Human dopamine transporter gene: variations and transcriptional regulation

描述（由申请人提供）：本提案的总体目标是确定人类多巴胺转运蛋白基因（hDAT）启动子区域的多态性，以及这些多态性影响转录因子结合和启动子活性的机制。多巴胺转运体（dopamine transporter， DAT）通过多巴胺的再摄取和释放调节多巴胺神经传递的时空域，参与运动、动机、认知和注意、工作记忆、行为组织和激素释放。众所周知，DAT基因在大脑中的表达是高度受限的，在个体受试者中是不同的，并且可以受到内源性和外源性因素的调节。DAT表达的改变可能导致hdat相关的病理生理状态，如注意缺陷/多动障碍、酗酒、吸烟、药物滥用、妥瑞氏综合征和帕金森病。然而，关于启动子区域的DNA序列变化如何影响hDAT的调控启动子活性，我们知之甚少。待验证的假设是hDAT启动子序列因个体而异，从而导致启动子活性和hDAT启动子活性调控的差异。我们的初步研究表明，高加索hDAT启动子区域具有高度多态性，许多单倍型具有不同的启动子活性，部分原因是核蛋白与内含子1结合。因此，本项目的目的是：1)揭示不同人群中hDAT启动子的共同单倍型；2)体外克隆结合内含子1的核蛋白cdna；3)确定以单倍型依赖的方式调节hDAT表达的细胞内信号通路。我们建议开发一种新的基于F质粒的方法来评估高达300 kb DNA片段的启动子活性，这对于研究大型启动子区域是至关重要的。这些发现将有助于我们理解外部刺激对hDAT的转录调控以及不同脑区选择性DAT表达的机制。可以想象，我们将确定新的药物靶点，并定义启动子单倍型，这可能成为工程多巴胺能神经元的工具。

DESCRIPTION (provided by applicant): The overall goals of this proposal are to identify polymorphisms in the promoter region of the human dopamine transporter gene (hDAT) and the mechanisms by which these polymorphisms influence transcription factor binding and promoter activity. The dopamine transporter (DAT) regulates the spatio- temporal domains of dopamine neurotransmission by reuptake and release of dopamine and contributes to locomotion, motivation, cognition and attention, working memory, behavioral organization and hormone release. It is well recognized that expression of the DAT gene in the brain is highly circumscribed, varies in individual subjects and can be regulated by endogenous and exogenous factors. Altered DAT expression may contribute to hDAT-associated pathophysiological states such as attention deficit/hyperactivity disorder, alcoholism, smoking, drug abuse, Tourette's syndrome and Parkinson's disease. However little is known about how DNA sequence variations in the promoter region influence the regulated promoter activity of hDAT. The hypothesis to be tested is that the hDAT promoter sequence varies from individual to individual, conferring differences in promoter activity and in regulation of hDAT promoter activity. Our preliminary studies show that the Caucasian hDAT promoter region is highly polymorphic, conferring many haplotypes with varying promoter activity partly due to nuclear protein binding to Intron 1. Therefore, the aims of this project are to: 1) reveal hDAT promoter common haplotypes in various populations; 2) clone cDNAs for nuclear proteins that bind to Intron 1 in vitro; and 3) identify intracellular signaling pathways that regulate hDAT expression in a haplotype-dependent manner. We propose to develop a new F plasmid-based methodology to assess promoter activity of up to 300 kb DNA fragments, which is critically needed for studying large promoter regions. These findings will contribute to our understanding of transcriptional regulation of hDAT by external stimuli and the mechanism of selected DAT expression in different brain regions. Conceivably, we will identify novel pharmaceutical targets and define promoter haplotypes that could become tools for engineering dopaminergic neurons.