Abstract
The rat phenylethanolamine N-methyltransferase (PNMT) gene contains overlapping consensus elements for the Sp1 and Egr-1 transcription factors located at -45 bp and -165 bp in the PNMT promoter. In the present study, we show that Sp1 and Egr-1 can specifically bind to these overlapping elements, that this binding appears to be mutually exclusive, and that binding site occupancy is dependent upon the concentration of each factor and its binding affinity for each site. Egr-1 binds to the -165 bp site with relatively high affinity (IC50 = 14 nM) and to the -45 bp site with relatively low affinity (IC50 = 1360 nM), whereas Sp1 binds to both sites with intermediate affinities (IC50 = 210 and 140 nM, respectively). Consistent with the DNA-binding data, Egr-1 stimulates PNMT promoter activity primarily through interaction with the -165 bp site, while Sp1 stimulates PNMT promoter activity by interacting with both the -45 bp and the -165 bp sites. These results show that Sp1 and Egr-1 are capable of differentially activating PNMT gene expression, thereby suggesting that different stimuli may control the activity of the PNMT gene by selectively regulating Sp1 and/or Egr-1.
Highlights
From The Nancy Pritzker Laboratory of Developmental and Molecular Neurobiology, Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, California 94305-5485
The rat phenylethanolamine N-methyltransferase (PNMT) gene contains overlapping consensus elements for the Spl and Egr-l transcription factors located at -45 bp and -165 bp in the PNMT promoter
Egr-l binds to the -165 bp site with relatively high affinity and to the -45 bp site with relatively low affinity (ICso = 1360 na), whereas Spl binds to both sites with intermediate affinities (ICso = 210 and 140 nM, respectively)
Summary
Oligonucleotides-Eight duplex 21-mer oligonucleotides were prepared by annealing individually synthesized complimentary DNA fragments as described previously (Ebert et al, 1994). These duplex oligonucleotides represent wild-type and mutant forms of the overlapping Spl/Egr-I DNA-binding elements from the rat PNMT promoter. Wild-type DNA sequences are designated with the prefix "wt" while mutants have the prefix "mut" followed by an "A" or "B" to indicate which of the overlapping elements from the rat PNMT promoter are represented by each duplex oligomer (A, -159 to -179 bp; B, -39 to -59 bp) (see Fig. 2). Statistical significance was determined by one way analysis of variance
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