Parsons D.W. Prior T.W. Androphy E.J. Burghes A.H. McPherson J.D. Hum. Mol. Genet. 1999; 8: 1177-1183Crossref PubMed Scopus (713) Google Scholar, 13Cartegni L. Krainer A.R. Nat. Genet. 2002; 30: 377-384Crossref PubMed Scopus (570) Google Scholar). Thus, SMN1 produces a majority of full-length SMN transcript, whereas SMN2 generates mostly an isoform lacking exon 7. The protein product of the Δ7 transcript is thought to be unstable and rapidly degraded (14Lorson C.L. Androphy E.J. Hum. Mol. Genet. 2000; 9: 259-265Crossref PubMed Google Scholar, 15Le T.T. Coovert D.D. Monani U.R. Morris G.E. Burghes A.H. Neurogenetics. 2000; 3: 7-16Crossref PubMed Scopus (32) Google Scholar). SMA patients who lack SMN1 but carry varying copies of SMN2 do not produce sufficient SMN for motor neuron survival. There is a tight correlation between clinical severity of SMA, SMN2 copy number, and the SMN protein level (2McAndrew P.E. Parsons D.W. Simard L.R. Rochette C. Ray P.N. Mendell J.R. Prior T.W. Burghes A.H. Am. J. Hum. Genet. 1997; 60: 1411-1422Abstract Full Text PDF PubMed Scopus (448) Google Scholar, 16Coovert D.D. Le T.T. McAndrew P.E. Strasswimmer J. Crawford T.O. Mendell J.R. Coulson S.E. Androphy E.J. Prior T.W. Burghes A.H. Hum. Mol. Genet. 1997; 6: 1205-1214Crossref PubMed Scopus (571) Google Scholar, 17Lefebvre S. Burlet P. Liu Q. Bertrandy S. Clermont O. Munnich A. Dreyfuss G. Melki J. Nat. Genet. 1997; 16: 265-269Crossref PubMed Scopus (854) Google Scholar).The 38-kDa SMN protein is ubiquitously expressed (16Coovert D.D. Le T.T. McAndrew P.E. Strasswimmer J. Crawford T.O. Mendell J.R. Coulson S.E. Androphy E.J. Prior T.W. Burghes A.H. Hum. Mol. Genet. 1997; 6: 1205-1214Crossref PubMed Scopus (571) Google Scholar, 17Lefebvre S. Burlet P. Liu Q. Bertrandy S. Clermont O. Munnich A. Dreyfuss G. Melki J. Nat. Genet. 1997; 16: 265-269Crossref PubMed Scopus (854) Google Scholar, 18Liu Q. Dreyfuss G. EMBO J. 1996; 15: 3555-3565Crossref PubMed Scopus (636) Google Scholar) and often localizes in the nuclei as dotlike structures termed gems (18Liu Q. Dreyfuss G. EMBO J. 1996; 15: 3555-3565Crossref PubMed Scopus (636) Google Scholar, 19Young P.J. Le T.T. thi Man N. Burghes A.H. Morris G.E. Exp. Cell Res. 2000; 256: 365-374Crossref PubMed Scopus (171) Google Scholar). SMN is important in small nuclear ribonucleoprotein biogenesis (20Fischer U. Liu Q. Dreyfuss G. Cell. 1997; 90: 1023-1029Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar, 21Liu Q. Fischer U. Wang F. Dreyfuss G. Cell. 1997; 90: 1013-1021Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar, 22Pellizzoni L. Kataoka N. Charroux B. Dreyfuss G. Cell. 1998; 95: 615-624Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar, 23Buhler D. Raker V. Luhrmann R. Fischer U. Hum. Mol. Genet. 1999; 8: 2351-2357Crossref PubMed Scopus (218) Google Scholar, 24Terns M.P. Terns R.M. Curr. Biol. 2001; 11: R862-R864Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) and has been shown to bind a series of other protein partners (25Monani U.R. Coovert D.D. Burghes A.H. Hum. Mol. Genet. 2000; 9: 2451-2457Crossref PubMed Scopus (115) Google Scholar, 26Rossoll W. Kroning A.K. Ohndorf U.M. Steegborn C. Jablonka S. Sendtner M. Hum. Mol. Genet. 2002; 11: 93-105Crossref PubMed Scopus (244) Google Scholar). However, it is not understood which function(s) of SMN is critical specifically for motor neurons. Consistent with the housekeeping functions of SMN, Smn knockout mice are embryonic lethal (27Schrank B. Gotz R. Gunnersen J.M. Ure J.M. Toyka K.V. Smith A.G. Sendtner M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9920-9925Crossref PubMed Scopus (542) Google Scholar). An animal model of SMA was created by introducing the SMN2 gene into Smn–/– mice (28Hsieh-Li H.M. Chang J.G. Jong Y.J. Wu M.H. Wang N.M. Tsai C.H. Li H. Nat. Genet. 2000; 24: 66-70Crossref PubMed Scopus (587) Google Scholar, 29Monani U.R. Sendtner M. Coovert D.D. Parsons D.W. Andreassi C. Le T.T. Jablonka S. Schrank B. Rossol W. Prior T.W. Morris G.E. Burghes A.H. Hum. Mol. Genet. 2000; 9: 333-339Crossref PubMed Scopus (622) Google Scholar). Introduction of one or two copies of the SMN2 gene in Smn–/– mice exhibits a type I SMA phenotype, whereas 8–16 copies of SMN2 completely ameliorate the disease phenotype (29Monani U.R. Sendtner M. Coovert D.D. Parsons D.W. Andreassi C. Le T.T. Jablonka S. Schrank B. Rossol W. Prior T.W. Morris G.E. Burghes A.H. Hum. Mol. Genet. 2000; 9: 333-339Crossref PubMed Scopus (622) Google Scholar). The presence of SMN2 in all SMA patients, the ability of more copies of SMN2 to modify the SMA phenotype, and the rescue of SMA mice by multiple copies of SMN2 make it an attractive therapeutic candidate. Molecules capable of inducing SMN2 expression (30Andreassi C. Patrizi A.L. Monani U.R. Burghes A.H. Brahe C. Eboli M.L. Neurogenetics. 2002; 4: 29-36Crossref PubMed Scopus (18) Google Scholar, 31Baron-Delage S. Abadie A. Echaniz-Laguna A. Melki J. Beretta L. Mol. Med. 2000; 6: 957-968Crossref PubMed Google Scholar) or altering the splicing of SMN2 such that more full-length SMN transcript is produced have been identified (32Andreassi C. Jarecki J. Zhou J. Coovert D.D. Monani U.R. Chen X. Whitney M. Pollok B. Zhang M. Androphy E. Burghes A.H. Hum. Mol. Genet. 2001; 10: 2841-2849Crossref PubMed Scopus (194) Google Scholar, 33Chang J.G. Hsieh-Li H.M. Jong Y.J. Wang N.M. Tsai C.H. Li H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9808-9813Crossref PubMed Scopus (352) Google Scholar, 34Lim S.R. Hertel K.J. J. Biol. Chem. 2001; 276: 45476-45483Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 35Zhang M.L. Lorson C.L. Androphy E.J. Zhou J. Gene Ther. 2001; 8: 1532-1538Crossref PubMed Scopus (115) Google Scholar, 36Cartegni L. Krainer A.R. Nat. Struct. Biol. 2003; 10: 120-125Crossref PubMed Scopus (246) Google Scholar, 37Skordis L.A. Dunckley M.G. Yue B. Eperon I.C. Muntoni F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4114-4119Crossref PubMed Scopus (225) Google Scholar). At the present time, there is limited information on the mode of action of these compounds as well as the protein complexes that interact with the SMN promoter. In this study, we identified two CRE sites in the SMN promoter, demonstrated that the CREB-1 protein binds to the CRE-II site, and showed that cAMP-stimulating agents activate SMN expression.MATERIALS AND METHODSCell Culture and Treatment—Cultures of mouse embryonic hybrid motor neuron (EHMN) cells (38Salazar-Grueso E.F. Kim S. Kim H. Neuroreport. 1991; 2: 505-508Crossref PubMed Scopus (81) Google Scholar) and HeLa cells were maintained in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal calf serum (Atlas Biologicals). Human SMA type 3813 fibroblasts and mouse SMA fibroblasts were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 2 mm glutamine. All of the cell cultures contained penicillin/streptomycin and were incubated at 37 °C in a 5% CO2 humidified atmosphere. For all of the experiments, the cells were plated the day preceding treatment with the cAMP elevators Bt2cAMP and forskolin and harvested at the indicated time.Preparation of Nuclear Extract—The nuclei were isolated from HeLa and EHMN cells, and nuclear extracts were prepared in buffer containing 0.35 m KCl following the protocol of Ref. 39Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9142) Google Scholar. The protein concentration in the nuclear extracts was measured with Bio-Rad reagent according to Bradford's method using bovine serum albumin as a standard.In Vivo Genomic Footprinting—In vivo genomic footprinting of the human SMN promoter was performed as described (40Ghoshal K. Majumder S. Li Z. Dong X. Jacob S.T. J. Biol. Chem. 2000; 275: 539-547Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 41Ghoshal K. Majumder S. Zhu Q. Hunzeker J. Datta J. Shah M. Sheridan J.F. Jacob S.T. Mol. Cell. Biol. 2001; 21: 8301-8317Crossref PubMed Scopus (55) Google Scholar). The human SMN promoter was amplified by ligation-mediated PCR (LM-PCR) according to the procedure of Mueller and Wold (42Mueller P.R. Wold B. Science. 1989; 246: 780-786Crossref PubMed Scopus (792) Google Scholar). Briefly, intact nuclei isolated (43Gorski K. Carneiro M. Schibler U. Cell. 1986; 47: 767-776Abstract Full Text PDF PubMed Scopus (971) Google Scholar) from the brain and liver of Smn–/– mice with eight copies of the human SMN2 gene (29Monani U.R. Sendtner M. Coovert D.D. Parsons D.W. Andreassi C. Le T.T. Jablonka S. Schrank B. Rossol W. Prior T.W. Morris G.E. Burghes A.H. Hum. Mol. Genet. 2000; 9: 333-339Crossref PubMed Scopus (622) Google Scholar) were exposed to limited dimethyl sulfate treatment (1 μl/ml, 2 min at room temperature) in phosphate-buffered saline, pH 7.4. The genomic DNA was isolated from the cells, purified, and subjected to piperidine cleavage (10%) at 90 °C for 30 min. The purified cleaved DNA (2 μg) was then subjected to LM-PCR to amplify SMN promoters. The following primers were used to amplify the region between +210 to +283 of the SMN promoter: SMN/5′-1, 5′-AACACAGTGAAATGAAAGGATTGAG-3′; SMN/5′-2, 5′-GATAACCACTCGTAGAAAGCGTGAG-3′; SMN/5′-3, 5′-CCACTCGTAGAAAGCGTGAGAAGTTACTAC-3′.The annealing temperatures for this set of primers were 58.8, 60.6, and 63.5 °C, respectively.The following primers were used to amplify the region between –312 and –443 of the SMN promoter: SMN/3′-1, 5′-TGTGTGTAGATATTTATTCCCCCTC-3′; SMN/3′-2, 5′-TATTCCCCCTCCCCCTTG-3′; SMN/3′-3, 5′-CCCCCTCCCCCTTGGAAAAG-3′.The annealing temperatures for the 3′-primers were 57.6, 60, and 66.6 °C, respectively.In Vitro DNase I Footprinting Analysis—In order to generate the labeled probe for in vitro DNase I footprinting; the plasmid p750 (44Monani U.R. McPherson J.D. Burghes A.H. Biochim. Biophys. Acta. 1999; 1445: 330-336Crossref PubMed Scopus (78) Google Scholar) was digested with HindIII. To label the lower strand, the HindIII fragment was end-labeled with [γ-32P]ATP. To label the upper strand, the HindIII fragment was filled in using Klenow in the presence of [α-32P]dGTP. The 32P-labeled p750 linear DNA was digested with PstI, and the probes were gel-purified for DNase I footprinting assays. To perform the binding reaction, 25–75 μg of HeLa nuclear extract was added to 40 μl of the reaction buffer (48 mm Hepes, pH 7.9, 240 mm KCl, 2mm dithiothreitol, 48% glycerol, and 20 mm MgCl2) on ice. The binding was initiated by the addition of 1 μl of probe containing ∼20,000 cpm and was incubated at room temperature for 40 min. For competition experiments, unlabeled HindIII/PstI fragment at concentrations of 50× and 100× were added to the reaction mixture prior to the addition of the probe. The DNA protein complexes were then subjected to DNase I digestion at room temperature for 2 min with an optimum amount of DNase I to generate a ladder both in the presence and absence of binding protein. The DNase I digestion was terminated by the addition of 50 μl of stop buffer containing 100 mm Tris, pH 8.0, 600 mm NaCl, 50 mm EDTA, 1% SDS, and proteinase K (0.4 mg/ml). Samples were then incubated at 37 °C for 30 min for proteinase K digestion, phenol-extracted, and ethanol-precipitated. Labeled coding and noncoding strands were chemically sequenced (45Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 13.96-13.97Google Scholar) to generate combined purine (A + G) ladders, which were separated alongside the DNase I-treated samples on a 6% sequencing gel. Gels were dried and exposed to x-ray film at –80 °C.Overexpression of CREB and Western Blot Analysis—For Western blot analysis of CREB, whole cell extracts from cells overexpressing CREB-1 protein were resolved by SDS-PAGE and transferred to ECL membrane (Amersham Biosciences). The membrane was blocked in 0.05% TBST (0.05% Tween-20 in Tris-buffered saline, pH 7.5) containing 5% milk, followed by incubation with human anti-mouse CREB/ATF-1 IgG (1:500 dilution) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in the blocking buffer for 1 h at room temperature. After incubation with anti-mouse IgG-peroxidase conjugate (1:5000 dilution), overexpression of CREB was confirmed with ECL-TM Western blot detection reagents (Amersham Biosciences) following the manufacturer's protocol.Electrophoretic Mobility Shift Assay—Nuclear extracts used for the DNA binding activities of the CREB family of proteins were prepared as described (39Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9142) Google Scholar). A typical binding reaction contained 5 μg of HeLa or 10 μg of EHMN nuclear extract, 0.1 pmol of labeled DNA, 2 μg of Escherichia coli DNA, and 5× Ficoll binding buffer (50 mm Tris-HCl, pH 7.5, 5 mm EDTA, 20% Ficoll, 5 mm dithiothreitol, 375 mm KCl) in a final volume of 20 μl. The binding reaction was initiated by the addition of 1 μl of the reaction buffer containing ∼50,000 cpm of end-labeled double-stranded oligonucleotide and incubated at room temperature for 30 min. ATF-1 antibody (Santa Cruz Biotechnology) or excess double-stranded oligonucleotides wild type CRE-II (5′-GGCGGCGGAAGTCGTCACTCTTAAGAAGG-3′), mutated CRE-II (5′-GGCGGCGGAAGTCGTGTCTCTTAAGAAGG-3′), and the CREB consensus (5′-AGAGATTGCCTGACGTCAGAGAGCTAG-3′) were added to the reaction buffer 30 min prior to the addition of the labeled oligonucleotide as indicated. Samples were then chilled on ice, and the entire volume was loaded onto a 5% polyacrylamide gel containing 0.5× TBE and electrophoresed at 4 °C.UV Cross-linking and SDS-PAGE—UV cross-linking and identification of the DNA-binding protein were performed according to the published protocol (46Liu Z. Jacob S.T. J. Biol. Chem. 1994; 269: 16618-16625Abstract Full Text PDF PubMed Google Scholar, 47Niu H. Jacob S.T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9101-9105Crossref PubMed Scopus (29) Google Scholar). For this purpose, the CRE-II oligonucleotide (5′-GGCGGCGGAAGTCGTCACTCTTAAGAAGG-3′) was annealed with a 10-bp oligonucleotide (5′-CCTTCTTAAG-3′) and internally labeled using Klenow (fill-in reaction) in the presence of [α-32P]dCTP. The labeled probe was purified on a Sephadex G-50 spin column to remove unincorporated nucleotides. Binding reactions were performed as described for EMSA using EHMN nuclear extracts and 0.05 pmol of labeled oligonucleotide in a final volume of 80 μl (4× reactions). The entire reaction mixture was separated on a 5% acrylamide gel in 0.5× TBE. The wet gel was exposed to a short wave UV light from a distance of 2–3 cm at 4 °C for 30 min. The gel was then exposed overnight to x-ray film to locate the complexes. The region of the gel containing the desired complexes were excised and eluted overnight at room temperature in the elution buffer (0.5 mm ammonium acetate, 5 mm dithiothreitol, 1 mm EDTA, pH 8.0, 0.1% SDS). The eluted proteins were precipitated with two volumes of ethanol, washed with 70% ethanol and were separated by SDS-PAGE. The labeled proteins were visualized by autoradiogram.Site-directed Mutagenesis—Site-directed mutation at CRE-II site was introduced into the p750 by overlap extension PCR (48Aiyar A. Leis J. BioTechniques. 1993; 14: 366-369PubMed Google Scholar, 49Majumder S. Ghoshal K. Gronostajski R.M. Jacob S.T. Gene Expr. 2001; 9: 203-215Crossref PubMed Scopus (26) Google Scholar). Plasmid p750 (44Monani U.R. McPherson J.D. Burghes A.H. Biochim. Biophys. Acta. 1999; 1445: 330-336Crossref PubMed Scopus (78) Google Scholar) contains –450 to +300 bp of the SMN2 gene in pGL3-basic vector (Promega). The primers used for mutagenesis are as follows: mut CRE-II oligo-F, 5′-GGCGGAAG TCGTGTCTCTTAAGAAGG-3′; mut CRE-II oligo-R, 5′-CCTTCTTAAGAGACACGACTTCCGCC-3′; SstI primer, 5′-TCGCTTGAGCTCTGGAGGTCGAGGCTG-3′; NcoI primer, 5′-TTACCCATGGAGGCTTTACCAACAGTACCG-3′. Two sets of PCRs were run using the mut CRE-II oligo-R/SstI primer and mut CRE-II oligo-F/NcoI primer pairs. The second PCR was carried out using the gel-purified PCR products from the first set of PCR as templates and SstI primer/NcoI primer. The condition for the PCR was 94 °C for 5 min and then 30 cycles of 94 °C for 1 min, 64 °C for 1 min, 72 °C for 1 min, and final extension at 72 °C for 10 min. The final PCR product was gel-purified, blunt-ended with Pfu polymerase, digested with SstI and NcoI, and cloned back into pGL3-Basic (Promega) vector to produce p750mCRE. Mutation was confirmed by sequence analysis.Transient Transfection Assay—EHMN or HeLa cells were seeded onto 6-well plates 24 h prior to transfection. Cells were transfected with 0.150 μg (for EHMN cells) and 0.500 μg (for HeLa cells) of p750 DNA or p750mCRE, using 2 μl of LipofectAMINE (Invitrogen) according to the manufacturer's protocol. For overexpression studies, 2 μg of pSG-RSVCREB plasmid (a gift from Dr. Tsonwin Hai, Ohio State University) or the corresponding empty vector DNA was transfected along with 0.5 μg of p750. Cells were harvested 48 h after transfection in lysis buffer, and luciferase activity was assayed using the dual luciferase assay kit (Promega, Madison, WI). Normally each transfection assay mixture consists of 2.5 μg of DNA along with the reporter plasmid RLTK (Renilla luciferase gene with thymidine kinase promoter; Promega, Madison, WI) used as an internal control. To see the effect of cAMP on the p750 promoter, HeLa cells were transfected with p750 in 100-mm dishes, and cells were split after 6 h of transfection and seeded into 6-well plates. After 24 h of transfection, cells were treated with 0.5 mm Bt2cAMP or 20 μm forskolin as indicated. Cells were then harvested after 24 h of treatment and assayed for luciferase activity as described previously.In Vivo Treatment of Animals and Hepatocytes—Smn+/– mice expressing two copies of the human SMN2 gene (29Monani U.R. Sendtner M. Coovert D.D. Parsons D.W. Andreassi C. Le T.T. Jablonka S. Schrank B. Rossol W. Prior T.W. Morris G.E. Burghes A.H. Hum. Mol. Genet. 2000; 9: 333-339Crossref PubMed Scopus (622) Google Scholar) received intraperitoneal injection of epinephrine (2 mg/kg body weight) every 2 h for 6 h and were sacrificed 2 h after the last injection. The mice were sacrificed by cervical dislocation, and the livers were snap frozen in liquid nitrogen for RNA isolation.Primary mouse hepatocytes were isolated as described by Matsuda et al. (50Matsuda M. Korn B.S. Hammer R.E. Moon Y.A. Komuro R. Horton J.D. Goldstein J.L. Brown M.S. Shimomura I. Genes Dev. 2001; 15: 1206-1216Crossref PubMed Scopus (262) Google Scholar), washed, and resuspended in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum, 100 units/ml penicillin G sodium, and 100 μg/ml streptomycin sulfate. Cell viability determined by trypan blue dye exclusion was found to be 85–90%. The cells were plated in the above medium at a density of 1.5 × 106 on 60-mm dishes coated with rat tail type I collagen (Sigma). After incubation for 14–16 h, fresh medium was added to the cells and was either left untreated or treated for 8 h with 20 mm forskolin alone or in combination with 0.5 mm dibutyryl cAMP.RT-PCR and Semiquantitative RT-PCR Analysis of SMN Transcripts—Total RNA was isolated from untreated and treated HeLa cells, mouse and human fibroblasts, primary hepatocytes, and liver using the guanidine isothiocyanate method (51Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62983) Google Scholar). First strand cDNA was synthesized from 3 μg of total RNA using an RT-PCR kit (PerkinElmer Life Sciences). One-tenth of the reaction mixture was used for amplification of SMN gene. To amplify the different splice variants of SMN transcripts, a multiplex PCR was performed as described previously (8Parsons D.W. McAndrew P.E. Monani U.R. Mendell J.R. Burghes A.H. Prior T.W. Hum. Mol. Genet. 1996; 5: 1727-1732Crossref PubMed Scopus (127) Google Scholar, 32Andreassi C. Jarecki J. Zhou J. Coovert D.D. Monani U.R. Chen X. Whitney M. Pollok B. Zhang M. Androphy E. Burghes A.H. Hum. Mol. Genet. 2001; 10: 2841-2849Crossref PubMed Scopus (194) Google Scholar), where different splice variants of the SMN gene were amplified along with the HPRT (hypoxanthine phosphoribosyltransferase) gene as an internal control. PCR primers used for amplification of exons 4–8 of the SMN gene (4 forward, 5′-GTGAGAACTCCAGGTCTCCTGG-3′;8 reverse, 5′-CTACAACACCCTTCTCACAG-3′), yielding four possible RT-PCR products (derived from the full-length SMN transcripts and isoforms lacking exon 5 and/or 7). Primers selected for amplification of HPRT (forward, 5′-TGTAATGACCAGTCAACAGG-3′; reverse, 5′-ATTGACTGCTTCTTACTTTTCT-3′) generated a product that is similar in size to (but distinguishably different from) the full-length SMN transcript (32Andreassi C. Jarecki J. Zhou J. Coovert D.D. Monani U.R. Chen X. Whitney M. Pollok B. Zhang M. Androphy E. Burghes A.H. Hum. Mol. Genet. 2001; 10: 2841-2849Crossref PubMed Scopus (194) Google Scholar). The forward primers of mouse and human HPRT and SMN were end-labeled with [γ-32P]ATP. cDNA was amplified by PCR in a 25-μl reaction mixture containing 0.5 mm dNTP, 1 unit of Taq polymerase, 30 ng of each SMN primer, 7.5 ng of each HPRT primer, 2.5 mm MgCl2 in 1× PCR buffer. Cycling conditions consisted of an initial denaturation step at 95 °C for 4 min, followed by 22 cycles of 95 °C for 1 min, 55 °C for 2 min, and 72 °C for 1 min, with a final extension step at 72 °C for 8 min. Ten microliters of the resulting PCR products was combined with 5 μl of loading dye (95% formamide, 10 mm EDTA, 0.1% bromphenol blue, and 0.1% xylene cyanol) and was electrophoresed on a 6% denaturing polyacrylamide gel. The gel was dried and exposed to hyperfilm (Amersham Biosciences) or to a PhosphorImager screen.RESULTSIn Vivo Genomic Footprinting Studies Demonstrate Occupancy of a CRE/ATF Site on the Human SMN2 Promoter— Transient transfection studies have identified a 750-bp segment spanning from –450 to +300 bp with respect to transcription start site on both human SMN1 and SMN2 gene that demonstrated maximal transcriptional activity (44Monani U.R. McPherson J.D. Burghes A.H. Biochim. Biophys. Acta. 1999; 1445: 330-336Crossref PubMed Scopus (78) Google Scholar). There was minimal difference in the sequence between the SMN1 and SMN2 promoters that is reflected in the comparable promoter activity (12Monani U.R. Lorson C.L. Parsons D.W. Prior T.W. Androphy E.J. Burghes A.H. McPherson J.D. Hum. Mol. Genet. 1999; 8: 1177-1183Crossref PubMed Scopus (7" @default.
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