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

多巴胺β-羟基酶(DBH)将多巴胺转化为去甲肾上腺素，在去甲肾上腺素能和肾上腺素能神经元中特异表达。人DBH基因的环状AMP反应元件(Cre，TGACGTCC)是启动子区域必需的转录元件。在该CRD下游有三个重要的DNA元件，如YY-1结合序列(CCAT)、E-box(GATGTG)和AP-1样序列(TGTGTCA)，它们可能调节许多基因的转录。为了鉴定DNA结合蛋白，用26个碱基的寡核苷酸(Actga TGACGTCCATGGTGTCAtagt)作为探针，进行了凝胶迁移率改变分析(EMSA)。根据定点突变工作，我们发现Cre和YY-1结合序列都与蛋白质形成了复合物，而E-box和AP-1类序列没有。添加酪氨酸羟基酶(TH)或生长抑素基因的Cre寡核苷酸作为竞争对手，消除了与Cre和Cre结合蛋白(可能是Cre…)形成的上移带更多或ATF)。此外，作为竞争对手的小鼠c-fos基因的YY-1结合序列，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，它调节多种基因的转录水平，如病毒基因、免疫球蛋白基因(kappa和u)、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，其正面或负面影响取决于启动子和细胞内环境。为了评价与人DBH CCAT基序结合的YY-1的功能，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。定点突变实验表明，YY-1通过加入佛波酯(TPA)正向调控人胸腺激素基因的转录水平。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基因启动子中的TPA反应DNA区(TRR)，制备了-604bp~-171bp的缺失突变载体，并将这些荧光素酶报告基因(Luc)导入SK-N-SH细胞。TRR位于人胸腺激素基因Cre上游-223bp~-187bp之间，在其他已知的TPA反应元件(TPA Response Element，TrE)序列中没有类似序列。此外，我们还尝试在Oli-A(Oli-A)、Oli-D(Oli-D)和Oli-B(Oli-B)三个DNA区域分别定位了未知的TrE、TRR，分别为-223bp~-199bp、-213bp~-183bp和-206bp~-181bp。首先，Oli-D消除了Oli-63(-224-162区域)和核蛋白复合体。第二，Oli-D利用tk启动子应答TPA的诱导。此外，OLi-D和Cre的人胸径组合增强了TPA的荧光素酶活性。

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