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• W2056565319 abstract "The norepinephrine transporter (NET) is responsible for the rapid NaCl-dependent uptake of norepinephrine into presynaptic noradrenergic nerve endings. Recently, we have characterized the structural organization of the 5′ upstream promoter region of the human NET (hNET) gene. A new intron of 476 base pairs was found in the middle of the 5′-untranslated leader sequence and was shown to robustly enhance the promoter activity. Here, we show that the first hNET intron enhances both the homologous hNET and the heterologous thymidine kinase promoter activities in an orientation- and position-dependent manner. The first hNET intron exhibited a similar promoter-enhancing effect in both SK-N-BE(2)C (NET-positive) and HeLa (NET-negative) cell lines, showing that its function is not cell-specific. Transient transfection assays of a series of deletional constructs show that the first hNET intron contains subdomains with either positive or negative regulatory functions. Furthermore, DNase I footprinting analysis demonstrated that the 5′ side of the intron, encompassing the splice donor site, is prominently protected by nuclear proteins isolated from both SK-N-BE(2)C and HeLa cells. The protected nucleotide sequence contains a consensus E-box motif, known to regulate diverse eukaryotic genes, which overlaps with the splice donor site of the first intron. We demonstrate that two basic helix-loop-helix proteins, upstream stimulatory factors 1 and 2, are major proteins interacting at this site and that the E-box is at least in part responsible for the promoter-enhancing activity of the first intron. Furthermore, site-directed mutagenesis of the splice donor site of the first intron affects both correct splicing and transcriptional activity. Taken together, our results indicate that acis-element residing at the first exon/intron junction, encompassing an E-box motif, has a unique dual role in basal transcriptional activation and splicing of hNET mRNA. The norepinephrine transporter (NET) is responsible for the rapid NaCl-dependent uptake of norepinephrine into presynaptic noradrenergic nerve endings. Recently, we have characterized the structural organization of the 5′ upstream promoter region of the human NET (hNET) gene. A new intron of 476 base pairs was found in the middle of the 5′-untranslated leader sequence and was shown to robustly enhance the promoter activity. Here, we show that the first hNET intron enhances both the homologous hNET and the heterologous thymidine kinase promoter activities in an orientation- and position-dependent manner. The first hNET intron exhibited a similar promoter-enhancing effect in both SK-N-BE(2)C (NET-positive) and HeLa (NET-negative) cell lines, showing that its function is not cell-specific. Transient transfection assays of a series of deletional constructs show that the first hNET intron contains subdomains with either positive or negative regulatory functions. Furthermore, DNase I footprinting analysis demonstrated that the 5′ side of the intron, encompassing the splice donor site, is prominently protected by nuclear proteins isolated from both SK-N-BE(2)C and HeLa cells. The protected nucleotide sequence contains a consensus E-box motif, known to regulate diverse eukaryotic genes, which overlaps with the splice donor site of the first intron. We demonstrate that two basic helix-loop-helix proteins, upstream stimulatory factors 1 and 2, are major proteins interacting at this site and that the E-box is at least in part responsible for the promoter-enhancing activity of the first intron. Furthermore, site-directed mutagenesis of the splice donor site of the first intron affects both correct splicing and transcriptional activity. Taken together, our results indicate that acis-element residing at the first exon/intron junction, encompassing an E-box motif, has a unique dual role in basal transcriptional activation and splicing of hNET mRNA. norepinephrine norepinephrine transporter human NET chloramphenicol acetyltransferase thymidine kinase polymerase chain reaction upstream stimulatory factor reverse transcription base pair(s) kilobase(s) Norepinephrine (NE)1 is directly involved in mood stabilization, sleep regulation, expression of aggression, and the general degree of alertness and arousal, as well as in exerting central control over the endocrine and autonomic nervous system. NE neurotransmission is terminated by the norepinephrine transporter (NET), located on NE nerve terminals, which is responsible for the rapid reuptake of NE into the presynaptic neurons (1Axelrod J. Kopin I.J. Prog. Brain Res. 1969; 31: 21-32Crossref PubMed Scopus (77) Google Scholar). The clinical importance of noradrenergic transmission is suggested by the therapeutic usefulness of tricyclic antidepressant drugs, which enhance the synaptic availability of NE through the inhibition of NE transport into presynaptic terminals. These findings provided support for the catecholamine hypothesis that depression is associated with a decrease in the levels of catecholamines, particularly norepinephrine (2Schildkraut J.J. Am. J. Psychiatry. 1965; 122: 509-522Crossref PubMed Scopus (2314) Google Scholar). Consistent with this catecholamine hypothesis, a recent analysis showed that the levels of norepinephrine and its metabolites dihydroxyphenylglycolaldehyde and monohydroxyphenylglycolaldehyde are significantly reduced in the internal jugular venous plasma of depression patients (3Lambert G. Johansson M. Agren H. Friberg P. Arch. Gen. Psychiatry. 2000; 57: 787-793Crossref PubMed Scopus (242) Google Scholar). Furthermore, a missense mutation (G to C) in exon 9 of NET gene has recently been identified and found to cause a 98% loss of function (4Shannon J.R. Flattem N.L. Jordan J. Jacob G. Black B.K. Biaggioni I. Blakely R.D. Robertson D. N. Engl. J. Med. 2000; 342: 541-549Crossref PubMed Scopus (467) Google Scholar). It is of great interest that this mutation is associated with hyperadrenergic states, leading to orthostatic intolerance (4Shannon J.R. Flattem N.L. Jordan J. Jacob G. Black B.K. Biaggioni I. Blakely R.D. Robertson D. N. Engl. J. Med. 2000; 342: 541-549Crossref PubMed Scopus (467) Google Scholar). Collectively, regulation of NET expression may significantly affect NE neurotransmission, and its abnormal expression may play a role in the pathophysiology of major depression and cardiovascular disorder (4Shannon J.R. Flattem N.L. Jordan J. Jacob G. Black B.K. Biaggioni I. Blakely R.D. Robertson D. N. Engl. J. Med. 2000; 342: 541-549Crossref PubMed Scopus (467) Google Scholar,5Klimek V. Stockmeier C. Overholser J. Meltzer H.Y. Kalka S. Dilley G. Ordway G.A. J. Neurosci. 1997; 17: 8451-8458Crossref PubMed Scopus (323) Google Scholar). NET gene expression is controlled by various physiological and pharmacological signals in noradrenergic neurons and neurosecretory cells. Levels of NET mRNA in the locus coeruleus and the adrenal medulla were decreased by the treatment with reserpine or glucocorticoid but were increased by nerve growth factor (6Cubells J.F. Kim K.S. Baker H. Volpe B.T. Chung Y. Houpt T.A. Wessel T.C. Joh T.H. J. Neurochem. 1995; 65: 502-509Crossref PubMed Scopus (63) Google Scholar, 7Wakade A.R. Wakade T.D. Poosch M. Bannon M.J. J. Physiol. (Lond.). 1996; 494: 67-75Crossref Scopus (31) Google Scholar). Several studies suggest that both short and long term function of NET may be modulated by metabolic hormones, such as insulin and thyroid hormone (8Figlewicz D.P. Epilepsy Res. 1999; 37: 203-210Crossref PubMed Scopus (45) Google Scholar, 9Tejani-Butt S.M. Yang J. Neuroendocrinology. 1994; 59: 235-244Crossref PubMed Scopus (42) Google Scholar, 10Yang S.P. Pau K.Y. Spies H.G. J. Neuroendocrinol. 1997; 9: 763-768Crossref PubMed Scopus (17) Google Scholar). In addition, angiotensin II has been shown to play an important role in the stimulation of NET gene transcription mediated by Ras-Raf-MAP kinase and PKC pathways in neuronal cultures (11Lu D., Yu, K. Paddy M.R. Rowland N.E. Raizada M.K. Endocrinology. 1996; 137: 763-772Crossref PubMed Scopus (62) Google Scholar, 12Yang H. Raizada M.K. J. Neurosci. 1999; 19: 2413-2423Crossref PubMed Google Scholar). In the superior cervical ganglia, leukemia inhibitory factor and ciliary neurotrophic factor suppressed the level of NET mRNA, whereas retinoic acid increased NET mRNA expression (13Matsuoka I. Kumagai M. Kurihara K. Brain. Res. 1997; 776: 181-188Crossref PubMed Scopus (20) Google Scholar). The NET mRNA is transiently elevated in locus coeruleus neurons following either kainic acid-induced status epilepticus (14Bengzon J. Hansson S.R. Hoffman B.J. Lindvall O. Brain Res. 1999; 842: 239-242Crossref PubMed Scopus (15) Google Scholar) or penytylenetetrazol-induced seizures (15Szot P. White S.S. Veith R.C. Brain Res. Mol. Brain Res. 1997; 44: 46-54Crossref PubMed Scopus (37) Google Scholar). At present, the molecular mechanisms of NET gene regulation by these physiological and pharmacological stimuli are poorly understood. The human NET (hNET) gene is a highly dispersed locus with ∼2 kb of coding exons spread across 45 kb of genomic DNA and located at chromosome 16q12.2 (16Porzgen P. Bonisch H. Bruss M. Biochem. Biophys. Res. Commun. 1995; 215: 1145-1150Crossref PubMed Scopus (73) Google Scholar). Recently, we have characterized the structural organization of the 5′ upstream promoter region of the hNET gene (17Kim C.H. Kim H.S. Cubells J.F. Kim K.S. J. Biol. Chem. 1999; 274: 6507-6518Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). A new intron of 476 bp was shown to reside in the middle of the 5′-untranslated leader sequence, and two major transcription initiation sites were determined. Whereas the 5′ upstream 9.0-kb sequence contains important regulatory information for the cell-specific expression of the hNET gene, the first intron increases the transcriptional activity by ∼10–40-fold, depending on the cell lines tested. These observations suggest that the first hNET intron may play an important role as a typical enhancer in NET gene regulation. To further elucidate its regulatory mechanisms, we initiated a systematic functional analysis of the first hNET intron. In addition to showing that the first hNET intron enhances the promoter activity in an orientation- and position-dependent manner, this study identifies a cis-regulatory element, an E-box motif residing in the junction of the first exon and intron, that is critical for basal transcriptional activation of NET mRNA via interaction with transcription factors USF1 and USF2. Furthermore, we demonstrate that the same cis-element directly regulates correct splicing of hNET mRNA because it coincides with the splice donor site of the first intron. Human neuroblastoma SK-N-BE(2)C and HeLa cell lines were used as the NET-positive and NET-negative cell lines respectively. Cell lines were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Hyclone), 100 μg/ml streptomycin, and 100 units/ml penicillin in a CO2 incubator. Transfection was performed by the calcium phosphate coprecipitation as previously described (18Ishiguro H. Kim K.T. Joh T.H. Kim K.S. J. Biol. Chem. 1993; 268: 17987-17994Abstract Full Text PDF PubMed Google Scholar, 19Kim K.S. Ishiguro H. Tinti C. Wagner J. Joh T.H. J. Neurosci. 1994; 14: 7200-7207Crossref PubMed Google Scholar). Plasmids for transfection were prepared using Qiagen columns (Qiagen Co., Santa Clarita, CA). For the SK-N-BE(2)C cell line, each 60-mm dish was transfected with an equimolar amount (0.5 pmol) of each reporter construct, 1 μg of pRSV-β-galactosidase, and pUC19 plasmid to a total of 5 μg of DNA. For the HeLa cell line, twice as much DNA was used in transfection. Cells were harvested 72 h after transfection, lysed by freeze-thaw cycles, and assayed for CAT activities. To correct for differences in transfection efficiencies among different DNA precipitates, CAT activity was normalized to that of β-galactosidase. CAT and β-galactosidase activities were assayed as previously described (18Ishiguro H. Kim K.T. Joh T.H. Kim K.S. J. Biol. Chem. 1993; 268: 17987-17994Abstract Full Text PDF PubMed Google Scholar,19Kim K.S. Ishiguro H. Tinti C. Wagner J. Joh T.H. J. Neurosci. 1994; 14: 7200-7207Crossref PubMed Google Scholar). The constructions of pNET1400CAT and pNET1400(i)CAT were described previously (17Kim C.H. Kim H.S. Cubells J.F. Kim K.S. J. Biol. Chem. 1999; 274: 6507-6518Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In order to investigate whether the first intron functions in conjunction with a heterologous promoter, the 540-bp BamHI fragment containing the first intron was also cloned into the BglII site of pBLCAT2 (20Luckow B. Schutz G. Nucleic Acids Res. 1987; 15: 5490Crossref PubMed Scopus (1401) Google Scholar) containing the herpes simplex virus thymidine kinase (TK) promoter to form TK(i)CAT. The same BamHI fragment was made blunt with Klenow fragment and subcloned into either upstream NET promoter or downstream CAT encoding sequence to generate pNET(i)1400CAT and pNET1400CAT(i), respectively. Similarly, the blunt-ended fragment was cloned into either upstream TK promoter or downstream CAT encoding sequences to generate (i)TKCAT and TKCAT(i), respectively. The plasmid pNET1400(i-rev)CAT was constructed by subcloning the SmaI-SmaI fragment covering positions +230 ∼ +586 within the first intron in the reverse orientation. The orientation of the first intron was confirmed by restriction enzyme mapping and sequence analysis. For the internal deletion constructs, intronic sequence was amplified by PCR using sense primer (5′-CCGGACACGTGAGTGCGCACTAGTCCTGAGCGCGGGACAGGGCTAGGT-3′) containing restriction enzyme SpeI and antisense primer (catoligo2; 5′-CATTTTAGCTTCCTTAGC-3′), digested with PmlI andXhoI, and subcloned into pNET1400(i)CAT that had been digested with PmlI and XhoI to yield pNET1400(i-spe)CAT. The 120-bp fragment including the intact splicing acceptor site was also amplified by PCR using sense primer (5′-GGATAACTAGTTTATCCAAGCAGAGCCTCGGCGTG-3′) and catoligo2 primer, cut with SpeI and XhoI, and subcloned into pNET1400- (i-spe)CAT, which was digested with SpeI andXhoI to generate pNET1400(iΔ190–645)CAT. In order to subclone the PCR fragments into the pNET1400(iΔ190–645)CAT, the 5′ primers were designed to contain a SpeI site, and the 3′ primers were designed to contain a XbaI site. PCR products that were digested by SpeI and XbaI were then subcloned into the SpeI site of the pNET1400(iΔ190–645)CAT. Nucleotide sequences of primers used in PCR were 5′-TGCCCACTAGTTCGGTGAGTTCAATCCCAGC-3′ and 5′-TGCTTTCTAGAAGGGAAAAGAGGTGGTTACC-3′ for pNET1400(iΔ381–645)CAT, 5′-CCGGACACGTGAGTGCGCACTAGTCCTGAGCGCGGGACAGGGCTAGGT-3′ and 5′-TGCCCTCTAGAGCTGGGATTGAACTCACCGA-3′ for pNET1400(iΔ190–362)CAT, 5′-CCGGACACGTGAGTGCGCACTAGTCCTGAGCGCGGGACAGGGCTAGGT-3′ and 5′-TTGAGACTAGTCGTGCCCCAACCTCTGTTTC-3′ for pNET1400(iΔ190–482)CAT, and 5′-CCGGACACGTGAGTGCGCACTAGTCCTGAGCGCGGGACAGGGCTAGGT-3′ and 5′-GGAAATCTAGAGAAACAGAGGTTGGGGCACG-3′ for pNET1400(iΔ190–362/ 501–645)CAT. The PCR-derived fragments were sequenced to ensure that no errors had been introduced. Base substitutions in E-box and splicing donor site were generated in the context of the 1400 bp upstream plus intron sequence using the TransformerTM site-directed mutagenesis kit (CLONTECH, Palo Alto, CA) according to the manufacturer's procedure. The following oligonucleotides were used in the mutagenesis procedure: 5′-GATCCCCTCGCCGCCGGAaAgGTGAGTGCGCCCTGAGCG-3′ for E-box mutation unaffecting the splice donor site and 5′-GATCCCCTCGCCGCCGGACACGacAGTGCGCCCTGAGCG-3′ for E-box mutation affecting the splice donor site (lowercase letters indicate the substitutions in nucleotides). The mutation was confirmed by sequence analysis. Nuclear extracts were prepared from SK-N-BE(2)C and HeLa cells according to the procedure described by Dignam et al. (21Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9131) Google Scholar). The pellet was resuspended in Buffer D (20 mm HEPES, pH 7.9, 20% glycerol, 0.1 m KCl, 0.2 mmphenylmethylsulfonyl fluoride, and 0.5 mm dithiothreitol) and dialyzed against the same buffer. The extracts were quick-frozen in liquid nitrogen, stored in aliquots at −70 °C, and used within 3 months of extraction. Protein concentrations of the nuclear extracts were determined by the Bio-Rad protein assay method using bovine serum albumin as a standard (22Bradford M.M. Anal. Biochem. 1976; 112: 195-203Google Scholar). The first intron fragment was prepared by PCR and used as a probe in the DNase I footprinting experiment. For the coding strand probe, a primer (5′-AGCTCTTCCCCGGCCCCGCCCGAACGCCACACGGCGGA-3′) representing the coding nucleotide sequence from +47 to +84 bp of the hNET gene was labeled by polynucleotide kinase using [γ-32P]ATP. This was used in PCR, together with an unlabeled nucleotide, 5′-CCCTACTTGCAACTCCCAAGACCACCCGGGAGCGCCTTAG-3′, representing the noncoding nucleotide sequence from +574 to +613 of the hNET gene. PCR was performed with the denaturation, annealing, and DNA synthesis at 95 °C (40 s), 55 °C (30 s), and 72 °C (1 min), respectively, for a total of 30 cycles using the plasmid pNET1400(i)CAT as a template,. The end-labeled probe was isolated from a 7% polyacrylamide gel, as described previously (23Kim K.S. Febraio M. Han T.H. Wessel T.C. Park D.H. Joh T.H. Rickwood D. Hames B.D. A Practical Approach: Gene Probes 2. Oxford University Press, Oxford, United Kingdom1995: 151-182Google Scholar). After incubating 30,000 cpm of labeled probe with 10–20 μl of nuclear extracts in 40 μl of binding buffer for 25 min at room temperature, DNase I digestion was carried out using freshly diluted DNase I in 1× binding buffer, which contained 20 mm HEPES (pH 7.9), 2 mmMgCl2, 50 mm NaCl, 1 mmdithiothreitol, 0.1 mm EDTA, and 10% glycerol. 2 μg of poly(dI-dC) was included in the reaction as a nonspecific competitor. The amount of DNase I was empirically adjusted for each extract to produce an even pattern of partially cleaved products. The DNase I reaction was stopped by adding 100 μl of stop buffer (50 mm Tris (pH 8.0), 1% SDS, 10 mm EDTA (pH 8.0), 0.4 mg/ml proteinase K, and 100 mm NaCl). Samples were then extracted twice with phenol-chloroform, and the DNA was precipitated with 3 volumes of ethanol. The DNA pellet was dried and resuspended in sequencing stop buffer (0.05% xylene cyanol, 0.05% bromphenol blue, 10 mm Na2 EDTA, and 90% deionized formamide) and incubated at 95 °C for 3 min. An aliquot of sample was then loaded onto a 6% polyacrylamide-8 m urea sequencing gel. The same probe was subjected to parallel digestion without prior incubation with nuclear extracts, typically using 5–10% of the DNase I used in the presence of nuclear extracts. Location of cleaved products was determined by comparison with sequencing ladders run in adjacent lanes on the gel. Sense and antisense oligonucleotides corresponding to the sequences protected by DNase I were synthesized (Gene Link, Inc., Thornwood, NY) with the following nucleotide sequences: 5′-GCCGCCGGACACGTGAGTGCGCCC-3′ and 5′-CGGGCGCACTCACGTGTCCGGCGG-3′ for NI, 5′-GCACCCGGTCACGTGGCCTACACC-3′ and 5′-GGGTGTAGGCCACGTGACCGGGTG-3′ for the consensus binding site for USF1 (UI). Nucleotide sequences for mutant oligonucleotides were 5′-GCCGCCGGAAAGGTGAGTGCGCCC-3′ and 5′-CGGGCGCACTCACCTTTCCGGCGG-3′ for NIm. The consensus AP2 and Sp1 oligonucleotides were previously described (24Seo H. Yang C. Kim H.S. Kim K.S. J. Neurosci. 1996; 16: 4102-4112Crossref PubMed Google Scholar, 25Kim H.S. Seo H. Brunet J.F. Kim K.S. J. Neurosci. 1998; 18: 8247-8260Crossref PubMed Google Scholar). The sense and antisense oligonucleotides were annealed, gel-purified, and32P-labeled by T4 DNA kinase and used as probes in electrophoretic mobility shift assays. Electrophoretic mobility shift assay and antibody coincubation experiments were performed using 30,000–50,000 cpm of labeled probe (∼0.05–0.1 ng) and nuclear extracts (10–30 μg) in a final volume of 20 μl of 12.5% glycerol, and 12.5 mm HEPES, pH 7.9, 4 mm Tris-HCl, pH 7.9, 60 mm KCl, 1 EDTA, and 1 mm dithiothreitol with 1 μg of poly(dI-dC) as described (26Yang C. Kim H.S. Seo H. Kim C.H. Brunet J.F. Kim K.S. J. Neurochem. 1998; 71: 1813-1826Crossref PubMed Scopus (118) Google Scholar). Competition binding assays were performed by adding nonradioactive competitor oligonucleotides in a molar excess before adding32P-labeled oligonucleotides. For the supershift assay, antibodies were coincubated with the nuclear extract mix for 30 min at room temperature before adding the radiolabeled probe. Antibodies against USF1, USF2, c-Myc, Sp1, and AP2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). SK-N-BE(2)C cells were transiently transfected with different CAT reporter gene constructs. Poly(A+) RNA was prepared by oligo(dT)-cellulose affinity column chromatography as previously described (23Kim K.S. Febraio M. Han T.H. Wessel T.C. Park D.H. Joh T.H. Rickwood D. Hames B.D. A Practical Approach: Gene Probes 2. Oxford University Press, Oxford, United Kingdom1995: 151-182Google Scholar). The poly(A+)RNA was reverse-transcribed with SUPERSCRIPT II RNase H− reverse transcriptase (Life Technologies, Inc.) by priming with the catoligo2 primer. The products were subjected to the PCR using sense primer 5′-AGCTCTTCCCCGGCCCCGCCCGAACGCCACACGGCGGA-3′ and antisense primer 5′-TCGCGGATCCGAATTCTGGCGAGAGGAACTTTACCGG-3′. The PCR product was analyzed on 7% polyacrylamide gel. RT-PCR was also performed using primers for β-actin gene messages as a control. Primers for the β-actin gene were 5′-GGTCAGAAGGACTCCTATGTG-3′ (sense) and 5′-TGTAGCCACGCTCGGTCAGG-3′ (antisense). In the native position (pNET1400(i)CAT construct), the first intron increased the reporter expression driven by the 1.4 kb NET upstream promoter by 12–15-fold in SK-N-BE(2)C and HeLa cell lines (Fig.1 A). However, the first hNET intron was unable to activate promoter activity when placed either in the 5′ upstream position (pNET(i)1400CAT) or in a position 3′ to the reporter gene (pNET1400CAT(i)). Thus, the promoter-enhancing function of the first hNET intron appeared to be dependent on its original position in the context of the homologous promoter. Notably, the first hNET intron by itself did not have any promoter activity, as the reporter gene expression driven by the first intron alone was no higher than that obtained with promoterless construct (data not shown). When placed downstream of the heterologous TK promoter (TK(i)CAT in Fig.1 B), which is analogous to the native position, the first hNET intron increased the transcriptional activity by 5- and 8-fold in SK-N-BE(2)C and HeLa cells, respectively. However, when placed either in the upstream position ((i)TKCAT) or in a position 3′ to the reporter gene (TKCAT(i)), the first hNET intron again failed to show enhancing activity (Fig. 1 B). In order to investigate whether orientation of the first hNET intron affects its function, we generated a reporter construct containing an internally inverted intron. To avoid the possibility that reporter gene expression is affected by an improper splicing of mRNA, the original donor and acceptor sites for splicing were kept intact. The inverted sequence somewhat stimulated promoter activity, but less effectively than that in the correct orientation (4.5-foldversus 11-fold; Fig.2 A). To confirm that the difference in reporter gene expression could be attributed to transcriptional regulation but not to a defective splicing mechanism, RT-PCR was performed. When the mRNAs isolated from SK-N-BE(2)C cells transfected with the forward or reverse intron-containing constructs were examined by RT-PCR, we detected only a mRNA species with a size expected from a proper splicing event (Fig. 2 B). Taken together, our results suggested that the first hNET intron robustly enhances the promoter activity, and its full activity requires the original position and orientation. Therefore, it appears that the regulatory elements within the first hNET intron fail to meet the criteria for a “classical enhancer” but require the native orientation and position for promoter-enhancing activity. To locate the regulatory domains within the first hNET intron, we next performed deletional analyses. To distinguish transcriptional regulation from improper splicing mechanisms, deletional constructs were generated such that the original splicing donor and acceptor sites were kept intact (Fig.3). Reporter gene activity of each construct was examined by transient transfection assays using SK-N-BE(2)C and HeLa cell lines. Deletion of a 5′-side domain between +190 and +362 bp (pNET1400(iΔ190–362)CAT) decreased the CAT expression by ∼40%, suggesting that this subdomain contains positive regulatory sequences. Interestingly, CAT expression driven by pNET1400(iΔ190–482)CAT, in which the first hNET intron was further deleted to +482 bp, was even higher than that by the wild type plasmid pNET1400(i)CAT, suggesting the possibility that an internal subdomain between +362 and +482 bp encompasses negative regulatory sequences. Deletion of a 3′-side domain between +381 and +645 bp (pNET1400(iΔ381–645)CAT) diminished CAT expression by about half, suggesting that a 3′ side subdomain between +483 and +645 bp contains positive regulatory sequences. The internal deletion of most nucleotides of the first intron (pNET1400(iΔ190–645)CAT) shows a reduction of approximately 50% of CAT activity. Finally, consistent with the concept that sequences between +362 and +501 contain negative regulatory sequences, insertion of this subdomain to pNET1400(iΔ190–645)CAT further decreased the CAT expression (construct pNET1400(iΔ190–362/501–645)CAT). The first hNET intron therefore appears to contain several regulatory subdomains with either positive or negative regulatory function, which collectively enhance the promoter activity. It is noteworthy that pNET1400(iΔ190–645)CAT, in which only 10 bases of each splicing junction area are retained, still can enhance the promoter activity 4–5-fold, which is approximately half the activity of the full first intron sequence. This indicates that a cis-element important for the enhancing activity may reside in or overlap with the splicing donor or acceptor site (see below). To identify specific transcription factor(s) responsible for the promoter enhancing activity of the first hNET intron, we investigated DNA-protein interactions by DNase I footprinting analysis using nuclear extracts isolated from SK-N-BE(2)C and HeLa cells. A prominent DNA-protein interaction was mapped at +173 to +190, using both the coding and noncoding strands (Fig. 4 and data not shown). This precisely overlaps with the splicing donor site, suggesting that this site may be important for basal transcriptional activation as well as correct splicing of NET mRNA. Nuclear proteins from both SK-N-BE(2)C and HeLa cells footprinted the same area. In contrast, no clear DNA-protein interaction was detected along the whole internal sequence of the intron (data not shown), suggesting that transcription factors involved in the transcriptional regulatory function of the first hNET intron may have weak DNA binding affinity and/or exist in a very low concentration. The protected area at +173 to +190 contains a consensus E-box (CACGTG), which is known to be a binding site for basic helix-loop-helix class transcription factors, such as c-Myc and the upstream stimulatory factors USF1 and USF2 (27Murre C. McCaw P.S. Vaessin H. Caudy M. Jan L.Y. Jan Y.N. Cabrera C.V. Buskin J.N. Hauschka S.D. Lassar A.B. Cell. 1989; 58: 537-544Abstract Full Text PDF PubMed Scopus (1289) Google Scholar, 28Murre C. McCaw P.S. Baltimore D. Cell. 1989; 56: 777-783Abstract Full Text PDF PubMed Scopus (1829) Google Scholar). When an oligonucleotide NI encompassing the protected nucleotides was used as the probe, a specific DNA-protein complex (C1) was prominently formed using nuclear extracts from SK-N-BE(2)C cell line (Fig.5 B). A similar DNA-protein complex was formed with an identical mobility when an oligonucleotide UI containing the consensus USF1 binding site (Santa Cruz Biotechnology) was used as a probe (Fig. 5 B). The formation of the C1 complex was completely abolished when the oligonucleotide NIm with two mutated bases within the core E-box motif was used as the probe. When the nuclear extracts from HeLa cell line were used, a similar pattern was observed except that another band with a faster mobility was also formed. Because this band was robustly formed even when the mutated NIm oligonucleotide was used as the probe, it may represent a nonspecific complex. In a competition assay, a 100-fold molar excess of unlabeled NI or UI oligonucleotide almost completely abolished formation of C1 (Fig. 5 C). However, its mutant form (NIm), the Sp1 oligonucleotide, or the AP1 oligonucleotide failed to compete for complex formation. In addition, formation of the second complex with HeLa nuclear extracts was not efficiently competed by the specific NI and UI oligonucleotides, supporting the notion that it is a nonspecific complex. These observations suggest that (i) C1 represents sequence-specific DNA-protein complex, (ii) the E-box motif is essential for formation of C1 complex, and (iii) the C1 complex may involve the USF1 or related factors. To identify protein factor(s) involved in formation of the C1 complex with the E-box motif, we performed antibody coincubation experiments using the oligonucleotide NI as the probe. When nuclear extracts from SK-N-BE(2)C or HeLa cells were coincubated with an antibody against c-Myc, formation of C1 was not affected, and no supershifted band was observed. In contrast, coincubation of nuclear extracts with an antibody against USF1 diminished formation of C1 and produced supershi" @default.
• W2056565319 created "2016-06-24" @default.
• W2056565319 creator A5000185396 @default.
• W2056565319 creator A5056462099 @default.
• W2056565319 creator A5078091681 @default.
• W2056565319 date "2001-01-01" @default.
• W2056565319 modified "2023-10-18" @default.
• W2056565319 title "An E-box Motif Residing in the Exon/Intron 1 Junction Regulates Both Transcriptional Activation and Splicing of the Human Norepinephrine Transporter Gene" @default.
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