Phenylethanolamine N-methyltransferase gene expression - SP1 and MAZ potential for tissue-specific expression

Phenylethanolamine N-methyltransferase (PNMT) promoter-luciferase reporter gene constructs (pGL3RP863, pGL3RP444, and pGL3RP392) transfected into COS1 , RS1, PC12, NIH/3T3, or Neuro2A cells showed the highest basal luciferase activity in the Neuro2A cells. DNase I footprinting with Neuro2A cell nucle ar extract identified protected PNMT promoter regions spanning the -168/-16 5 and -48/-45 base pair Sp1/Egr-1 binding sites. Gel mobility shift assays and transient transfection assays using site-directed mutant PNMT promoter- luciferase reporter gene constructs indicated that the elevated basal lucif erase activity in the Neuro2A cells was mediated by Sp-1. Furthermore, acti vation of the PNMT promoter by Sp1 depends on both its binding affinity for its cognate target sequences and its intracellular concentrations. When Sp 1 levels were increased through an expression plasmid, luciferase reporter gene expression rose well beyond basal wild-type levels, even with either S p1 binding element mutated. Finally, another transcription factor expressed in the Neuro2A cells competes with Sp1 by interacting with DNA sequences 3 ' to the -48 base pair Sp1 site to prevent Sp1 binding and induction of the PNMT promoter. The DNA consensus sequence, Southwestern analysis, and gel mobility shift assays with antibodies identify MAZ as the competitive facto r. These findings suggest that Sp1 may potentially contribute to the tissue -specific expression of the PNMT gene, with the competition between Sp1 and MAZ conferring additional tissue-specific control.