Lim J, Yang C, Hong S J, Kim K S
Molecular Neurobiology Laboratory, McLean Hospital, Harvard Medical School, Belmont, MA 02478, USA.
Mol Cell Biochem. 2000 Sep;212(1-2):51-60.
The conversion of L-tyrosine to 3,4-dihydroxy-L-phenylalanine by tyrosine hydroxylase (TH) is the first and rate-limiting step in biosynthesis of catecholamine neurotransmitters. TH gene expression is regulated in a cell type-specific and cAMP-dependent manner. Evidence from this laboratory and others indicates that the cAMP response element (CRE), residing at -45 to -38 bp upstream of the transcription initiation site, is essential for both basal and cAMP-inducible transcription of the TH gene. To understand the control mechanisms of TH gene transcription in greater detail, we sought to identify and characterize the transcription factors involved in recognition and activation of the CRE of the TH gene. Remarkably, electrophoretic mobility shift assay and antibody supershift experiments indicated that all three major CRE-binding protein factors, i.e. CREB, ATF1, and CREM, may participate in forming specific DNA/protein complexes with the CRE of the TH gene. To address the transcriptional activation function of individual factors, we replaced the TH CRE with a GAL4-binding site and cotransfected this modified TH promoter-reporter gene with an effector plasmid that encodes GAL4-fused transcription factor. Our results indicate that CREB but not ATF1 can support basal promoter activity while both can robustly induce the promoter activity in response to co-expression of the catalytic subunit of cAMP-dependent protein kinase (PKA). We further show that the coactivator CBP up-regulates PKA-mediated activation of the TH promoter and, if tethered to the TH promoter by a GAL4-fusion, can robustly transactivate the TH promoter even in the absence of PKA. Collectively, our results suggest that multiple CRE-binding factors interact with the CRE and regulate, in conjunction with the coactivator CBP, the transcriptional activity of the TH gene.
酪氨酸羟化酶（TH）将L-酪氨酸转化为3,4-二羟基-L-苯丙氨酸是儿茶酚胺神经递质生物合成的第一步，也是限速步骤。TH基因表达以细胞类型特异性和cAMP依赖性方式受到调控。来自本实验室及其他实验室的证据表明，位于转录起始位点上游-45至-38 bp处的cAMP反应元件（CRE）对于TH基因的基础转录和cAMP诱导的转录均至关重要。为了更详细地了解TH基因转录的调控机制，我们试图鉴定并表征参与识别和激活TH基因CRE的转录因子。值得注意的是，电泳迁移率变动分析和抗体超迁移实验表明，所有三种主要的CRE结合蛋白因子，即CREB、ATF1和CREM，可能都参与与TH基因的CRE形成特异性DNA/蛋白质复合物。为了研究各个因子的转录激活功能，我们用GAL4结合位点替换了TH CRE，并将这个修饰后的TH启动子-报告基因与编码GAL4融合转录因子的效应质粒共转染。我们的结果表明，CREB而非ATF1能够支持基础启动子活性，而二者均可在cAMP依赖性蛋白激酶（PKA）催化亚基共表达时强力诱导启动子活性。我们进一步表明，共激活因子CBP上调PKA介导的TH启动子激活，并且如果通过GAL4融合与TH启动子相连，即使在没有PKA的情况下也能强力反式激活TH启动子。总的来说，我们的结果表明多种CRE结合因子与CRE相互作用，并与共激活因子CBP一起调节TH基因的转录活性。
