Abstract
Arylamine N-acetyltransferase 1 (NAT1) plays an important role in the biotransformation of xenobiotics, and genetic variants have been implicated in susceptibility to cancer and birth defects. A specific and quantitative reverse transcription-polymerase chain reaction assay for transcription from the major NAT1 promoter detected high expression with limited variability in human tissues. A 213-base pair (bp) minimal promoter was identified by transfection of luciferase reporter constructs into MCF-7 and HepG2 cell lines. Alignment of the 213-bp region with paralogous and orthologous promoters revealed two conserved region segments, one of which overlaps a 16-bp perfect palindrome. Transfection of luciferase constructs with artificial mutations in the minimal promoter defined two sites important for promoter function. One of these sites included a close match to the Sp1 transcription factor binding consensus sequence. Electrophoretic mobility shift assays (EMSAs), followed by competitive and supershift analyses, confirmed the Sp1 binding. Mutation of the highly conserved portion of the 16-bp palindrome reduced promoter activity more than 3-fold, and an EMSA shift was detected with an oligonucleotide, 200L29, which spans this segment. The 200L29 EMSA shift could not be competed by consensus Sp1 or AP-2 oligonucleotides, and may represent binding of a transcription factor that is common to N-acetyltransferase genes in humans and other species.
Footnotes
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This work was partially supported by National Institutes of Health Grants CA34627 and ES12557. Portions of this work constituted partial fulfillment for the Ph.D. in Pharmacology and Toxicology awarded to A.H. from the University of Louisville.
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doi:10.1124/dmd.107.016485.
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ABBREVIATIONS: NAT, N-acetyltransferase; 5′-RACE, 5′ rapid amplification of cDNA ends; BAC, bacterial artificial chromosome; bp, base pair; EMSA, electrophoretic mobility shift assay; kb, kilobase pair; RT, reverse transcription; PCR, polymerase chain reaction; SNP, single-nucleotide polymorphism; TF, transcription factor; TSS, transcription start site.
- Received May 2, 2007.
- Accepted June 21, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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