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

多巴胺β-羟化酶基因转录调控的分子机制。

• 批准号: 07670156
• 负责人: ISHIGURO Hiroshi
• 金额: $ 1.22万
• 依托单位: Fujita Health University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07670156/
• 关键词: Dopamine beta-hydroxylase; gene expression; transcriptional regulation; protein kinase A; protein kinase C; YY-1; ドーパミンβ水酸化酵素

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

多巴胺β-羟化酶（DBH）将多巴胺转化为去甲肾上腺素，并在去甲肾上腺素能和肾上腺素能神经元中特异性表达。环磷酸腺苷反应元件（CRE，TGACGTCC）是人DBH基因启动子区的重要转录元件。在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），它们可以调节许多基因的转录。为了鉴定DNA结合蛋白，使用26 bp寡核苷酸（actga TGACGTCCATGTGTCAttagt）作为电泳迁移率变动分析（EMSA）的探针。通过定点突变，我们发现CRE和YY-1结合序列都能与蛋白质形成复合物，而E-box和AP-1样序列不能。加入酪氨酸羟化酶（TH）或生长抑素基因的CRE寡核苷酸作为竞争者，消除了CRE和CRE结合蛋白（可能是CREB）形成的上移条带 ...更多信息 或ATF）。过量的小鼠c-fos基因的YY-1结合序列寡核苷酸作为竞争剂，完全消除了中间移动的条带。YY-1是一种广泛表达的锌指型DNA结合蛋白，它调节多种基因的转录水平，如多种病毒基因、免疫球蛋白基因、（kappa和mu），癌基因（c-fos和c-myc），珠蛋白基因该蛋白的一个显著特征是具有双重功能，根据启动子背景和细胞内环境，该双重功能可以产生积极或消极的影响。为了评估YY-1与人DBH的CCAT基序结合的功能，在表达DBH的神经母细胞瘤细胞（SK-N-SH）中进行瞬时转染实验。定点突变实验表明，YY-1通过添加佛波醇酯（TPA）对人DBH基因的转录水平具有正调控作用。荧光素酶检测结果表明，CRE结合蛋白与YY-1相互作用需要特异性的方向。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TPA反应DNA区（TRR），制备了-604 ~-171bp的缺失突变体，并将这些荧光素酶报告基因（Luc）转染SK-N-SH细胞。TRR位于人DBH基因CRE上游-223 ~-187bp之间，在其它已知的TPA反应元件（TRE）序列中未发现类似序列。另外，我们还对未知的TRE、TRR进行了鉴定，将其分为-223 ~-199 bp（OLI-A）、-213 ~-183 bp（OLI-D）和-206 ~-181 bp（OLI-B）三个DNA区域。首先，OLI-D消除了OLI-63（-224至-162区域）和核蛋白复合物。第二，OLI-D通过使用tk启动子响应TPA诱导。此外，人DBH的OLI-D和CRE组合通过TPA增强荧光素酶活性。

项目成果

TakaHide Nomura, Hiroshi Ishiguro, Yasumichi Hagino, and Toshiharu Nagatsu.: "Noradrenaline, Adrenaline" Japanese Journal of Neuropsychopharmacology. 19. 94-113 (1997)

TakaHide Nomura、Hiroshi Ishiguro、Yasumichi Hagino 和 Toshiharu Nagatsu。：“去甲肾上腺素，肾上腺素”日本神经精神药理学杂志。

野村隆英: "ノルアドレナリン、アドレナリン" 神経精神薬理. 19. 94-113 (1997)

Takahide Nomura：“去甲肾上腺素，肾上腺素”神经精神药理学 19. 94-113 (1997)。

野村隆英: "ニューロトランスミッター・トゥディ" 神経精神薬理, 283 (1997)

Takahide Nomura：“今天的神经递质”神经精神药理学，283（1997）

石黒啓司: "Identification of a negative regulatory element in the 5'-flanking region of the human dopamine β-hydroxylasegene." Mol.Brain Res.34. 251-261 (1995)

Keiji Ishiguro：“人类多巴胺 β-羟化酶基因 5 侧翼区域的负调控元件的鉴定。Mol.Brain Res.34 (1995)。

Hiroshi Ishiguro, Kwang-Soo Kim, and Tong H.Joh: "Identification of a negative regulatory element in the 5'-flanking region of the human dopamine beta-hydroxylasegene." Mol.Brain Res.34. 251-261 (1995)

Hiroshi Ishiguro、Kwang-Soo Kim 和 Tong H.Joh：“鉴定人类多巴胺 β-羟化酶基因 5 侧翼区域的负调控元件。”