FUNCTIONAL PROMOTER AND POLYADENYLATION SITE MAPPING OF THE HUMAN SEROTONIN (5-HT) TRANSPORTER GENE

We have isolated and characterized the 5'-flanking region and the prox imal polyadenylation site of the human 5-HT transporter gene. The majo r gene transcript is 2,793 bp in length and it contains 208 bp of 5'-u ntranslated region (5'-UTR) and 694 bases of 3'-UTR. While only a sing le mRNA species occurs in rats and mice, the most proximal signal for polyadenylation in the human gene appears to be highly degenerate in c omparison to the rat and murine motif. This polyadenylation signal-lik e motif may lead to alternate usage of additional polyadenylation site s resulting in multiple mRNA species in humans. A TATA-like motif and several potential binding sites for transcription factors including AP 1, AP2, SP1, and a cAMP response element (CRE)-like motif are present in the 5'-flanking region. A similar to 1.7 kb fragment beginning 217 bp downstream from the transcription start site, which had been ligate d into a luciferase reporter vector and transiently expressed in JAR h uman placental choriocarcinoma cells, displayed both constitutive and forskolin/cholera toxin-induced promoter activity. Functional promoter mapping revealed that there are negative attenuating elements between bp -1,428 and -1,185 and positive elements between bp -1,184 and -78 from the transcription initiation site. Studies with deletional mutant s also indicated that core promoter sequences are contained within 78 bp of the transcription start site and that regulation of cAMP-inducib le promoter activity depends on multiple cis-acting elements including two AP1 binding sites and a single CRE-like element located at bp -99 . Our findings suggest that (1) the 5-HT transporter gene promoter is active in human JAR cells, but inactive in 5-HT transporter-deficient human SK-N-SH neuroblastoma and HeLa cells, (2) the information contai ned within 1.4 kb of 5'-flanking sequence is sufficient to confer its cell-specific expression, (3) the promoter responds to cAMP induction, and (4) the expression of the 5-HT transporter gene is regulated by a combination of positive and negative cis-acting elements operating th rough a basal promoter unit defined by a TATA-like motif.