Saur Dieter, Seidler Barbara, Paehge Heidi, Schusdziarra Volker, Allescher Hans-Dieter
Department of Internal Medicine II, Technische Universität München, Germany.
J Biol Chem. 2002 Jul 12;277(28):25798-814. doi: 10.1074/jbc.M109802200. Epub 2002 Apr 17.
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.
神经元型一氧化氮合酶（nNOS）在多种人体组织中表达，并且由于存在九个可变的第一外显子（1a - 1i）而呈现出复杂的转录调控，这导致nNOS转录本具有不同的5' - 非翻译区。我们之前证明，nNOS外显子1c是人类胃肠道中主要的转录本之一，由一个独立的启动子驱动（绍尔，D.，帕赫格，H.，舒斯齐亚拉，V.，和阿勒斯彻，H. D.（2000年）《胃肠病学》118卷，849 - 858页）。本研究聚焦于nNOS第一外显子变体在不同人体组织中的定量表达以及基础nNOS外显子1c启动子的特性。在人类大脑、骨骼肌、结肠和TGW - nu - I神经母细胞瘤细胞中，通过定量实时逆转录 - PCR分析第一外显子的表达模式。在这些组织/细胞中，外显子1c是nNOS最丰富的第一外显子之一。通过用包含外显子1c调控区一系列5'和3'缺失的报告质粒瞬时转染TGW - nu - I和HeLa细胞，最小的无TATA启动子定位于44个碱基对内。对这个顺式调控区的凝胶迁移率变动分析揭示了基础启动子的高度复杂性，有几种转录因子如Sp和锌指蛋白（ZNF）家族成员的协同结合。当最小启动子构建体的Sp结合位点发生突变时，两个细胞系中的启动子活性完全丧失，而ZNF76和ZNF143的共同结合位点发生突变导致TGW - nu - I细胞中启动子活性下降53%，HeLa细胞中下降37%。在果蝇施奈德细胞中，Sp1、长的Sp3异构体、ZNF76和ZNF143的表达有力地反式激活了nNOS外显子1c启动子。这些结果确定了nNOS外显子1c基础启动子的关键调控区，并强调了涉及Sp和ZNF转录因子家族的多种蛋白质复合物在调节nNOS外显子1c转录中的功能重要性。
