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J. Biol. Chem., Vol. 277, Issue 28, 25798-25814, July 12, 2002
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From the Department of Internal Medicine II, Technische
Universität München, Munich 81675, Germany
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AJ308545.
Complex Regulation of Human Neuronal Nitric-oxide Synthase Exon
1c Gene Transcription
ESSENTIAL ROLE OF Sp AND ZNF FAMILY MEMBERS OF TRANSCRIPTION
FACTORS*
,
*
This work was supported by Deutsche Forschungsgemeinschaft
Sonderforschungsbereich 391 C5, KKF TU Munich F71-98 and F47-01. Preliminary results were presented at the annual meeting of the American Gastroenterological Association in Atlanta (2001).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: II. Medizinische
Klinik und Poliklinik, Technische Universität München,
Ismaningerstrasse 22, München D-81675, Germany. Tel.:
49-89-4140-4377; Fax: 49-89-4140-4932; E-mail:
Dieter.Saur@lrz.tu-muenchen.de.
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