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Originally published In Press as doi:10.1074/jbc.M109802200 on April 17, 2002

J. Biol. Chem., Vol. 277, Issue 28, 25798-25814, July 12, 2002
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Complex Regulation of Human Neuronal Nitric-oxide Synthase Exon 1c Gene Transcription

ESSENTIAL ROLE OF Sp AND ZNF FAMILY MEMBERS OF TRANSCRIPTION FACTORS*

Dieter SaurDagger, Barbara Seidler, Heidi Paehge, Volker Schusdziarra, and Hans-Dieter Allescher

From the Department of Internal Medicine II, Technische Universität München, Munich 81675, Germany

Received for publication, October 10, 2001, and in revised form, April 2, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Neuronal nitric-oxide synthase (nNOS) is expressed in a variety of human tissues and shows a complex transcriptional regulation with the presence of nine alternative first exons (1a-1i) resulting in nNOS transcripts with differing 5'-untranslated regions. We previously demonstrated that nNOS exon 1c, one of the predominant transcripts in the human gastrointestinal tract, is driven by a separate promoter (Saur, D., Paehge, H., Schusdziarra, V., and Allescher, H. D. (2000) Gastroenterology 118, 849-858). The present study focused on the quantitative expression of nNOS first exon variants in different human tissues and the characterization of the basal nNOS exon 1c promoter. In human brain, skeletal muscle, colon, and TGW-nu-I neuroblastoma cells, first exon expression patterns were analyzed by quantitative real-time reverse transcription-PCR. In these tissues/cells exon 1c was one of the most abundant first exons of nNOS. By transient transfections of TGW-nu-I and HeLa cells with reporter plasmids containing a series of 5' and 3' deletions in the exon 1c regulatory region, the minimal TATA-less promoter was localized within 44 base pairs. Gel mobility shift assays of this cis-regulatory region revealed a high complexity of the basal promoter with a cooperative binding of several transcription factors, like Sp and ZNF family members. When the Sp binding site of the minimal promoter construct was mutated, promoter activity was completely abolished in both cell lines, whereas mutation of the common binding site of ZNF76 and ZNF143 resulted in a decrease of 53% in TGW-nu-I and 37% in HeLa cells. In Drosophila Schneider cells expression of Sp1, the long Sp3 isoform, ZNF76 and ZNF143 potently transactivated the nNOS exon 1c promoter. These results identify the critical regulatory region for the nNOS exon 1c basal promoter and stress the functional importance of multiple protein complexes involving Sp and ZNF families of transcription factors in regulating nNOS exon 1c transcription.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nitric oxide (NO),1 a ubiquitous multifunctional mediator, is synthesized by nitric-oxide synthases (NOS) during the oxidation of L-arginine to L-citrulline. In the central and peripheral nervous system, skeletal muscle, the macula densa of the kidney, testis, and neutrophils, neuronal NOS (nNOS) is the predominant enzyme for the generation of nitric oxide. NO, synthesized by nNOS, acts as neurotransmitter, neuromodulator, or intracellular signaling molecule. It is involved in synaptic plasticity, regulation of gene expression, development, differentiation, and regeneration and plays an important role in neurodegenerative disorders and stroke as a mediator of neurotoxicity (for review see Refs. 1-4). In the gastrointestinal tract NO generated by nNOS acts as an important mediator of the non-adrenergic non-cholinergic inhibitory innervation of intestinal smooth muscle (5) and as a neuromodulator within the enteric nervous system (6).

Although the transcriptional regulation of the other two NOS enzymes, the calcium-dependent endothelial NOS and the calcium-independent inducible NOS, are extensively studied (for review see Refs. 3, 7, 8), little is known about the transcriptional regulation of the nNOS gene (3, 4, 9, 10), which is considered to be responsible for the largest proportion of NO in the body (1). Although usually named constitutive, recent observations suggest a tightly regulated gene expression of nNOS in response to different physiological and pathophysiological stimuli, resulting in an up- or down-regulation of nNOS mRNA (3, 9).

Recently nine distinct first exons, called exons 1a-1i, of nNOS mRNA have been identified, leading to nNOS mRNA variants with different 5'-untranslated regions and translational efficiencies (11). The nNOS gene is therefore believed to be one of the most complex genes known in terms of first exon usage and alternative splicing (11-13). It has been shown that nNOS exons 1c (12) and 1f and 1g (13) (former called exons 15'3, 15'2, and 15'1, respectively), which show high abundant expression in the human gastrointestinal tract (12), are driven by separate promoters in HeLa cells. The use of multiple alternative promoters allows a cell-, tissue-, and site-specific transcriptional regulation of nNOS in different physiological and pathophysiological stages.

An altered expression or biological activity of nNOS has been linked to several physiological conditions, like aging and pregnancy, as well as different pathophysiological conditions and diseases such as ischemia/hypoxia and injuries of the central nervous system, inherited diabetes insipidus, heart failure, arteriosclerosis, achalasia, diabetic gastroparesis, and hypertrophic pyloric stenosis (1-4, 7, 14-17). nNOSalpha mutant mice, generated by targeted disruption of the nNOS gene by homologous recombination, showed a gastrointestinal phenotype resembling hypertrophic pyloric stenosis with delayed gastric emptying of solids and fluids (18, 19). In addition, these mice have a hypertensive lower esophageal sphincter with impaired relaxation (20). A recent study comprising 27 families with inherited infantile pyloric stenosis identified nNOS as a susceptibility gene for this disorder (15), and expression of nNOS exon 1c mRNA is significantly reduced in the pyloric sphincter of patients with infantile hypertrophic pyloric stenosis.2

Therefore it is of physiological and pathophysiological interest to investigate the molecular basis of nNOS exon 1c transcription. In addition, the distribution and quantitative expression of alternative first exon nNOS variants in different human tissues was determined.

Here we present evidence for a tissue-specific expression of nNOS first exon variants, with exon 1c being one of the predominant forms in human brain, skeletal muscle, gastrointestinal tissue, and neuroblastoma cells. We furthermore characterize the basal promoter of exon 1c, showing a high complexity with a cooperative binding of several Sp and ZNF family members of transcription factors.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- The cell lines TGW-nu-I (human neuroblastoma) and ME-180 (human cervix carcinoma) were kindly provided to our laboratory by Dr. Esumi (21) and Dr. E. R. Werner (22), respectively. The mammalian expression plasmids pcDNA3 ZNF76 and pcDNA3 143 were a gift from Dr. P. Carbon (23), and CB6-MZF-1 (alternative name CB6-ZNF42), under the control of the cytomegalovirus early promoter, was generously provided by Dr. R. Hromas (24). The plasmid pPac-Sp1, which expresses Sp1 from the Drosophila actin promoter, and the "empty" control plasmid pPac0, containing only the Drosophila actin promoter, were generously provided by Dr. R. Tjian (25). The expression vector for Sp2 (pPac-Sp2) was a gift from Dr. J. D. Noti (26), and the expression plasmids for the short isoforms of Sp3 (pPac-Sp3), the long isoform of Sp3 (pPacUSp3), Sp4 (pPac-Sp4), and the beta -galactosidase expression plasmid p97b were generously provided by Dr. G. Suske (27-30). All cell culture reagents were obtained from Invitrogen (Groningen, Netherlands). Polyclonal antibodies against Sp1 (PEP 2 X), Sp2 (K-20 X), Sp3 (D-20 X), Sp4 (V-20 X), NFkappa B p65 (C-20 X), NF-kappa B p50 (C-19 X), Ap-2a (C-17 X) were purchased from Santa Cruz Biotechnologies (Heidelberg, Germany). Consensus and mutant consensus oligonucleotides used in electrophoretic mobility shift assays (EMSAs) were obtained from Santa Cruz Biotechnologies (Ap2, Myc-Max, NF-I, YY1, NFkappa B, p53, Sp1, USF-1), or synthesized (Staf, Olf-1, ZNF42, MAZ) by MWG (Ebersberg, Germany). Primers were made by MWG and TaqMan-probes by Applied Biosystems (Weiterstadt, Germany). Restriction endonucleases were obtained from New England BioLabs (Mannheim, Germany). [gamma -32P]ATP was supplied from Amersham Biosciences, Inc. (Freiburg, Germany). The Escherichia coli strain TOP10 (Invitrogen) was used for transformation and amplification of DNA-containing plasmids.

Tissue Preparation-- Tissues from human rectum were obtained from surgical resections for malignant disease and prepared as previously described (12).

RNA Isolation-- Liquid nitrogen-frozen rectum muscle layer preparations and fresh TGW-nu-I, ME-180, and HeLa cells were homogenized with a Polytron homogenizer (Kinematica, Switzerland), and total RNA was isolated using the guanidine isothiocyanate/phenol/chloroform extraction method as previously described (12, 31). Pooled total RNA from human adult brain and skeletal muscle was purchased from CLONTECH (Heidelberg, Germany).

RT-PCR-- Reverse transcription and PCR amplification were carried out exactly as described before (12, 31). RT-PCR was performed with specific primer pairs for ZNF42, ZNF76, and ZNF143 (for all primers see Table I) for 35 cycles using random hexamer-primed cDNA from human brain, skeletal muscle, rectum, TGW-nu-I cells, ME-180 cells, and HeLa cells (annealing 58 °C, 30 s; extension 72 °C, 60 s; denaturation 94 °C, 30 s). Amplification products were cloned into pCRII plasmid (Invitrogen) and subjected to DNA sequence analysis (GATC, Konstanz, Germany).

Quantitative Real-time RT-PCR-- Real-time quantitative RT-PCR analyses for the alternative first exons 1a-1i and exon 6/7 of human nNOS mRNA were performed using an ABI Prism 7700 Sequence Detection System instrument (Applied Biosystems). cDNAs for quantitative analysis were generated with the TaqMan reverse transcription reagents (Applied Biosystems) as recommended by the manufacturer, using murine leukemia virus reverse transcriptase, random hexamer primers, and 5 µg of total RNA from human rectum and from TGW-nu-I, ME-180, and HeLa cells. In addition, total RNA obtained from CLONTECH and isolated from pooled human brain and skeletal muscle was used. nNOS transcripts were amplified with intron-spanning, isoform-specific primers and probes complementary to the alternative first exons 1a-1i (forward primers) and the common exon 2 (reverse primer, probe). As a parameter for total nNOS mRNA expression, an intron-spanning pair of exon 6- and exon 7-specific primers present in all known human nNOS cDNAs was used with an exon 7-specific internal probe. Primers and TaqMan probes were designed to meet specific criteria by using the Primer Express software (Applied Biosystems). The 5'-end of the probe was labeled with a fluorescent reporter (6-carboxy-fluorescein (FAM)) and the 3'-end with a quencher dye (6-carboxyltetramethylrhodamine). Sequences of primers and fluorescent probes used in the study are shown in Table I. GAPDH, HPRT, and TF2D primers and probes were purchased from Applied Biosystems.

The principle of real-time RT-PCR has been described in detail elsewhere (32, 33). Briefly, real-time PCR is based on a sequence-specific probe labeled with a 5' reporter and 3' quencher dye. When the probe is intact, reporter dye emission is quenched, but during the extension phase of the PCR, the nucleolytic activity of the Taq-DNA polymerase cleaves the hybridized probe, and due to the separation of reporter and quencher dye a fluorescence signal is released that is monitored by the sequence detector. A computer algorithm normalizes the signal to an internal reference (Delta Rn) and calculates the threshold cycle number (Ct), when Delta Rn becomes equal to 10 standard deviations of the baseline. Ct is used for quantification of the input mRNA number. For each amplicon, the amount of target and endogenous reference (glyceraldehyde-3-phosphate dehydrogenase, HPRT, TF2D) was determined from a standard curve generated by serial 5-fold dilutions (25,000 to 8 copies) of plasmids containing the respective target sequence. The standard curve was amplified in triplicate during every experiment, and the amount of target gene was normalized by the endogenous reference. Quantitative PCR was performed using the TaqMan Universal PCR Master Mix (Applied Biosystems), with cDNAs corresponding to 100 ng of total RNA, 200 nM probe, and 900 nM primers in a 25-µl final reaction mix (1 PCR cycle 50 °C, 2 min; 95 °C, 10 min; 50 PCR cycles 60 °C, 1 min; 95 °C, 15 s). Signals were analyzed by the ABI Prism Sequence Detection System software version 1.7 (Applied Biosystems).

Cloning of the 5'-Flanking Region of Exon 1c-- The 5'-flanking region of human nNOS exon 1c was determined by rapid amplification of genomic ends (RAGE) using the human GenomeWalker kit (CLONTECH) as previously described (12). Digested and adapter-ligated genomic DNA fragments obtained from CLONTECH were amplified in a first round of PCR (seven cycles 94 °C, 25 s; 72 °C, 7 min and 32 cycles 94 °C, 25 s; 67 °C, 7 min adding 5 s each cycle) using the antisense gene-specific primer GSP1-AS/ex 1c (10 pmol) (for all primers see Table I) and the sense generic primer AP1 from CLONTECH specific for the ligated adapters. The second round of PCR was performed with nested antisense gene-specific primer GSP2-AS/ex 1c (10 pmol) and sense generic primer AP2 (5 cycles 94 °C, 25 s; 72 °C, 7 min; 20 cycles 94 °C, 25 s; 67 °C 7 min adding 5 s each cycle). PCR products were cloned into pCRII plasmid and subjected to commercial sequence analysis (GATC). The BLASTn program was employed to search for sequence identity. Potential cis-acting DNA sequences and putative consensus elements were identified by analysis with the MatInspector professional software (Genomatix Software GmbH, Munich, Germany) using the Transfac data base.

                              
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Table I
Sense (S) and antisense strand (AS) primers and TaqMan probes
Sequences of primers and probes for RT-PCR, real-time RT-PCR, 5'-RAGE-PCR, constructions of pPac-ZNF42, pPac-ZNF76, and pPac-ZNF143, generation of 5' deletions of pGL3-5891/+49, point mutations of pGL3-90/+49, and site-directed mutagenesis of pGL3-5891/+49 and pGL3-332/+49. Boldface letters indicate mutated bases.

Plasmid Constructions-- The human genomic 5'-flanking region of nNOS exon 1c was obtained by 5'-RAGE PCR. To create a 5940-bp fragment containing the promoter of exon 1c (nt -5891 to +49), 100 ng of genomic DNA was PCR-amplified (30 cycles at 94 °C, 20 s; 60 °C, 30 s; 72 °C, 7 min) using a proofreading polymerase (Pwo, Roche Molecular Biochemicals, Mannheim, Germany) and the primers P-5891 (sense) and P+49 (antisense) (see Table I for primer sequences). The gel-purified PCR product was blunt end-cloned into the SmaI site of the promoter-/enhancerless firefly luciferase reporter gene vector pGL3-basic (Promega, Mannheim, Germany) in the forward (pGL3-5891/+49) and reverse (pGL3+49/-5891) orientation. Reporter gene constructs containing 5' and 3' deletions of the promoter region of nNOS exon 1c were generated by PCR, exonuclease III/S1 nuclease digestion, or restriction endonuclease digestion. pGL3-2774/+49, pGL3-1938/+49, pGL3-1520/+49 and pGL3-332/+49 were prepared by PCR and blunt end cloning similar to the preparation of pGL3-5891/+49, using the sense primers P-2774, P-1938, P-1520, and P-332 combined with the common antisense primer P+49. For construction of pGL3-5891/-279, sense primer P-5891 was used with antisense primer P-279. pGL3-226/+49 was prepared by restriction endonuclease digestion of pGL3-332/+49 with PflmI and MluI, followed by blunting and religation. Additional 5' deletions of pGL3-332/+49 (pGL3-278/+49, pGL3-241/+49, pGL3-174/+49, pGL3-131/+49, pGL3-90/+49, pGL3-83/+49, pGL3-63/+49, pGL3-48/+49, pGL3-34/+49, pGL3-24/+49, pGL3-14/+49, pGL3-4/+49, pGL3 + 18/+49), were prepared by successive exonuclease III and S1 nuclease digestions and re-ligations. pGL3-90/+49 Sp1/ZNF42-M1, containing a point mutation of the common Sp1 and ZNF42 binding site, pGL3-90/+49 Sp1-M2, containing only a mutated Sp1 element, pGL3-90/+49 Ap2/Olf-1-M, containing a mutation of the common Ap2 and Olf-1 binding site, and pGL3-90/+49 Staf-M, containing a mutated Staf consensus sequence (common binding site for ZNF76 and ZNF143), were prepared by PCR using the common antisense primer P+49 combined with the following sense primers: P-90 Sp1/ZNF42-M1, P-90 Sp1-M2, P-90 Ap2/Olf-1-M, and P-90 Staf-M. Gel-purified PCR products were blunt end-cloned into pGL3-basic in the forward orientation. Mutations and deletions of the common Sp1/ZNF42 binding site, the Staf binding site, and both the Sp1/ZNF42 and Staf binding sites in the longer promoter constructs pGL3-5981/+49 and pGL3-332/+49 were generated by using the QuikChange XL site-directed mutagenesis kit (Stratagene, Heidelberg, Germany) exactly as described by the manufacturer with the following primers: SDM-Sp1/ZNF42-M1 (mutation of the Sp1/ZNF42 binding site), SDM-Sp1/ZNF42-del (deletion of the Sp1/ZNF42 binding site), SDM-Staf-M (mutation of the Staf binding site), SDM-Staf-del (deletion of the Staf binding site), SDM-Staf/Sp1/ZNF42-M1 (mutation of the Staf and Sp1/ZNF42 binding sites), and SDM-Staf/Sp1/ZNF42-del (deletion of the Staf and Sp1/ZNF42 binding sites). To construct plasmids that express the transcription factors ZNF42, ZNF76, and ZNF143 from the Drosophila actin promoter, the eukaryotic expression vectors CB6-ZNF42 (alternative name CB6-MZF-1), pcDNA3 ZNF76, and pcDNA3 ZNF143 were used as templates for PCR to generate DNAs containing the complete coding sequences of these transcription factors. Primers for ZNF42 contained XhoI, and primers for ZNF76 and ZNF143 contained BamHI restriction sites and a Kozak consensus sequence upstream of the ATG start codon. The XhoI and BamHI restriction sites were used to clone the coding sequences of ZNF42, ZNF76, and ZNF143 into the respective XhoI or BamHI sites of the expression plasmid pPac0, which contains only the Drosophila actin promoter. Integrity of all cloned sequences was confirmed by automated DNA sequencing (GATC) using an ABI Prism 377 DNA sequencer (Applied Biosystems).

Cell Culture, Transient Expression, and Reporter Gene Assays-- HeLa cells were cultured and transiently cotransfected with the different nNOS exon 1c-pGL3 promoter gene constructs and the herpes simplex virus thymidine kinase promoter-driven Renilla luciferase expression vector pRL-TK (Promega) to normalize for transfection efficiency and cell number essentially as described previously (12). TGW-nu-I cells were cultured in minimum Eagle's medium containing 10% fetal bovine serum (FBS), 25 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin sulfate (37 °C/5%CO2). 50-60% confluent cells were transiently cotransfected by lipid-mediated transfer using 0.95 µg of plasmid DNA of each pGL3 construct and 0.05 µg of pRL-TK DNA with 6 µl of Plus reagent and 4 µl of LipofectAMINE (Invitrogen) per 3.5-cm dish. Cells were incubated in FBS and antibiotic-free minimum Eagle's medium containing the lipid-coated DNA complexes for 3 h and in complete medium for an additional 48 h. ME-180 cells were cultured in McCoy's 5A medium as described before (22). About 60% confluent cells were transfected with 0.38 µg of each pGL3 test plasmid DNA and 0.02 µg of pRL-TK DNA using 4 µl of Effectene and 3.2 µl of Enhancer (Qiagen) per 3.5-cm dish.

Approximately 2 × 106 Drosophila Schneider cells (SL2 cells) were plated the day prior to transfection onto six-well plates and cultured at 25 °C in DES Expression Media containing 10% FBS. After washing and addition of fresh medium without FBS, cells were transfected using 9 µl of Cellfectin (Invitrogen) per well along with 1 µg of each pGL3 construct, 0.5 µg of the beta -galactosidase expression plasmid p97b for normalization of transfection efficiencies, and variable amounts of the expression plasmids pPac-Sp1, pPac-Sp2, pPac-USp3, pPac-Sp3, pPac-Sp4, pPac-ZNF42, pPac-ZNF76, and pPac-ZNF143. Variations in the amount of the expression plasmids were compensated with the empty plasmid pPac0 to adjust the total DNA content of the transfection mix to 2 µg per well. After 24 h of transfection, medium was removed and cells were incubated for an additional 24 h in complete medium containing FBS. SV40 promoter/enhancer-directed pGL3-control and promoter-/enhancerless pGL3-basic plasmids (both Promega) were used as positive and negative controls in all experiments, respectively.

48 h after transfection, cells were harvested by treatment with lysis buffer (Promega). Total cellular protein was determined by Bio-Rad II assay and beta -galactosidase activity was assayed using the beta -Galactosidase Enzyme Assay System (Promega) essentially as recommended by the manufacturer. Firefly and Renilla luciferase activities were measured in a luminometer (EG&G Berthold, Bad Wildbad, Germany) by using the Dual Luciferase Reporter Assay System (Promega) as previously described (12). All values were determined from three independent transfection experiments, each done in triplicate, and are expressed as mean values ± S.D. Data of TGW-nu-I and HeLa cell transfections are presented as relative luciferase activity of firefly/Renilla luciferase. Values for SL2 cells are expressed as -fold induction of normalized firefly luciferase activity relative to that obtained following cotransfection of the pGL3 reporter plasmids with empty pPac0, which does not express Sp/ZNF proteins. In SL2 cells firefly luciferase activity was normalized in all samples for total protein content, because the transcription of the beta -galactosidase expression vector p97b, intended as internal control, was highly inducible by cotransfection with pPac-ZNF76 and pPac-ZNF143 (but not with pPac-Sp1-4 and pPac-ZNF42).

Electrophoretic Mobility Shift Assays-- Nuclear extracts were prepared from cultured untransfected TGW-nu-I and HeLa cells and from Drosophila Schneider cells transiently transfected with the expression plasmids pPac-Sp1, pPac-Sp2, pPac-USp3, pPac-Sp3, pPac-Sp4, pPac-ZNF42, pPac-ZNF76, and pPac-ZNF143. Cells were harvested from 10-cm dishes by scraping, washed once with ice-cold PBS, centrifuged, resuspended in 400 µl of ice-cold buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) and incubated for 20 min at 4 °C. After addition of 25 µl of Nonidet P-40 (10%), the probes were vortexed for 10 s at top speed, followed by a centrifugation step (10,000 × g, 5 min, 4 °C). The pelleted nuclei were resuspended in ice-cold high salt buffer C (20 mM HEPES (pH 7.9), 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) and vigorously mixed on a shaker for 30 min at 4 °C to extract nuclear proteins. The samples were subjected to centrifugation (10,000 × g, 5 min, 4 °C) and the supernatant was stored at -80 °C. For EMSAs 10 µg of the appropriate nuclear extract was incubated in 20 µl of binding buffer (25 mM Tris-HCl (pH 8.0), 50 mM KCl, 6.25 mM MgCl2, 10% glycerol, 50 µg/ml bovine serum albumin, 1 µg of poly(dI-dC) (Amersham Biosciences, Inc.)) with 20 fmol of the indicated 32P-labeled double-stranded oligonucleotide probe and in certain experiments with unlabeled competitor oligonucleotides (see Table II and under "Materials"). 32P-Labeled probes were prepared by 5'-end labeling using T4 polynucleotide kinase and [gamma -32P]ATP. Reactions were incubated for 30 min at 22 °C and loaded onto a non-denaturing, 5% polyacrylamide gel (37:1 ratio of acrylamide to N,N'-methylenebisacrylamide) in 0.5 × Tris borate EDTA (TBE) buffer. Electrophoresis was performed at 250 V for 3 h in 0.5× TBE buffer with buffer recirculation at 4 °C, followed by autoradiography at -80 °C with intensifying screens. For supershift assays, antibodies (2 µl) were added 30 min after addition of the labeled probe and incubated for an additional 30 min.

Western Blot Analysis-- Nuclear extracts (60 µg of protein per lane) from transfected SL2 cells and untransfected TGW-nu-I and HeLa cells were separated by 7.5% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad) as previously described (12, 31). Blots were probed with Sp1, Sp2, and Sp3 antibodies diluted 1:1000. Signal detection of the immunoreactive bands was facilitated by enhanced chemiluminescence using the ECL system (Amersham Biosciences, Inc.).

Data Analysis-- Unless otherwise indicated, all data were determined from three independent experiments, each done in triplicate, and are expressed as mean values ± S.D. Comparisons among data sets were made with analysis of variance, followed by Student's t test. Values of p < 0.05 or less were considered to be statistically significant.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression Pattern of nNOS First Exon mRNA Variants-- We analyzed mRNA expression of the nine alternative first exon variants of human nNOS (Fig. 1A) by real-time quantitative RT-PCR (5' nuclease assay) in human brain, skeletal muscle, rectum, TGW-nu-I neuroblastoma cells, ME-180 cervix carcinoma cells, and HeLa cells. In addition, as a parameter for total nNOS mRNA expression, a sequence between exon 6 and exon 7, encoding parts of the oxygenase domain of nNOS (hem binding site that is essential for NOS activity (34)), was amplified. As internal controls GAPDH, HPRT, and TF2D mRNA expression were quantified. Real-time RT-PCR of the different first exons revealed high expression of nNOS exon 1c in the investigated human tissues (Fig. 2, A-C) and the TGW-nu-I cell line (Fig. 2D), whereas ME-180 cells were nNOS exon 1c-negative (Fig. 2E). The other first exons showed varying expression patterns with a cell- and tissue-specific expression (see Fig. 2, A-F). Exon 1f and exon 1g are highly abundant in the brain and rectum and very lowly abundant in skeletal muscle (Fig. 2, A-C, F; where bars are not evident despite positive RT-PCR results in Fig. 2F, values are less than the resolution shown in the figure). In contrast exon 1a is highly expressed in skeletal muscle (Fig. 2B) but missing in TGW-nu-I cells (Fig. 2D) and lowly abundant in the rectum (Fig. 2C). HeLa cells were nNOS mRNA-negative but showed high expression of the investigated housekeeping genes (data not shown). Fig. 2F summarizes the distribution of alternative first exons of nNOS in the investigated tissues and cell lines. nNOS first exon expression was normalized against GAPDH (Fig. 2, A-E) and showed no significant difference when other housekeeping genes (HPRT, TF2D) were used as internal controls (data not shown).


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  		Fig. 1.   A, schematic exon structure of alternatively spliced mRNA transcripts of human nNOS (adapted from Wang et al. (11)). Nine distinct first exons (exon 1a-1i) driven by separate promoters and spliced to the common exon 2 of nNOS are shown. Arrows indicate the positions of sense and antisense strand primers used in quantitative real-time RT-PCR (Fig. 2). The position of the internal 6-carboxy-fluorescein (FAM)-labeled TaqMan Probe is marked above exon 2 by a line, and the translational initiation codon is noted by a vertical arrow. B, genomic organization of the alternative first exons 1a, 1b, and 1c of the nNOS gene. Exons 1a and 1b are located in the 5'-upstream genomic DNA region of exon 1c. The arrows below the nucleotide sequence depict the 5' deletion sites of the different nNOS pGL3 promoter constructs used in reporter gene assays. The nucleotide sequence reported in this study has been deposited in the EMBL/GenBankTM data base with accession number AJ308545. C, nucleotide sequence of human nNOS exon 1c (in boldface letters) and in part the 5'-flanking region including the minimal promoter. The transcription start site is indicated as +1 and underlined. Cis-acting elements as determined by gel shift assays (Fig. 4, A-F) are indicated by lines above the sequence. Arrows below the nucleotide sequence depict the 5' deletion sites of the different nNOS pGL3 promoter constructs used in reporter gene assays.


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  		Fig. 2.   Quantitative analysis of human nNOS mRNA expression by real-time RT-PCR in human brain (A), skeletal muscle (B), rectum (C), TGW-nu-I neuroblastoma cells (D), and ME-180 cervix carcinoma cells (E). A set of nine forward primers, specific for the nine alternative first exons of nNOS (exon 1a-1i) were used with a common exon 2-specific reverse primer and an internal exon 2-specific 6-carboxy-fluorescein (FAM)-labeled TaqMan probe. For primer locations see arrows in Fig. 1A. As a parameter for total nNOS mRNA expression, a pair of exon 6- and exon 7-specific primers present in all known nNOS cDNAs were used with an exon 7-specific internal FAM-labeled probe. Relative amounts of transcripts were calculated using standard curves and dividing the expression levels of the different nNOS variants by the expression levels of the GAPDH housekeeping gene measured in the same RNA preparation. Results shown are the mean ± S.D. of one (pooled RNA obtained from CLONTECH; A and B) and three independent RNA isolations (C-E). Individual cDNA samples were analyzed in triplicate with a given pair of primers. F, distribution of alternative first exon variants of human nNOS in the brain, skeletal muscle, rectum, TGW-nu-I cells, ME-180 cells, and HeLa cells as determined by real-time RT-PCR. +++ indicates a high, ++ a moderate, and + a low expression level; - indicates negative RT-PCR results (where bars are not evident (A-E) despite positive RT-PCR results, values are less than the resolution of the figure).

Cloning of the 5'-Flanking Region of nNOS Exon 1c-- To further determine the partially known promoter sequence of nNOS exon 1c (formerly called exon 15'3) (12), we used rapid amplification of genomic ends (RAGE) to obtain a 5939-bp DNA fragment of the 5'-flanking regulatory region of exon 1c spanning nucleotides (nt) -5891 to +49 (Fig. 1B). This nucleotide sequence has been deposited in the EMBL/GenBankTM data base with accession number AJ308545. The transcriptional start site of exon 1c was identified previously to be located 84 bp upstream of the exon 1c/intron 1 splice junction at an adenine (+1) (Fig. 1, B and C) (12). Computer-based analysis of the sequence immediately upstream of this region using the MatInspector professional software, revealed no canonical TATA or CAAT boxes as initiators of transcription, but a putative transcription initiation site with overlapping consensus sequences for Staf (common binding site for ZNF76 and ZNF143 (23)), p53 half site, Olf-1, Ap2, Myc/Max, ZNF42 (alternative title MZF-1 (24)), Sp1 and MAZ between nt -90 and -47 bp relative to the transcription start site. The obtained 5'-flanking region of exon 1c contains in addition the sequence of nNOS exon 1a (nt 3656-3755) derived from EMBL accession number AF049712 (11) and exon 1b (nt 5567-5635) derived from EMBL accession number AF049713 (11) (see Fig. 1B). Exon 1c is located at nt 5892-5975 of the submitted sequence with EMBL accession number AJ308545. Therefore exons 1a, 1b, and 1c are located in close proximity within 2400 bp, similar to the genomic structure of exons 1f and 1g (former called exons 15'2 and 15'1) of human (13) and exons 1b and 1c of rat nNOS (35). Furthermore, the BLASTn search revealed that the putative transcription initiation site of exon 1c is highly conserved between rat and human with a sequence homology of 100% between nt -84 and -41 relative to the transcription start site of human exon 1c and nt 320 and 363 of rat nNOS exon 1b (EMBL accession number AF008911) (35). Note that the nomenclature of alternative first exons of nNOS is different in rat and human with human exon 1c corresponding to rat exon 1b (11, 35).

Identification of the Basal Promoter Region of nNOS Exon 1c-- Previously, we showed that exon 1c (designated exon 15'3) of nNOS is driven by a separate promoter within 332 bp upstream of the transcription start site in HeLa cells (12). However, neither the minimal promoter, nor the major transcription factors that regulate basal transcriptional activation were elucidated. To examine the sequence that is necessary for basal transcription of nNOS exon 1c, we analyzed various 5' and 3' deletions of the 5'-flanking region of exon 1c by reporter gene assays. The 5.9-kb promoter fragment of exon 1c (see Fig. 1B), obtained by 5'-RAGE, was cloned 5' to the firefly luciferase reporter gene of the pGL3-basic plasmid, resulting in pGL3-5891/+49. Different 5' and 3' deletions of the exon 1c promoter were generated by PCR, or restriction endonuclease and exonuclease III digestion of the pGL3-5891/+49 and pGL3-332/+49 plasmids (Fig. 1, B and C). These constructs were transiently transfected into nNOS exon 1c-positive TGW-nu-I neuroblastoma cells (see Fig. 2, D and F) and nNOS-negative HeLa cells (see Fig. 2F), as determined by quantitative real-time RT-PCR. After transfections of TGW-nu-I and HeLa cells with pGL3-5891/+49, we obtained a 13.7- and 9.1-fold increase of the normalized promoter activity compared with that of promoter-/enhancerless pGL3-basic, respectively (Fig. 3, A and B). After deletion of 3117 bp from full-length pGL3-5891/+49, a decrease of 57% (p = 0.008) in promoter activity could be observed in TGW-nu-I cells (Fig. 3A). In contrast, an increase of 67% (p = 0.007) was evident in HeLa cells (Fig. 3B). For both cell lines, progressive deletions from -2774/+49 to -1520/+49 did not show a significant change of transcriptional activity. Further deletion to -332/+49 resulted in a decrease of ~44% (p = 0.012) in TWG-nu-I cells, whereas promoter activity showed no significant change in HeLa cells. These observations suggest the presence of cis regulatory sequences exhibiting positive effects on the promoter activity between -5891 and -2774 and between -1520 and -332 specifically in TGW-nu-I cells, whereas sequences between -5891 and -2774 negatively affect promoter activity in HeLa cells. This argues for a cell-specific regulation of nNOS exon 1c promoter activity by cis acting elements in the upstream 5'-flanking region of exon 1c.


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  		Fig. 3.   Functional analysis of the human nNOS exon 1c promoter in TGW-nu-I (A), HeLa (B), and ME-180 (C) cells. 5' and 3' deletions were introduced into the nNOS exon 1c 5'-flanking region, and the indicated DNA fragments were ligated into the promoter-/enhancerless firefly luciferase expression vector pGL3-basic. As positive control an SV40 promoter/enhancer-directed firefly luciferase control vector (pGL3-control) was used (data not shown for HeLa and ME-180 cells in B and C, respectively). pGL3-basic and an antisense construct of pGL3-5891/+49 named pGL3 + 49/-5891 were used as a negative control. Constructs were transiently cotransfected into TGW-nu-I (A), HeLa (B), and ME-180 (C) cells with the herpes simplex virus thymidine kinase promoter driven Renilla luciferase expression vector pRL-TK as internal control as described under "Experimental Procedures." To control for transfection efficiency, the firefly luciferase activity of the test plasmids was corrected for Renilla luciferase activity of pRL-TK. Data are expressed as means ± S.D. of three independent experiments in triplicate. Where bars and/or error bars are not evident, values are less than the resolution of the figure.

Up to 242 bp (to position -90) could be further deleted in HeLa cells without a significant change of transcriptional activity. In contrast, in TGW-nu-I cells, we observed a drop in activity of ~33% (not significant) between -278 and -241, an increase between -241 and -131 of ~72% (p < 0.01), and an additional profound rise of ~108% (p < 0.001) after deletion of sequences between -131 and -90. However, deletion of an additional 27 bp (to position -63) abolished promoter activity completely in both cell lines (p < 0.001). Values seen for this -63/+49 construct, as well as for further deletions up to +18/+49, essentially reflected those of pGL3-basic. The antisense construct of pGL3-5891/+49, named pGL3 + 49/-5891, showed only background activity. As positive control the SV40 promoter/enhancer-directed pGL3-control vector was used in TGW-nu-I (Fig. 3A) and HeLa cells (data not shown).

These data demonstrate that the basal promoter for exon 1c is disrupted by deletion to -63 relative to the exon 1c transcription start site, and therefore, they strongly suggest that the promoter resides within the predicted region between -90 and -47. Interestingly, adjacent upstream 5' regions regulate this basal promoter activity differentially in nNOS-positive TGW-nu-I cells and nNOS-negative HeLa cells, demonstrating distinct mechanisms of activation in these two cell lines.

When 350 bp was removed from the 3'-end of pGL3-5891/+49, a -5891/-279 construct resulted that lacks the minimal promoter and the transcription start site of exon 1c but contains the putative promoters and transcription start sites of exon 1a and exon 1b. After transient transfection of this construct, normalized promoter activity showed just background activity in exon 1a and exon 1b mRNA-negative TGW-nu-I cells and in nNOS mRNA-negative HeLa cells (Fig. 3, A and B). These results demonstrate that the upstream 5'-flanking region of exon 1c, containing exons 1a and 1b and their putative promoters, can influence transcription of exon 1c but does not activate transcription of exon 1a and/or exon 1b in both cell lines under the investigated conditions.

Because exon 1c promoter constructs are active in endogenous nNOS-negative HeLa cells, we further investigated this discrepancy by reporter gene assays using nNOS-positive but exon 1c-negative ME-180 cells. After transient transfection with different nNOS exon 1c pGL3 promoter constructs, we observed a moderate, but significant increase in relative luciferase activity over that of promoter-/enhancerless pGL3-basic (Fig. 3C). Therefore, endogenous nNOS-negative HeLa cells and nNOS exon 1c-negative ME-180 cells are able to transactivate nNOS exon 1c reporter constructs.

Identification of Transcription Factors Binding to the Basal nNOS Exon 1c Promoter-- Computer-based sequence inspection of the GC-rich (66%) and TATA-less basal promoter region between -90 and -47 of exon 1c indicated a variety of putative cis-regulatory elements, like Staf (consensus binding site for ZNF143 and ZNF 76 (23)) (-85 to -64), p53 half-site (-79 to -70), Olf-1 (-78 to -57), Ap2 (-73 to -62), Myc/Max (-61 to -48), ZNF42 (alternative title MZF-1) (-70 to -63), and a low affinity GC box with binding sites for Sp1 (-69 to -57) and MAZ (-67 to -59). To assess transcription factor binding, a double-stranded 44-bp 32P-labeled oligonucleotide (-90/-47) (see Table II for all oligonucleotides used in gel shift experiments, except consensus and mutant consensus oligonucleotides for Ap2, Myc-Max, NF-I, YY1, NFkappa B, p53, Sp1, USF-1, which were purchased from Santa Cruz Biotechnologies), including these elements, was used in electrophoretic mobility shift assays (EMSAs). A series of five shifted protein-DNA complexes were observed after incubation of TGW-nu-I nuclear extracts with the labeled probe (Fig. 4A, lane 2), whereas only two protein-DNA complexes were evident using HeLa nuclear extracts (Fig. 4B, lane 2). These complexes were competed with a 100-fold molar excess of unlabeled -90/-47 probe, establishing binding specificity (Fig. 4, A and B, lane 3). Shifted bands without competition after addition of the unlabeled -90/-47 probe were considered as nonspecific binding and were marked by an asterisk in Fig. 4 (A-D). Using a 100-fold molar excess of an unlabelled Sp1 consensus oligonucleotide (containing the binding site for the transcription factors Sp1, Sp3, and Sp4) complexes I/III (TGW-nu-I cells, Fig. 4A, lane 7) and complex I (HeLa cells, Fig. 4B, lane 7) were completely competed, and complex II (both cell lines) was partially competed. There was no further competition of complex II, when a 200- and 500-fold molar excess of unlabeled Sp1 consensus oligonucleotides was used (both cell lines, data not shown). No competition of the complexes was observed with a 100- and 200-fold molar excess of a commercial mutant Sp1 oligonucleotide (Fig. 4, A and B, lane 8). Using TGW-nu-I nuclear extracts a slight reduction in the protein-DNA complexes I, II, and III was seen with a -90/-63 oligonucleotide where the GC box is disrupted (Fig. 4A, lane 4) and with a -90/-47 oligonucleotide in which the Sp1 binding site was mutated (Fig. 4A, lane 5), whereas complexes IV and V were completely competed by these two oligonucleotides. With HeLa cell nuclear extracts complex I was slightly and complex II was completely competed using the -90/-63 and -90/-47 Sp1-Mut competitors (Fig. 4B, lanes 4 and 5). Furthermore, no competition of any complex was observed using an unrelated YY1 consensus oligonucleotide (TGW-nu-I cells, Fig. 4A, lane 23; HeLa cells, Fig. 4B, lane 21). When protein-DNA complexes were incubated with antibodies against Sp1, Sp2, Sp3, and Sp4 or combinations of these antibodies, supershifts of different complexes were observed with TGW-nu-I and HeLa cell nuclear extracts (Fig. 4, C and D). For the Sp1 antibody a supershift of complex I/III (TGW-nu-I cells) and complex I (HeLa cells), for the Sp2 antibody a supershift of complex I (TGW-nu-I and HeLa cells), for the Sp3 antibody a supershift of complex I/II/III (TGW-nu-I cells) and complex II (HeLa cells) was observed whereas for the Sp4 antibody no supershift was seen. A combination of Sp1, Sp2, and Sp3 antibodies led to different supershifts as shown in Fig. 4C for TGW-nu-I cells and Fig. 4D for HeLa cells. When all three antibodies were added to the binding reaction, complexes I/II/III (TGW-nu-I cells) and complexes I/II (HeLa cells) were supershifted with a nearly complete abrogation. Using antibodies against goat and rabbit IgG, no supershift of any complex was evident (data not shown). Taken together these results demonstrate that Sp1, Sp2, and Sp3 can interact with the 44-bp minimal promoter of exon 1c. To further characterize the retarded complex II obtained with TGW-nu-I and HeLa cells, which was not completely competed by an Sp1 consensus oligonucleotide, and the unaffected complexes IV and V (TGW-nu-I cells), consensus oligonucleotides for the transcription factors Staf (ZNF76 and ZNF143), p53, Olf-1, Ap2, Myc/Max, ZNF 42, MAZ, NF-kappa B, NF-1, YY1, and USF were used. Myc/Max, NF-1, USF, p53 (data not shown), and YY1 (Fig. 4A, lane 23; Fig. 4B, lane 21) had no effects on nucleoprotein complex formation. Unspecific competition of the retarded protein-DNA complexes V (MAZ, Fig. 4A, lane 20) and IV/V (Olf-1, Fig. 4A, lanes 11 and 12) was evident using TGW-nu-I cell nuclear extracts and a 100-fold excess of unlabelled consensus and mutated consensus oligonucleotides for MAZ and Olf-1. In contrast, oligonucleotides containing Staf, ZNF42, Ap2, and NF-kappa B binding sites were specific in competition studies. An oligonucleotide with a Staf binding site (consensus sequence for ZNF76 and ZNF143) competed partially complex II (both cell lines, Fig. 4, A and B, lane 9), whereas ZNF42 consensus oligonucleotides competed complexes I/III/IV/V (TGW-nu-I cells, Fig. 4A, lane 15) and complex I (HeLa cells, Fig. 4B, lane 15). Addition of oligonucleotides containing mutations in the Staf and ZNF42 binding sites failed to compete protein-DNA complexes (Fig. 4, A and B, lane 16). A combination of Sp1 and Staf consensus oligonucleotides resulted in a complete competition of complex II (both cell lines, Fig. 4, A and B, lane 13), whereas Sp1 combined with other consensus oligonucleotides like Olf-1 (Fig. 4, A and B, lane 14) and ZNF42 (data not shown) showed the same degree of competition as Sp1 alone. This observation suggests a cooperative binding of Sp and ZNF76/143 transcription factors to the basal nNOS exon 1c promoter.

                              
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Table II
Double-stranded oligonucleotides used for electrophoretic mobility shift assays
DNA sequences for double-stranded oligonucleotides used as probes and/or competitors in gel shift assays. Boldface letters indicate mutated bases. Note: Sequences of oligonucleotides purchased from Santa Cruz Biotechnologies (see "Materials") are not listed.


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  		Fig. 4.   Identification of multiple DNA-protein complexes in the nNOS exon 1c basal promoter. Electromobility shift assays (EMSAs) were performed using nuclear extracts from TGW-nu-I (A, C, E) and HeLa (B, D, F) cells as described under "Experimental Procedures." A and B, 32P-labeled oligonucleotide probes spanning the -90 to -47 region of the basal nNOS exon 1c promoter were incubated with 10 µg of TGW-nu-I (A) and HeLa (B) cell nuclear extracts resulting in five (panel A) and two (panel B) specific shifted protein-DNA complexes, respectively. Nonspecific bands are indicated by an asterisk. In lanes 3, 4, and 5 a 100-fold molar excess of unlabeled -90/-47, -90/-63, and -90/-47 Sp1 Mut oligonucleotides were used as competitors. To characterize shifted protein-DNA complexes, unlabeled consensus and mutated consensus oligonucleotides for the indicated transcription factors were used, as well as combinations of these oligonucleotides (panel A: lanes 7-16, 20-23; panel B: lanes 7-21). Lane 1 represents labeled probe alone. C and D, detection of protein-DNA complexes using antibodies against Sp1, Sp2, Sp3, Sp4, and combinations of these antibodies. EMSAs were done as described above using TGW-nu-I (C) and HeLa (D) cell nuclear extracts and the 32P-labeled -90/-47 spanning probe of the basal nNOS exon 1c promoter. Supershift assays were performed by incubation of the binding reaction with the labeled probe for 30 min followed by the addition of 2 µl of each antibody and an additional incubation for 30 min at room temperature. Nonspecific bands are indicated by an asterisk. E and F, EMSAs of TGW-nu-I (E) and HeLa (F) cell nuclear extracts binding to the -90/-63 region of the nNOS exon 1c promoter. Gel shift assays were performed as described above (A and B), except that a 32P-labeled oligonucleotide probe spanning the -90 to -63 region of the minimal exon 1c promoter was used. Lane 1 represents probe alone, and 10 µg of the respective nuclear extracts was added to the binding reaction in lane 2, resulting in four (panel E) and six (panel F) specific shifted protein-DNA complexes, respectively. A 100-fold molar excess of various unlabeled oligonucleotides were added as competitors in lanes 3-10 as indicated.

Consensus oligonucleotides for Ap2 and NF-kappa B showed a clear reduction in the protein-DNA complexes I/II/III (TGW-nu-I cells, Fig. 4A, lanes 21 and 22) and complex I (HeLa cells, Fig. 4B, lanes 17 and 20), whereas mutated oligonucleotides failed to compete (Fig. 4B, lane 18 and data not shown). However, using Ap2 and NF-kappa B (subunits p50 and p65) antibodies, we were unable to observe a supershift or abrogation of any complex (data not shown). The causal mechanism of this observation is unclear. Ap2 and NF-kappa B may not be able to bind to the minimal promoter of exon 1c autonomously and thus do not play a direct role in exon 1c basal promoter activation. Therefore, competition of the retarded bands in the gel shift assays could be due to protein-protein interactions of Ap2/NF-kappa B with other nuclear factors whose DNA binding affinity and specificity could be increased by the presence of Ap2 or NF-kappa B. Such mechanisms have been demonstrated recently (36, 37), and therefore Ap2 and NF-kappa B could participate in the formation of multiple protein complexes that enhance or repress the transactivation potential of other transcription factors like Sp1.

To determine transcription factor binding in the absence of Sp and GC box activity, a 22-bp 32P-labeled -90/-63 oligonucleotide with a disrupted GC box and Sp1 binding site was used in EMSAs. A series of four and six shifted protein-DNA complexes was observed after incubation with TGW-nu-I (Fig. 4E, lane 2) and HeLa (Fig. 4F, lane 2) cell nuclear extracts, respectively. These complexes were completely competed with a 100-fold molar excess of unlabeled -90/-63 probe (Fig. 4, E and F, lane 3). Using a 100-fold molar excess of an unlabelled Sp1 consensus oligonucleotide, the signal intensity of the protein-DNA complexes II and III were amplified, and the signal intensity of complex IV was reduced with TGW-nu-I cell nuclear extracts (Fig. 4E, lane 4). In contrast, complete competition of complex I was observed using HeLa cell nuclear extracts (Fig. 4F, lane 4). When a ZNF42 consensus oligonucleotide was used, complex IV was completely competed and complexes II and III were shifted or competed resulting in a retarded band between complex II and III in TGW-nu-I cells (Fig. 4E, lane 5), whereas no competition was observed with a mutated ZNF42 consensus oligonucleotide (Fig. 4E, lane 6). The significance of the described observation remains unclear but demonstrates that ZNF42 is able to modulate transcription factor binding to the -90/-63 sequence. In HeLa cells ZNF42 consensus oligonucleotides resulted in only an unspecific competition of complex III (Fig. 4F, lanes 5 and 6). A Staf consensus oligonucleotide competed complex I/II/III (TGW-nu-I cells, Fig. 4E, lane 9) and complex II/III/IV/V (HeLa cells, Fig. 4F, lane 9). However, complex VI of Fig. 4F (HeLa cells) could not specifically be competed by any of the used consensus oligonucleotides, indicating that an as yet unknown transcription factor present in HeLa cells is able to bind to the -90 to -63 sequence of the exon 1c promoter. No competition was observed with mutant Staf (Fig. 4, E and F, lane 10), mutant Sp1 (data not shown), and unrelated YY1 (Fig. 4E, lane 7, Fig. 4F, lane 8) consensus oligonucleotides using nuclear extracts from both cell lines.

Collectively, these results identify a multiplicity of protein-DNA complexes within the nNOS exon 1c basal promoter involving Sp1, Sp2, Sp3, and members of the ZNF family of transcription factors. These factors have differential effects on nucleoprotein complex formation. Some of them increase, whereas others decrease, band intensities, suggesting modulatory effects on protein-DNA interactions.

Confirmation of Transcription Elements and Promoter Transactivation by Mutagenesis and Transient Transfections-- To further clarify the role of the different cis regulatory elements in the basal nNOS exon 1c promoter, reporter gene constructs for pGL3-90/+49 containing point mutations in the Sp1/ZNF42 (pGL3-90/+49 Sp1/ZNF42-M1), Sp1 (pGL3-90/+49 Sp1-M2), Ap2/Olf-1 (pGL3-90/+49 Ap2/Olf-1-M), and Staf (pGL3-90/+49 Staf-M) binding sites were constructed (see Fig. 5, A and B). These constructs were transiently expressed in TGW-nu-I (Fig. 5A) and HeLa cells (Fig. 5B), and promoter activities were compared with that of the wild type plasmid pGL3-90/+49. Furthermore, 5'-deleted pGL3-83/+49, pGL3-48/+49, and promoter-/enhancerless pGL3-basic were used as controls. Mutation of the common Sp1/ZNF42 binding site, as well as the Sp1 binding site, alone abolished promoter activity of nNOS exon 1c to that of pGL3-basic in both cell lines (Fig. 5, A and B). In TGW-nu-I cells, the pGL3-90/+49 promoter construct containing a mutated Staf element resulted in a 53% decrease in luciferase activity compared with the pGL3-90/+49 wild type construct (Fig. 5A), whereas a decrease of 37% was detected in HeLa cells (Fig. 5B). In TGW-nu-I and HeLa cells, constructs of pGL3-90/+49 with mutations in the Ap2/Olf-1 binding site were 94% and 98.5% as active as wild type pGL3-90/+49, respectively, indicating that the Ap2 and Olf-1 consensus binding sites in the exon 1c minimal promoter play no role for basal transcription (Fig. 5, A and B). Taken together, these data clearly demonstrate the critical cis-acting roles for Sp and ZNF binding motifs in the exon 1c minimal promoter.



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  		Fig. 5.   Mutagenesis of transcription factor binding sites decreases transcription from the nNOS exon 1c minimal promoter. TGW-nu-I (A, C, E) and HeLa (B, D, F) cells were transiently cotransfected with pRL-TK and different wild type or mutant reporter gene constructs containing targeted substitutions or deletions in transcription factor binding sites as described under "Experimental Procedures." To adjust for transfection efficiency, the firefly luciferase activity of the test plasmids was corrected for Renilla luciferase activity (pRL-TK). Data are expressed as percent luciferase activity relative to the respective "full-length" wild type pGL3 construct and represent the means ± S.D. of three independent experiments in triplicate. A and B, the percent luciferase activities of wild type pGL3-90/+49, 5'-deleted pGL3-83/+49, and pGL3-48/+49, the promoter-/enhancerless pGL3-basic vector, or mutant pGL3-90/+49 reporter gene constructs containing the indicated targeted substitutions in the binding site of Sp1 (pGL3-90/+49 Sp1-M2), the common binding sites of Sp1 and ZNF42 (pGL3-90/+49 Sp1/ZNF42-M1), Ap2 and Olf1 (pGL3-90/+49 Ap2/Olf1-M), ZNF76 and ZNF143 (called Staf binding site; pGL3-90/+49 Staf-M) are plotted for TGW-nu-I (A) and HeLa (B) cells. C and D, the percent luciferase activities of wild type pGL3-5891/+49, 5'-deleted pGL3-48/+49, pGL3-basic vector, and the indicated mutant pGL3-5891/+49 constructs containing targeted substitutions or deletions in the Sp1/ZNF42, Staf, and both the Sp1/ZNF42 and Staf binding element are plotted for TGW-nu-I (C) and HeLa (D) cells. E and F, the percent luciferase activities of wild type pGL3-332/+49, 5'-deleted pGL3-48/+49, pGL3-basic vector, and the indicated mutant pGL3-332/+49 constructs containing targeted substitutions or deletions in the Sp1/ZNF42, Staf, and both the Sp1/ZNF42 and Staf binding element are plotted for TGW-nu-I (E) and HeLa (F) cells.

To investigate the importance of this basal promoter region for the transactivation of the longer reporter constructs pGL3-5891/+49 and pGL3-332/+49, site-specific mutations and deletions in the Sp and Staf binding sites were introduced into the wild type plasmids. After transient expression in TGW-nu-I cells, promoter activities of all mutated/deleted pGL3-5891/+49 plasmids reflected that of pGL3-basic (Fig. 5C). In HeLa cells, the construct pGL3-5891/+49 Staf/Sp1/ZNF42-M1, which contains a double mutation of the Staf and Sp1/ZNF42 binding sites, abolished promoter activity completely, and the construct pGL3-5891/+49 Staf/Sp1/ZNF42-del, with a deletion of both sites, was 11% as active as wild type pGL3-5891/+49 (Fig. 5D). Plasmids containing a mutation (pGL3-5891/+49 Sp1/ZNF42-M1) or deletion (pGL3-5891/+49 Sp1/ZNF42-del) of the Sp1/ZNF42 site displayed a drop of 87 and 79% in functional promoter activity relative to the wild type construct, respectively, and mutation (pGL3-5891/+49 Staf-M) and deletion (pGL3-5891/+49 Staf-del) of the Staf element resulted in a decrease of 68 and 71%, respectively (Fig. 5D). The same mutations and deletions were incorporated in to the pGL3-332/+49 construct. After transient expression in TGW-nu-I and HeLa cells, relative functional reporter activities revealed no important differences compared with that of the mutations/deletions in the setting of the full-length -5891/+49 construct (Fig. 5, E and F).

Collectively, these data clearly indicate the major importance of the GC-rich region between -90 and -47 relative to the exon 1c transcription start site to promote nNOS exon 1c expression.

Activation of the nNOS Exon 1c Promoter in Drosophila Schneider Cells-- To further determine whether members of the Sp and ZNF families of transcription factors functionally interact with the basal promoter of exon 1c, transient transfection experiments were performed with Drosophila Schneider cells (SL2 cells), which lack many mammalian transcription factors, like Sp-related proteins (25, 26), ZNF76 and ZNF143 (23, 38). Expression constructs under the control of an SL2-specific promoter for Sp1 (pPac-Sp1), Sp2 (pPac-Sp2), the long (pPac-USp3) and short isoforms of Sp3 (pPac-Sp3), Sp4 (pPac-Sp4), ZNF42 (pPac-ZNF42), ZNF76 (pPac-ZNF76), ZNF 143 (pPac-ZNF143) and empty pPac0 were cotransfected along with reporter vectors (pGL3) under the control of different nNOS exon 1c promoters. Normalized luciferase activities for the pGL3 reporter constructs were compared with that in cotransfections with the empty SL2 expression vector pPac0. As shown in Fig. 6A cotransfection of pGL3-90/+49 with pPac-Sp1 induced a 10.4-fold increase in relative luciferase activity over cotransfection with pPac0. In contrast, values slightly above those of pGL3-basic (representing basal luciferase activity) were observed after cotransfection of SL2 cells with pPac-Sp1 and pGL3-48/+49, which lacks the GC box of the basal exon 1c promoter (Fig. 6A). A 14.8-fold increase was seen, when the long isoform of Sp3 (pPac-USp3) was cotransfected with pGL3-90/+49, whereas cotransfection experiments with pGL3-48/+49 again showed only background activity (Fig. 6A). Cotransfection of pPac-ZNF76 and pPac-ZNF143 along with pGL3-90/+49 resulted in a 11.6- and 6.8-fold transactivation of pGL3-90/+49, respectively, whereas induction of pGL3-48/+49 by ZNF76 and ZNF143 reflected that of pGL3 basic (Fig. 6A). No transactivation of pGL3-90/+49 and pGL3-48/+49 promoter activity was seen after cotransfection with pPac-ZNF42, indicating that ZNF42 is not able to induce the basal nNOS exon 1c promoter autonomously (Fig. 6A). Likewise, there was no significant stimulation of normalized luciferase activity over that of pGL3-basic, when either pPac-Sp2, pPac-Sp3 encoding the short isoforms of Sp3, or pPac-Sp4 was cotransfected with pGL3-90/+49 or pGL3-48/+49 (data not shown). Fig. 6B shows the effects of increasing amounts of pPac-Sp1, pPac-USp3, pPac-ZNF76, pPac-ZNF143, and pPac-ZNF42 on pGL3-90/+49 luciferase activity. Sp1, the long isoform of Sp3, ZNF76, and ZNF143 exhibited a dose-dependent transactivation of pGL3-90/+49, whereas pGL3-48/+49 was again not activated. Increasing amounts of pPac-ZNF42 had no effect on transactivation of pGL3-90/+49 (Fig. 6B).


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  		Fig. 6.   Sp1, the long isoform of Sp3, ZNF76, and ZNF143 transactivate the human nNOS exon 1c promoter in Drosophila Schneider cells. SL2 cells were cotransfected with the indicated nNOS exon 1c-promoter/pGL3-luciferase reporter gene constructs and different combinations and amounts of pPac-Sp1, pPac-Sp2, pPac-Sp3, pPac-USp3, pPac-Sp4, pPac-ZNF76, pPac-ZNF143, pPac-ZNF42, or empty pPac0 expression vector as described under "Experimental Procedures." Luciferase activity was assayed 48 h after transfection and normalized for total cellular protein values of lysed SL2 cells. Data are expressed as -fold induction of normalized luciferase activity relative to that obtained following cotransfection of the pGL3 reporter plasmids with empty pPac0, which does not express Sp/ZNF proteins. Values represent means ± S.D. of three independent experiments in triplicate. Where bars and/or error bars are not evident, values are less than the resolution of the figure. Variations in the amount of the expression plasmids were compensated with the empty plasmid pPac0 to keep the total DNA content of pPac plasmids constant (0.5 µg per transfection). A, Drosophila Schneider cells were cotransfected with 1.0 µg of pGL3-90/+49, 5'-deleted pGL3-48/+49, and the promoter-/enhancerless pGL3-basic luciferase reporter plasmid along with 0.5 µg of the expression plasmids pPac-Sp1, pPac-USp3, pPac-ZNF76, pPac-ZNF143, and pPac-ZNF42. B, cotransfection of 1.0 µg of pGL3-90/+49 along with increasing concentrations (0.025-0.5 µg) of the expression plasmids pPac-Sp1, pPac-USp3, pPac-ZNF76, pPac-ZNF143, and pPac-ZNF42 into SL2 cells reveals a dose-dependent transactivation of the minimal nNOS exon 1c promoter for Sp1, the long isoform of Sp3, ZNF76, and ZNF143. C, specific Sp and ZNF family members of transcription factors exert a differential activation of the nNOS exon 1c promoter. Combinations of the expression plasmids pPac-Sp1, pPac-Sp2, pPac-Sp3, pPac-USp3, pPac-Sp4, pPac-ZNF76, pPac-ZNF143, pPac-ZNF42, and empty pPac0 were cotransfected along with 1.0 µg of wild type pGL3-90/+49 into SL2 cells. 0.25 µg of DNA of each indicated pPac expression plasmid was used for transfections.

In combination experiments (Fig. 6C), the stimulatory effects of Sp1 and the long Sp3 isoform (21.3-fold induction), as well as Sp1 and ZNF143 (15.3-fold induction) were additive, whereas Sp1 combined with ZNF76 (28.4-fold induction) and full-length Sp3 combined with ZNF76 (27.2-fold induction) resulted in a potentiation of nNOS exon 1c basal promoter transactivation. Cotransfection of pPac-Sp1 with pPac-Sp3 or pPac-Sp4, as well as cotransfection of pPac-USp3 with Sp2, Sp4, ZNF143, or ZNF42, had no effect on promoter activation of pGL3-90/+49 (Fig. 6C). However, combining pPac-Sp1 with pPac-Sp2 or pPac-ZNF42, pPac-USp3 with pPac-Sp3, pPac-ZNF76 with pPac-ZNF42, and pPac-ZNF143 with pPac-ZNF42 resulted in a slight, but statistically significant decrease of nNOS exon 1c promoter activity (Fig. 6C). Thus ZNF and Sp family members are able to exert positive and negative effects on the transactivation of the nNOS exon 1c minimal promoter.

Expression of Sp1, Sp2, isoforms of Sp3, and Sp4 in SL2 cells after transient transfection with the different pPac plasmids was verified by Western blot analysis of SL2 cell nuclear extracts using selective antibodies against Sp1, Sp2, Sp3, and Sp