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Papers In Press, published online ahead of print April 17, 2002
Internal Medicine 2, Technische Universität München, Munich 81675
Corresponding Author: Dieter.Saur{at}lrz.tum.de
Neuronal nitric oxide synthase (nNOS) is expressed in a variety of human tissues and shows a complex transcriptional regulation with 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 RT-PCR. In these tissues/cells exon1c was one of the most abundant first exons of nNOS. By transient trans-fections of TGW-nu-I and HeLa cells with reporter plasmids containing a series of 5´and 3´deletions in the exon1c 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 complexicity 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.
J. Biol. Chem, 10.1074/jbc.M109802200
Submitted on October 10, 2001
Revised on April 2, 2002
Accepted on April 17, 2002
Complex regulation of human neuronal NO synthase exon 1c gene transcription. Essential role of Sp and ZNF family members of transcription factors
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