Molecular mechanism for transcriptional regulation of dopamine beta-hydroxylase gene.

多巴胺-羟化酶（DBH）将多巴胺转化为去甲肾上腺素，在去甲肾上腺素和肾上腺素能神经元中特异性表达。人DBH基因的环AMP应答元件（Cyclic AMP response element， CRE，TGACGTCC）是启动子区必不可少的转录元件。在这个CRD的下游，有三个重要的DNA元件，如YY-1结合序列（CCAT）、E-box序列（GATGTG）和AP-1样序列（TGTGTCA），它们可以调节许多基因的转录。为了鉴定DNA结合蛋白，用26bp寡核苷酸（actga TGACGTCCATGTGTCAttagt）作为探针进行了电泳迁移转移试验（EMSA）。根据定点诱变工作，我们发现CRE和YY-1结合序列都与蛋白形成复合物，而E-box和AP-1样序列则不形成复合物。酪氨酸羟化酶（TH）或生长抑素基因的CRE寡核苷酸作为竞争者的加入消除了由CRE和CRE结合蛋白（可能是CREB…More或ATFs）形成的上移带。此外，作为竞争对手的小鼠c-fos基因YY-1结合序列中过量的寡核苷酸完全消除了中间移位的条带。YY-1是一种普遍表达的锌指型DNA结合蛋白，调控多种基因的转录水平，如几种病毒基因、免疫球蛋白基因（kappa和mu）、癌基因（c-fos和c-myc）、珠蛋白基因（gamma和epsilon）等。该蛋白的显著特征是具有双重功能，根据启动子环境和细胞内环境的不同，具有积极或消极的作用。为了评估YY-1结合人DBH CCAT基元的功能，我们在表达DBH的神经母细胞瘤细胞（SK-N-SH）中进行了瞬时转染实验。定点诱变实验表明，YY-1通过添加phorbol酯（TPA）正向调节人DBH基因的转录水平。此外，荧光素酶检测结果表明，CRE结合蛋白与YY-1之间的相互作用需要特定的方向。这些数据表明YY-1在TPA诱导的DBH基因转录调控中起重要作用。为了鉴定人DBH基因启动子的TPA应答DNA区（TRR），制备了-604bp ~ -171bp的缺失突变体，并将这些荧光素酶报告基因（Luc）转染到SK-N-SH细胞中。TRR位于人类DBH基因CRE上游-223bp ~ -187bp之间，在其他已知的TPA应答元件（TRE）序列中均未发现类似序列。此外，我们试图鉴定未知的TRR，TRR分割到三个DNA区域，分别为-223bp至-199bp (OLI-A)， -213bp至-183 （OLI-D）和-206bp至-181bp （OLI-B）。首先，OLI-D去除OLI-63（-224至-162区域）和核蛋白复合物。其次，OLI-D响应tk启动子对TPA的诱导。此外，OLI-D和人DBH的CRE组合通过TPA增强了荧光素酶的活性。Int少

Dopamine beta-hydroxylase (DBH) converts dopamine to norepinephrine and is specifically expressed in noradrenergic and adrenergic neurons. Cyclic AMP response element (CRE,TGACGTCC) of human DBH gene is an essential transcriptional element in the promoter region. Downstream of this CRD,there are three important DNA elements such as YY-1 binding sequence (CCAT), E-box (GATGTG), and AP-1 like sequence (TGTGTCA) that may regulate the transcription of many genes. To identify the DNA binding proteins, 26 bp oligonucleotide (actga TGACGTCCATGTGTCAttagt) was used as a probe for the electrophoretic mobility shift assay (EMSA). According to the site-directed mutagenesis work, we found that both CRE and YY-1 binding sequence formed complexes with proteins, but E-box and AP-1 like sequences did not. The addition of CRE oligonucleotides for tyrosine hydroxylase (TH) or somatostatin genes as competitors eliminated upper-shifted bands that were formed with CRE and CRE binding protein (possibly CREB … More or ATFs). Furthermore, an excess amount of oligonucleotide which was the YY-1 binding sequence of mouse c-fos gene as a competitor, completely eliminated the intermediate-shifted band. YY-1, a ubiquitously expressed zinc finger type DNA binding protein, regulates the transcriptional level of many genes, such as several viral genes, immunoglobuline gene (kappa and mu), oncogene (c-fos and c-myc), globin gene (gamma and epsilon), and so on. Astriking characteristic of this protein is a dual function which either positive or negative effects, depending on the promoter context and the intracellular circumstance. In order to assess the function of YY-1 that bound to the CCAT motif of human DBH,transient transfection experiments were performed in the DBH-expressing neuroblastoma cells (SK-N-SH). Site-directed mutagenesis experiments showed that YY-1 positively regulated the transcriptional level of human DBH gene by adding phorbol ester (TPA). In addtion, CRE binding proteins and YY-1 needed specific direction to interact with each other by the results of luciferase assay. These data suggest that YY-1 is important in the transcriptional regulation of DBH gene by the TPA induction. To identify the TPA response DNA region (TRR) in human DBH gene promoter, deletion mutant constructs from -604bp to -171bp were prepared and these luciferase reporter genes (Luc) were transfected to SK-N-SH cells. TRR resided in the upstream of CRE between -223bp and -187bp of human DBH gene, and there were no similar sequences in other known TPA response element (TRE) sequences. In addition, we tried to identifed unknown TRE,TRR disected to three DNA regions that were -223bp to -199bp (OLI-A), -213bp to -183 (OLI-D) and -206bp to -181bp (OLI-B). First, OLI-D eliminates the OLI-63 (region from -224 to -162) and nucler protein complex. Second, OLI-D responses the TPA induction by the use of tk promoter. Furthermore OLI-D and CRE of human DBH combination enhanced the luciferase activity by TPA.Int Less