Promoter and intronic variants affect the transcriptional regulation of the human dopamine transporter gene Tiffany A. Greenwood and John R. Kelsoe* Department of Psychiatry, University of California, San Diego and San Diego VA Health Care System, San Diego, CA 92093, USA Received 12 March 2003; accepted 29 April 2003 Abstract We have attempted to identify regions involved in the transcriptional regulation of the DAT1 (HUGO approved symbol SLC6A3) gene that may harbor functional variants predisposing to several neuropsychiatric disorders by examining haplotypes of various 5' and intronic regions for their effect on expression in a dopaminergic cell line. A 1.5-fold difference in regulatory activity was observed between haplotypes of the proximal promoter/intron 1 region, representing the two previously identified 5' clades. Although we found no effect on transcription with inclusion of the 9- and 10-repeat alleles of the 3' VNTR, introns 9, 12, and 14 appear to contain enhancer elements capable of increasing expression approximately 2-fold with respect to the promoter constructs. Differences in expression were also observed between two alleles of intron 14. These results thus suggest that it may be the particular combination of polymorphisms in a haplotype across the gene that ultimately affects DAT1 gene expression. © 2003 Elsevier Inc. All rights reserved. Keywords: Dopamine transporter; Dopamine; Bipolar disorder; Genetic variation; Single-nucleotide polymorphism; Gene expression; Transcriptional regulation The dopamine transporter (DAT) mediates the active reuptake of dopamine from the synapse and thereby plays a key role in the regulation of dopaminergic neurotransmis- sion. Dopamine is an important mediator of motor behav- iors through projections to the striatum and of emotional and attentional behaviors largely through projections to a variety of limbic targets. Cocaine, amphetamine, and other stimulants block the action of DAT, thus increasing synap- tic dopamine concentrations. The neuropsychiatric effects of these drugs, as well as a wide array of other data, argue for the importance of dopamine in many neuropsychiatric illnesses. A better understanding of the elements controlling the expression of this gene may provide insight into the pathophysiology of these disorders. The DAT gene is expressed exclusively in the central nervous system, primarily in midbrain dopaminergic neu- rons of the substantia nigra and ventral tegmental regions [1]. This highly restricted pattern of DAT gene expression is presumably regulated by a unique combination of positive and negative regulatory factors, which have thus far re- mained elusive. Characterization of the 5' flanking se- quence of the human DAT gene (DAT1; HUGO approved symbol SLC6A3) has provided some insight as far as which sequences or regions may be involved in transcriptional regulation. In vitro experiments have demonstrated that proximal DAT1 sequences constitute a strong core promoter capable of initiating expression without obvious neurospeci- ficity [2,3]. The presence of a strong nonspecific promoter suggests the existence of silencing elements that define the specific expression of DAT1, an idea supported by the ob- served silencing of basal proximal promoter activity in reporter gene constructs with the inclusion of 5' flanking sequences [2,3]. Enhancer elements also appear to play a role in regulating the expression of the DAT1 gene. The transcriptional activity of DAT1 gene constructs has been shown to be enhanced in the presence of the nuclear recep- tor nurr1, an interaction that appears to be mediated by an as yet unidentified nurr1-responsive element located within 1 kb upstream [3,4]. Although regulatory elements have pre- * Corresponding author. Fax: +1-858-534-5527. E-mail address: jkelsoe@ucsd.edu (J.R. Kelsoe). R Available online at www.sciencedirect.com Genomics 82 (2003) 511–519 www.elsevier.com/locate/ygeno 0888-7543/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0888-7543(03)00142-3