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. ? ?
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