Cloning and functional characterization of the human norepinephrine transporter gene promoter

The norepinephrine transporter (NET) plays a critical role in brain norepinephrine homeostasis and is a target for antidepressants and drugs of abuse. We have analyzed the 5'-flanking regulatory region of the human NET gene (SLC6A2). Primer extension and 5' RACE revealed a single transcription start site, alternative splicing of exon 1 due to alternate splice donor usage, and a variable splice acceptor of intron 1. A TA-rich motif 35 bp upstream of the transcription start and several potential binding sites for transcription factors including a cAMP response element (CRE)-like motif are present in the 5'-flanking region. A 4.0-kb fragment, which had been fused to the luciferase reporter gene and transiently expressed in a NET+ cell line, displayed both constitutive and inducible promoter activity. Functional analysis by serial deletions revealed several clusters of cell-selective enhancer elements. Our findings indicate that (1) the NET gene promoter is active in NET-expressing cells and the information contained within approximately 4 kb of the 5'-flanking sequence is required to confer its cell-selective expression, (2) the expression of NET is regulated by a combination of positive cis-acting elements operating through a basal promoter defined by a TA-rich motif, and (3) the promoter responds to cAMP-dependent induction. Fusion of the human NET gene promoter to selected genes will facilitate their cell-selective expression in gene transfer strategies.