An E-box motif residing in the exon/intron 1 junction regulates both transcriptional activation and splicing of the human norepinephrine transporter gene
Abstract
The norepinephrine transporter (NET) is responsible for the rapid NaCl-dependent uptake of norepinephrine into presynaptic noradrenergic nerve endings. Recently, we have characterized the structural organization of the 5' upstream promoter region of the human NET (hNET) gene. A new intron of 476 base pairs was found in the middle of the 5'-untranslated leader sequence and was shown to robustly enhance the promoter activity. Here, we show that the first hNET intron enhances both the homologous hNET and the heterologous thymidine kinase promoter activities in an orientation- and position-dependent manner. The first hNET intron exhibited a similar promoter-enhancing effect in both SK-N-BE(2)C (NET-positive) and HeLa (NET-negative) cell lines, showing that its function is not cell-specific. Transient transfection assays of a series of deletional constructs show that the first hNET intron contains subdomains with either positive or negative regulatory functions. Furthermore, DNase I footprinting analysis demonstrated that the 5' side of the intron, encompassing the splice donor site, is prominently protected by nuclear proteins isolated from both SK-N-BE(2)C and HeLa cells. The protected nucleotide sequence contains a consensus E-bo...Continue Reading
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Molecular cloning and organization of the coding region of the human norepinephrine transporter gene
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