A proximal promoter domain containing a homeodomain-binding core motif interacts with multiple transcription factors, including HoxA5 and Phox2 proteins, and critically regulates cell type-specific transcription of the human norepinephrine transporter gene.

Expression of the norepinephrine transporter (NET), which mediates the reuptake of norepinephrine into presynaptic nerve terminals, is restricted to noradrenergic (NA) neurons. We have demonstrated previously that the 9.0 kb upstream sequences and the first intron residing in the 5' untranslated area are critical for high-level and NA cell-specific transcription. Here, using transient transfection assays, we show that 4.0 kb of the 5' upstream sequences contains sufficient genetic information to drive reporter gene expression in an NA cell type-specific manner. Three functional domains appear to be potentially important for the regulation of human NET (hNET) gene transcription: an upstream enhancer region at -4.0 to -3.1 kb, a proximal domain at -133 to -75 bp, and a middle silencer region between these two domains. DNase I footprinting analysis of the proximal promoter region shows that a subdomain at -128 to -80 bp is protected in a cell-specific manner. We provide evidence that multiple protein factors interact with the proximal promoter domain to critically regulate the transcriptional activity of the hNET gene. In the middle of this proximal subdomain resides a homeodomain (HD)-binding core motif, which interacts with HD factors, including Phox2a and HoxA5, in an NA-specific manner. Cotransfection analyses suggest that HoxA5 and Phox2a may transactivate the hNET gene promoter. Together with previous studies indicating direct activation of dopamine beta-hydroxylase transcription by Phox2a/2b, the present results support a model whereby Phox2 proteins may coordinately regulate the phenotypic specification of NA neurons by activating both NA biosynthetic and reuptake genes.