Differential Activation of the Rat Phenylethanolamine N-Methyltransferase Gene by Sp1 and Egr-1

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.

Protein kinase A and protein kinase C signaling pathway interaction in phenylethanolamine N‐methyltransferase gene regulation

“…PC12-derived RS1 cells (Ebert et al 1994) were cultured in DMEM supplemented with 5% equine serum, 5% bovine calf serum, gentamycin sulfate (50 lg/mL) and hygromycin (100 units/mL). All cells were maintained in a humidified incubator at 37°C in an atmosphere of 5% CO 2 ) 95% air.…”

“…Site-directed mutagenesis of the )165 bp Egr-1 and the ) 168 and ) 48 bp Sp1 binding site was performed using the Transformer site-directed mutagenesis kit (Clontech, Palo Alto, CA, USA) to generate pGL3RP893mutEgr-1, pGL3RP392mutSp1A, pGL3RP392mutSp1B and pGL3RP392mut-Sp1A/B (Ebert et al 1994;Ebert and Wong 1995;Her et al 1999). The pGL3RP893mutSp1A, pGL3RP893mutSp1B and pGL3RP893-mutSp1A/B plasmids were constructed by excising the DNA sequences spanning ) 893 to ) 393 bp from pGL3RP893 using NheI, followed by re-ligation into pGL3RP392mutSp1A, pGL3RP392mutSp1B or pGL3RP392mutSp1A/B, respectively, linearized with NheI.…”

“…The wild-type and nested deletion mutant PNMT promoterluciferase reporter gene constructs pGL3RP893, pGL3RP442, pGL3RP392, pGL3RP60 were generated as described previously (Ebert et al 1994;Her et al 1999). Site-directed mutagenesis of the )165 bp Egr-1 and the ) 168 and ) 48 bp Sp1 binding site was performed using the Transformer site-directed mutagenesis kit (Clontech, Palo Alto, CA, USA) to generate pGL3RP893mutEgr-1, pGL3RP392mutSp1A, pGL3RP392mutSp1B and pGL3RP392mut-Sp1A/B (Ebert et al 1994;Ebert and Wong 1995;Her et al 1999).…”

“…Recent studies on the proximal rat PNMT gene promoter/regulatory sequences have identified a number of key transcription factors involved in trans-activation of the promoter, including Egr-1 (Ebert et al 1994), Sp1 , AP-2 , MAZ (Her et al 1999) and the glucocorticoid receptor (GR) (Ross et al 1990). Three of these factors, Egr-1, AP-2 and the GR interact synergistically to activate the PNMT gene , and it has further been shown that stimulation of the rat PNMT promoter via the PKC (Morita et al 1995) and PKA pathways (Tai et al 2001) is in part mediated by the immediate early gene transcription factor, Egr-1.…”

Chromaffin cell function and structure is impaired in corticotropin-releasing hormone receptor type 1-null mice

“…34 In addition, the PNMT gene contains overlapping consensus elements for the promoter selective transcription factor (Sp 1) and the immediate early gene transcription factor Egr-1 that are capable of differentially activating PNMT gene expression. 35 Sp 1, Egr-1 and the glucocorticoid receptor and AP-2 function cooperatively to stimulate PNMT promoter activity through poorly delineated mechanisms. 36 Glucocorticoid control of PNMT gene transcription and protein synthesis do not fully account for changes in PNMT expression in the present study.…”