Abstract
Previously, the existence of dual promoters was reported in mouse mu-opioid receptor (mor) gene, with mor transcription in the mouse brain predominantly initiated by the proximal promoter. In this study, we further analyzed the proximal promoter region, base pairs -450 to -249, to identify cis-DNA regulatory elements and trans-acting protein factors that are important for mor promoter activity. The results revealed that a mor inverted GA (iGA) motif and a canonical Sp1 binding site are required for the promoter activity. Using electrophoretic mobility shift analysis, we identified nuclear proteins that specifically bind to the mor iGA motif and that are immunologically related to Sp1 and Sp3. Mutation of the mor iGA motif, resulting in a loss of Sp binding, led to a 50% decrease in activity. Mutation of the canonical Sp1 binding site yielded a lesser (approximately 25%) loss of activity. Mutation of both motifs together resulted in an approximately 70% decrease in activity. In cotransfection assays using Drosophila SL2 cells, Sp1 trans-activated the promoter in a manner dependent on the presence of mor iGA and canonical Sp1 binding motifs. Sp3 can also trans-activate the promoter, and furthermore, Sp1 and Sp3 can trans-activate the mor promoter additively. Our results suggest that combined or cooperative interaction of Sp transcription factors within the proximal promoter is necessary for activation of mor gene transcription.
Highlights
Mainly expressed in the central nervous system, with receptors varying in densities in different regions and perhaps playing different roles [5,6,7]
Identification of the Minimum Sequences Required for the Proximal Promoter Activity of the Mouse -Opioid Receptor Gene—We previously showed that deletional constructs containing only the proximal promoter exhibited activities similar to the activities of constructs containing both distal and proximal promoters in SH-SY5Y, morexpressing cells [18]
Because the pL450 construct contains the entire region of multitranscription initiation sites (Fig. 1A), these results implied that elements important for the basal proximal activity are positioned in the Ϫ450 to Ϫ249 bp region
Summary
Mainly expressed in the central nervous system, with receptors varying in densities in different regions and perhaps playing different roles [5,6,7]. Using deletional and transient transfection assays, a distal and a proximal promoter of mouse mor gene were identified in the cell lines endogenously expressing mor [18, 19]. The proximal promoter initiated mor transcription from four major transcription initiation sites, located in a region 291–268 bp upstream of the translation initiation site [17]. We report here that a mor iGA motif-containing region, positioned near the cluster of transcription start sites, is essential for mor gene expression. This site binds Sp1 and Sp3 or antigenically related proteins. A canonical Sp1 binding site, located 40 bp upstream of the mor iGA motif, contributes to mor promoter activity.
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