Regulation of the Rat PhenylethanolamineN-Methyltransferase Gene by Transcription Factors Sp1 and MAZ

Abstract

The rat phenylethanolamine N-methyltransferase (PNMT) gene promoter contains 1-base pair (bp) overlapping consensus sequences for Sp1 and MAZ transcription factors at Ϫ48 and Ϫ38 bp, respectively. Gel mobility assays using PC-12-derived RS1 cell nuclear extracts or in vitro translated proteins showed that Sp1 and MAZ specifically bind to these elements, that MAZ displaces/prevents Sp1 binding, and that Sp1 and MAZ binding is mutually exclusive, with occupancy dependent on each factor's concentration and affinity for its consensus element. In transfection assays, PNMT promoter activation by Sp1 and MAZ depends on promoter length, with Ϫ893 bp of sequence yielding greatest activation. Although MAZ has higher affinity for its binding element, it is a less effective activator. Changes in PNMT promoter activity for the constructs pGL3RP60 or pGL3RP893 using a fixed amount of MAZ expression construct and a variable amount of Sp1 expression construct or vice versa confirmed the latter. Mutation of the MAZ or Sp1 sites in pGL3RP60 attenuated but did not eliminate PNMT promoter activity, even though the proteins no longer bind to their consensus elements. Phosphatase treatment of RS1 cell nuclear extracts prevented MAZ-and Sp1-DNA binding complex formation. Although MAZ and Sp1 elevate endogenous PNMT mRNA in RS1 cells, MAZ preferentially increases intron-retaining whereas Sp1 preferentially increases intronless mRNA. Thus, expression of the PNMT gene seems to be modulated through competitive binding of phosphorylated Sp1 and MAZ to their consensus elements in the promoter. In addition, posttranscriptional regulation seems to be another important mechanism controlling PNMT expression.

Quantitative Proteomic Identification of MAZ as a Transcriptional Regulator of Muscle-Specific Genes in Skeletal and Cardiac Myocytes

“…Furthermore, since MAZ is only one of a plethora of factors that recognize GC-rich sequences, transcriptional regulation of its target promoters is likely to be complex and highly dynamic. Sp1 and MAZ, for instance, have been shown to compete for binding to the same or overlapping sites (25,49,50,57), which are often present more than once within a given promoter. Interestingly, phosphorylation by casein kinase II has been shown to enhance DNA binding of MAZ (64), whereas it inhibits DNA binding of Sp1 (2).…”

“…In an in vitro coupled transcription-polyadenylation assay, the MAZ binding site promotes pausing of RNA polymerase II and the activation of polyadenylation (73). MAZ has been implicated in the activation of a number of genes, including RAG-2, Adenovirus major late, (9,14,25,37,49,50,66,70,71,72). In addition, MAZ has been shown to repress its own gene, as well as c-myc, eNOS, Sp4, telomerase, and eosinophil-derived neurotoxin (28,34,57,60,62,69).…”

Nerve Growth Factor Regulates Adrenergic Expression

“…Radioactive reverse transcription-polymerase chain reaction (RT-PCR) was performed as described previously (Her et al, 2003). In brief, total RNA was extracted from control and drug-treated PC-12 cells using Tri-Reagent (Sigma-Aldrich) as per the manufacturer.…”

“…Wild-type (pGLRP893) and nested deletion PNMT promoter-luciferase reporter gene constructs (pGL3RP442, pGL3RP392, and pGL3RP60) were generated as described previously (Ebert et al, 1994;Tai et al, 2001). PNMT promoter-luciferase reporter gene constructs containing mutations of the Ϫ165 bp Egr-1 (pGL3RP-893mutEgr-1) and the Ϫ168 and/or Ϫ48 bp Sp1 (pGL3RP893mutS-p1A, pGL3RP893mutSp1B and pGL3RP893mutSp1A/B) binding sites were generated by site-directed mutagenesis Her et al, 2003;Tai and Wong, 2003). The pRSV-LacZ plasmid containing the ␤-galactosidase gene was used as a normalization control to correct for variable transfection efficiency.…”

“…Functional Sp1 binding elements are located at Ϫ48 and Ϫ168 bp in the rat PNMT promoter , and we have shown that the Ϫ48 bp Sp1 site contributes to the cooperative activation of the PNMT promoter by PKA and PKC . Although NGF did not alter Sp1 protein levels in PC-12 cells, Sp1 must be phosphorylated to bind to its consensus element and activate transcription (Kadonaga et al, 1987;Her et al, 2003;Tai and Wong, 2003), and recent findings indicate that NGF does increase Sp1-DNA binding (Liu et al, 2001;Melnikova and Gardner, 2001). The present study showed that site-directed mutation of the Ϫ168-bp Sp1 site attenuated NGF activation of the PNMT promoter, whereas mutation of the Ϫ48 bp Sp1 site or both Sp1 sites eliminated NGF activation.…”