An E-box Motif Residing in the Exon/Intron 1 Junction Regulates Both Transcriptional Activation and Splicing of the Human Norepinephrine Transporter Gene*

Chun-Hyung KimDagger §, Paul ArdayfioDagger , and Kwang-Soo KimDagger

From the Dagger  Molecular Neurobiology Laboratory, McLean Hospital, Harvard Medical School, Belmont, Massachusetts 02478 and the § Department of Anatomy and Neurobiology, University of Tennessee, College of Medicine, Memphis, Tennessee 38163

Received for publication, February 9, 2000, and in revised form, March 20, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 a cis-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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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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 (1). 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 (2). 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 (3). 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 (4). It is of great interest that this mutation is associated with hyperadrenergic states, leading to orthostatic intolerance (4). 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 (4, 5).

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 (6, 7). Several studies suggest that both short and long term function of NET may be modulated by metabolic hormones, such as insulin and thyroid hormone (8-10). 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 (11, 12). 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 (13). The NET mRNA is transiently elevated in locus coeruleus neurons following either kainic acid-induced status epilepticus (14) or penytylenetetrazol-induced seizures (15). 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 (16). Recently, we have characterized the structural organization of the 5' upstream promoter region of the hNET gene (17). 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.

    EXPERIMENTAL PROCEDURES
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INTRODUCTION
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Cell Culture and Transient Transfection Assays-- 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 (18, 19). 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-beta -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 beta -galactosidase. CAT and beta -galactosidase activities were assayed as previously described (18, 19).

Plasmid DNA Constructions-- The constructions of pNET1400CAT and pNET1400(i)CAT were described previously (17). 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 (20) 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 and XhoI, 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 and XhoI to generate pNET1400(iDelta 190-645)CAT. In order to subclone the PCR fragments into the pNET1400(iDelta 190-645)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(iDelta 190-645)CAT. Nucleotide sequences of primers used in PCR were 5'-TGCCCACTAGTTCGGTGAGTTCAATCCCAGC-3' and 5'-TGCTTTCTAGAAGGGAAAAGAGGTGGTTACC-3' for pNET1400(iDelta 381-645)CAT, 5'-CCGGACACGTGAGTGCGCACTAGTCCTGAGCGCGGGACAGGGCTAGGT-3' and 5'-TGCCCTCTAGAGCTGGGATTGAACTCACCGA-3' for pNET1400(iDelta 190-362)CAT, 5'-CCGGACACGTGAGTGCGCACTAGTCCTGAGCGCGGGACAGGGCTAGGT-3' and 5'-TTGAGACTAGTCGTGCCCCAACCTCTGTTTC-3' for pNET1400(iDelta 190-482)CAT, and 5'-CCGGACACGTGAGTGCGCACTAGTCCTGAGCGCGGGACAGGGCTAGGT-3' and 5'-GGAAATCTAGAGAAACAGAGGTTGGGGCACG-3' for pNET1400(iDelta 190-362/ 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.

Preparation of Nuclear Extracts-- Nuclear extracts were prepared from SK-N-BE(2)C and HeLa cells according to the procedure described by Dignam et al. (21). The pellet was resuspended in Buffer D (20 mM HEPES, pH 7.9, 20% glycerol, 0.1 M KCl, 0.2 mM phenylmethylsulfonyl 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 (22).

DNase I Footprinting Analysis-- 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 [gamma -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 (23). 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 mM MgCl2, 50 mM NaCl, 1 mM dithiothreitol, 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.

Electrophoretic Mobility Shift Assay-- 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 (24, 25). The sense and antisense oligonucleotides were annealed, gel-purified, and 32P-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 (26). Competition binding assays were performed by adding nonradioactive competitor oligonucleotides in a molar excess before adding 32P-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).

Reverse Transcription-PCR (RT-PCR)-- 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 (23). 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 beta -actin gene messages as a control. Primers for the beta -actin gene were 5'-GGTCAGAAGGACTCCTATGTG-3' (sense) and 5'-TGTAGCCACGCTCGGTCAGG-3' (antisense).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The First Intron of the Human NET Gene Increases Activities of Both the Homologous NET and the Heterologous TK Promoters in a Position- and Orientation-dependent Manner-- 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. 1A). 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. 1B), 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. 1B).


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Fig. 1.   Position effect of the first hNET intron. A, the intron was placed in different positions in the context of the homologous hNET promoter. The bent arrow represents the major transcription start site of hNET, which had been determined previously (17). The boldface line denotes the 5'-untranslated leader sequence of the hNET gene. The first intron is shown in a dotted box. The structure of each promoter construct is shown at the left side of the figure. Constructs were transiently expressed in SK-N-BE(2)C (black bar) and HeLa cells (open bar). CAT activities were standardized relative to beta -galactosidase activity driven by Rous sarcoma virus-LacZ plasmid as an internal control for variation in transfection efficiencies. The normalized CAT activities are expressed relative to pNET1400CAT containing 1400 bp of hNET promoter, assigned a value of 1.0. This experiment was repeated in triplicate using independently prepared plasmid DNAs, resulting in similar patterns. In the native position (pNET1400(i)CAT construct), the first intron increased the transcription of the NET promoter in both SK-N-BE(2)C and HeLa cell lines, but not when placed in the 5' upstream site of the hNET promoter or in the 3' position of the CAT gene. B, the first intron was fused to different positions in conjunction with the heterologous thymidine kinase promoter. The CAT constructs were co-transfected with Rous sarcoma virus LacZ plasmid into SK-N-BE(2)C (black bar) and HeLa cells (open bar). The CAT activities normalized by beta -galactosidase activity are expressed relative to pBLCAT2, assigned a value of 1.0. The first intron exerted a prominent enhancer function to the TK promoter when placed in the untranslated region. However, it again failed to show any enhancer activity either in the 5' or in the 3' location. Comparable patterns were observed in SK-N-BE(2)C and HeLa cell lines.

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-fold versus 11-fold; Fig. 2A). 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. 2B). 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.


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Fig. 2.   Orientation effect of the first hNET intron. A, the intron was placed in different orientations relative to the hNET promoter. The bent arrow represents the major transcription start site of hNET. The boldface line denotes the 5'-untranslated leader sequence of the hNET gene. The first intron is shown in a dotted box. The arrow indicates the orientation of the first intron. Levels of CAT reporter gene activity were determined by transient transfection assays into SK-N-BE(2)C cells and expressed relative to that of pNET1400CAT, assigned a value of 1.0. When residing in the 5'-untranslated area, the first intron exerted a prominent enhancer function only in the correct orientation. B, SK-N-BE(2)C cells were transfected with pNET1400(i)CAT and pNET1400(i-rev)CAT plasmids, and the resultant mRNAs were subjected to RT-PCR, as described under "Experimental Procedures." The size of amplified products was ascertained by electrophoresis on a 7% polyacrylamide gel. Lane 1, cells transfected with pNET1400(i)CAT plasmid; lane 2, cells transfected with pNET1400(i-rev)CAT plasmid. The beta -actin gene was used as a control. RT-PCR detected an mRNA with a size of 176 bp, as expected from a proper splicing event.

Potential Regulatory Domains Residing in the First hNET Intron-- 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(iDelta 190-362)CAT) decreased the CAT expression by ~40%, suggesting that this subdomain contains positive regulatory sequences. Interestingly, CAT expression driven by pNET1400(iDelta 190-482)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(iDelta 381-645)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(iDelta 190-645)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(iDelta 190-645)CAT further decreased the CAT expression (construct pNET1400(iDelta 190-362/501-645)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(iDelta 190-645)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).


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Fig. 3.   The activity of plasmids bearing deletions in the area of the hNET first intron. Several 5' and 3' first intron deletion mutants in the context of the upstream 1400-bp sequence were transiently transfected into SK-N-BE(2)C (black bar) and HeLa cells (open bar). The normalized CAT activity driven by pNET1400CAT in each cell line was set to a value of 1.0 to compare the effect of each mutation. Transfections were performed in triplicate in at least three independent experiments. The positive regulatory region consists of at least two distinct subdomains, which are present at +181 to +190 bp and +482 to +656 bp. In addition, the sequences between +362 and +481 bp appear to contain a subdomain mediating negative control on transcription.

Upstream Stimulatory Factors 1 and 2 Specifically Interact at the Splicing Junction Area of the First hNET Intron-- 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.


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Fig. 4.   DNase I footprinting analysis of the first intron region of the hNET gene. Nuclear extracts were prepared from SK-N-BE(2)C and HeLa cells and used for DNase I footprinting of the first intron area of the hNET gene. The coding strand probe was labeled as described under "Experimental Procedures." The sequencing reaction mixtures (lanes 1-4) were run on the left side of the autoradiogram. Each labeled probe was digested with DNase I in the absence (lane 5) or presence of nuclear extracts prepared from SK-N-BE(2)C (lane 6) and HeLa cells (lane7), as described under "Experimental Procedures." The nucleotide sequence and number on the right side represent the protected region. Uppercase letters indicate the nucleotide sequences in exon, and lowercase letters represent the nucleotide sequences in intron. Nuclear proteins from both SK-N-BE(2)C and HeLa cells footprinted the same area, which contains a consensus E-box (CACGTG) and splicing donor site.

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 (27, 28). 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. 5B). 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. 5B). 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. 5C). 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.


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Fig. 5.   Sequence-specific binding of protein to the region protected by DNase I footprinting analysis. A, oligonucleotides used in gel shift assays to identify factors binding to the first intron. The oligonucleotide NI contained the sequence from +169 to +192 of the hNET gene, including the consensus E-box (underscored). E-box consensus oligonucleotide (UI) was used as unlabeled competitors. The oligonucleotide NIm contained the same sequence as NI except for 2 base pairs mutated as indicated by lowercase. B, electrophoretic mobility shift assays were conducted using nuclear extracts from SK-N-BE(2)C cells (left) or HeLa cells (right) with labeled probes for the NI (lanes 1 and 4), UI (lanes 2 and 5), and NIm oligonucleotides (lanes 3 and 6). A sequence-specific complex (C1) was formed by both nuclear extracts as indicated by the arrow at left. Mutant oligonucleotide NIm failed to form the sequence-specific complex with the nuclear extracts from both SK-N-BE(2)C cells or HeLa cells. A nonspecific complex was observed with the nuclear extracts from HeLa cells, as shown by the arrow at right. Unbound free probe (F) is indicated by an arrowhead. C, electrophoretic mobility shift assays were performed using nuclear extracts from SK-N-BE(2)C cells (left) or HeLa cells (right) with 32P-labeled NI oligonucleotide. DNA-protein complex formed with nuclear extracts and the NI oligonucleotide was competed by molar excesses of NI or UI unlabeled nucleotides but not by the mutant form of NI (NIm), SP1, or AP2 consensus sequence.

USF1 and USF2 Interact with a Consensus E-box Site Located in the First hNET Intron-- 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 supershifted complexes (Fig. 6, lanes 2 and 8). When nuclear extracts were incubated with anti-USF2 antibody (Fig. 6, lanes 3 and 9), complex C1 was modestly diminished, and supershifted complexes were formed to a lesser degree than the USF1 antibody. Coincubation with antibodies against Sp1 or AP2 neither diminished the complex C1 nor generated a supershifted band (Fig. 6, lanes 4-6 and 10-12). Based on these results, we conclude that USF1 and USF2, but not c-Myc, participate in formation of the C1 complex via interacting with the E-box motif residing at the junction area of the first hNET intron.


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Fig. 6.   Both USF1 and USF2 bind to the E-box in the exon/intron 1 junction of the hNET gene. A mobility shift assay was performed using NI as a radiolabeled probe and incubated with 10 µg of SK-N-BE(2)C nuclear extract (lanes 1-6) and 14 µg of HeLa nuclear extract (lanes 7-12). 1 µg of anti-USF1 antibody was added to lanes 2 and 8, and 1 µg of anti-USF2 antibody was added to lanes 3 and 9. Anti-c-Myc, anti-Sp1, and anti-AP2 antibodies were added to lanes 4 and 10, lanes 5 and 11, and lanes 6 and 12, respectively. The main band (C1) resulting from the interaction between the oligonucleotide NI and nuclear extract was partially supershifted (asterisks) by anti-USF1 and anti-USF2 antibodies but not by anti-c-Myc, anti-Sp1, or anti-AP2 antibodies.

A Single cis-Regulatory Element Encompassing the E-box Motif and the Splicing Donor Site of the First hNET Intron Regulates Both Splicing and Transcriptional Enhancement of hNET mRNA-- Given that the E-box motif precisely overlaps with the splice donor site of the first intron, the cis-element encompassing these sites may regulate both splicing and basal transcriptional activation of the hNET gene. To address this possibility, we generated two different mutant reporter gene constructs and examined them by transient transfection assay and RT-PCR analysis of mRNAs isolated from transfected cells. In one of these mutant constructs (pNET1400(iCaCg)CAT), a double point mutation was introduced into the E-box core motif (CACGTG to aAgGTG). Whereas this mutation completely disrupted the interaction with USF1 and USF2 (Fig. 5A, lanes 3 and 6), the splicing donor motif was still intact. In contrast, the other construct (pNET1400(iTaGc)CAT) harbors a double mutation right at the consensus splicing donor site, which disrupted its interaction with USF1 and USF2 (data not shown) and, presumably, also affected the proper splicing of the first intron. By analyzing and comparing mRNAs from cell lines transfected with the wild type and mutant reporter constructs, we sought to address the importance of different nucleotides of the E-box motif on the enhancer activity and/or on splicing mechanisms (Fig. 7). As shown in Fig. 7B, reporter gene expression driven by either mutant construct was ~40% of that driven by the wild type construct. This observation clearly demonstrates that interaction of the E-box motif with USF1 and USF2 is important for the promoter enhancing activity of the first hNET intron. When mRNAs isolated from transfected cells were analyzed by RT-PCR, it was shown that only a properly spliced transcript (170 bp) was detected from mRNAs isolated from both wild type and pNET1400(iCaCg)CAT constructs (Fig. 7C). In contrast, pNET1400(iTaGc)CAT generated an aberrant transcript of 360 bp that resulted from retention of 184 bp of intronic sequence. Sequence analysis of this transcript showed that the mutation in the donor splice site at E-box led to the use of a cryptic donor splice site (gt) starting at nucleotide position +365 (Fig. 7C).


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Fig. 7.   Effect of mutating the conserved E box binding in exon/intron 1 junction on enhancer activity. A, schematic diagram showing the cis-element residing at the junction area of the first intron and exon, encompassing the E-box motif and splice donor site, that was identified by DNase I footprinting analysis. Nucleotides of the first exon are indicated by uppercase letters, and those of the intron are indicated by lowercase letters. The consensus splice donor site is denoted by an open box, and the E-box motif is underlined. B, SK-N-BE(2)C cells were transiently transfected with the wild type (pNET1400(i)CAT) and mutated constructs as indicated. CAT activities were normalized to the beta -galactosidase activity driven by the co-transfected Rous sarcoma virus-LacZ plasmid. The normalized CAT activity driven by the intron-less construct (pNET1400CAT) was set to 1 to compare the enhancing activity of the wild type and mutated intron. Reporter gene expression driven by either mutant construct was ~40% of that by the wild type construct, indicating that interaction of the E-box motif with USF1 and USF2 is important for the promoter enhancing activity of the first hNET intron. B, SK-N-BE(2)C cells were transfected with wild type and mutated plasmids, and the resultant mRNAs were subjected to RT-PCR as described under "Experimental Procedures." Amplified products were subjected to 7% polyacrylamide gel electrophoresis. Lane 1, cells transfected with wild type plasmid; lane 2, cells transfected with the pNET1400(iCaCg)CAT plasmid; lane 3, cells transfected with the pNET1400(iTaGc)CAT plasmid. An mRNA species was detected with a size exactly matching that expected from a proper splicing event from both wild type and E-box motif mutant constructs (pNET1400(iCaCg)CAT). In contrast, the other construct (pNET1400(iTaGc)CAT), which harbors a double mutation that also targeted the consensus splicing donor site, prevented proper splicing of the first intron and led to the use of a cryptic donor splice site, which is indicated in boldface.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have previously reported that ~9 kb of the 5' flanking sequences of the hNET gene can confer cell specificity but require the presence of an intronic enhancer for high level reporter gene expression (17). In the present study, we investigated the functional and structural properties of the first hNET intron to further delineate control mechanisms affecting hNET gene regulation. Our results reveal several salient features of the first intron in NET gene regulation, as follows.

Our analysis of the first hNET enhancer in the context of either the homologous NET promoter or the heterologous TK promoter demonstrated that it enhances the transcriptional activity of both promoters in an orientation- and position-dependent manner (Figs. 1 and 2). This sequence therefore does not belong to the category of classical enhancers, the action of which are independent of position, orientation, or distance in relation to the transcription start site (29). It is increasingly recognized that transcription of some mammalian genes can be influenced by intronic sequences (30-35), and many of them contain important regulatory sequences in the first intron. For example, the first intron (1.8 kb) of the aldolase B gene was shown to be crucial for tissue-specific expression, independent of position and orientation (31). On the other hand, in many other genes the first intron was shown to enhance or silence the transcriptional activity in an orientation- and position-dependent manner (36-41). It may be presumed that correct and efficient transcriptional regulation of these genes may require specific spatial and directional arrangement between protein factors binding to the intronic sequences and other regulatory elements residing in the 5' region or near the transcription start site. In this scenario, misplacement of the intronic sequences may disrupt the normal spatial geometry of protein-protein interactions required for correct transcriptional regulation. In addition, if an important cis-element resides exactly in the exon/intron junction, the structural integrity of the first intron may be crucial for intact enhancer (or silencer) activity. The latter possibility appears to apply to the first hNET intron.

Deletional analysis identified two subdomains with positive regulatory function at +181 to +190 bp and at +482 to +656 bp (Fig. 3). Surprisingly, a middle subdomain at +362 to +501 bp was found to have a negative regulatory function. Therefore, the robust enhancing activity of the first hNET intron seems to be a net effect of several subdomains with either positive or negative regulatory function. At present, the exact physiological role of the negative subdomain remains largely a matter of speculation. In several genes that are known to have both positive and negative elements in the first intron, the combined action of these elements has been shown to be important for cell type-specific gene expression (42-44).

Although several subdomains appear to participate in the regulatory function of the first intron, only one domain was prominently protected in our DNase I footprinting assay using nuclear proteins from SK-N-BE(2)C and HeLa cells (Fig. 4). The protected area encompassed a consensus E-box motif (Fig. 4), and detailed analyses of DNA-protein interaction showed that the basic helix-loop-helix transcription factors USF1 and USF2 interact with this site (Figs. 5 and 6). Site-directed mutational analyses demonstrated that interaction of this E-box motif with USF1/USF2, at least in part, underlies the enhancer activity of the first hNET intron. USF, originally purified as a complex from HeLa cells (45-47), is composed of two polypeptides of 43 and 44 kDa. Protein factors belonging to the USF family homodimerize and heterodimerize with each other and thus are able to generate diverse sets of complexes with potentially different transcriptional capacity (48, 49). However, it is to be noted that an in vivo footprinting analysis is required to absolutely prove that the E-box motif is bound in the endogenous NET gene, which awaits further investigation. In addition, interaction of USF1/USF2 with the E-box has been shown to alter the transcriptional activity of the eukaryotic genes in response to various physiological stimuli. For example, the interaction of USF and E-box is required for insulin regulation of the fatty acid synthase gene transcription (50). In USF knockout mice, USF factors have been shown to be involved in transcriptional regulation of liver-specific genes in response to glucose (51, 52). Thus, it is likely that USF1 and USF2 may co-operatively regulate NET gene expression in the presence and/or absence of physiological stimuli.

The E-box motif interacting with USF1 and USF2 resides at the first exon/intron junction and overlaps with the consensus splice donor site. This observation suggested the intriguing possibility that this region may control not only transcriptional activation but also splicing of hNET mRNA. To address this possibility, we generated mutant reporter constructs in which different nucleotides of the E-box motif were mutagenized. When the mutation blocked binding of USF factors without affecting the consensus splice donor site, the transcription enhancing activity was diminished, but splicing was intact. There was no evidence for an altered splicing pattern with the USF binding site mutations. This result indicates that reduced CAT activity does not result from the defective splicing event. However, when the mutation changed the splicing donor site, not only was the transcriptional enhancing activity similarly diminished, but an aberrant transcript of 360 bp was generated instead of the correct 174-bp transcript. Sequencing analysis of this transcript showed that the mutation in the splice donor site, which resides within the E-box motif, led to the use of a cryptic donor splice site (gt) starting at nucleotide position +365. Taken together, these results demonstrate that a cis-element residing at the junction of the first exon and intron, encompassing the splice donor site and an E-box motif, has a unique dual role in splicing and transcriptional activation of NET mRNA and that the latter involves the interaction with transcription factors USF1 and USF2. To our knowledge, this is the first report that a single cis-element regulates both transcription and splicing of mRNA. However, based on the potential of the consensus splicing donor site (5'-GT-3') to coincide with critical cis-elements, such as E-box (5'-CACGTG-3') and consensus binding site for TEF-2 (5'-GGGTGTGG-3') (53), it is possible that more genes may contain a similar sequence motif(s) with regulatory functions in both transcription and splicing.

    ACKNOWLEDGEMENT

We thank Dr. Thaddeus Nowak at the College of Medicine, University of Tennessee, for critically reading the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant MH48866 and a National Alliance for Research on Schizophrenia and Depression (NARSAD) Independent Award (to K. S. K.), a Ford Foundation Predoctoral fellowship (to P. A.), and an LG Chem. Inc. fellowship (to C. H. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Molecular Neurobiology Laboratory, McLean Hospital, Harvard Medical School, 115 Mill St., Belmont, MA 02478. Tel.: 617-855-2024; Fax: 617-855-2023; E-mail: kskim@mclean.harvard.edu.

Published, JBC Papers in Press, May 1, 2001, DOI 10.1074/jbc.M101279200

    ABBREVIATIONS

The abbreviations used are: NE, norepinephrine; NET, norepinephrine transporter; hNET, human NET; CAT, chloramphenicol acetyltransferase; TK, thymidine kinase; PCR, polymerase chain reaction; USF, upstream stimulatory factor; RT, reverse transcription; bp, base pair(s); kb, kilobase(s).

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