Vol. 60, Issue 2, 331-340, August 2001

-Opioid Receptor Gene: Effect of Sp1 Factor on Transcriptional Regulation in Vivo

Dmitri Smirnov, Hee-Jeong Im, and Horace H. Loh

Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota

-Opioid receptor (DOR) promoter exhibited a cell-type-specific expression pattern. Protein-DNA interactions in this promoter were identified by dimethyl sulfate in vivo footprinting analysis of NG108-15 cells, expressing endogenous DOR. Complete protection of the putative Sp1 cis-element and partial protection of the sequence defined as X-NotI in the basal promoter were observed only in the G₀/G₁ phase of the cell cycle. No protection was detected in Neuro2A cells that do not express DOR. In vivo formaldehyde cross-linking confirmed Sp1 factor binding to its cis-acting element during the G₀/G₁ phase. The functional significance of these Sp1 and X-NotI sites was evaluated by transient transfection analysis. Northern blot analysis and nuclear run-off assays revealed maximum DOR mRNA level and transcription rate, respectively, during the G₀/G₁ phase of NG108-15 cells. In summary, the protein-DNA interactions at the Sp1 and X-NotI sites are necessary for cell cycle-dependent and cell-type-specific up-regulated DOR gene expression.