Functional analysis of a novel human serotonin transporter gene promoter in immortalized raphe cells

To investigate the structural basis for genetic regulation of the human ser otonin transporter gene, a 1.8 kb fragment upstream to the cap site was clo ned and sequenced. The promoter possesses a polymorphic repeat region with 16 and 14 repeats, respectively. Both were cloned and characterized. The pr omoter sequence revealed an internal 379 bp fragment not reported in previo us publications. This novel fragment contains consensus sequences for sever al transcription factors including SpI and GATA. DNA from 48 unrelated indi viduals was PCR amplified, in this region, to test for allelic variations. All were found to possess the additional 379 bp fragment. The integrity of the promoter was furthermore confirmed by genomic Southern blotting. The pr omoter activity was analyzed by reporter gene assays in neuronal and non-ne uronal serotonergic cell lines. In immortalized serotonergic raphe neurons, RN46A, three cis-acting, cell specific, activating elements and a silencer were located. One of the activators and the silencer are located in the re peat region and one activator is positioned in the novel fragment. A fourth activating element was found to be active in both RN46A cells and in a non -neuronal serotonergic cell line, JAR. A 3.5 kb fragment from intron 1 was cloned and found to possess cell specific activity in JAR cells indicating the presence of an alternative promoter in intron 1. (C) 1999 Elsevier Scie nce B.V. All rights reserved.