Received for publication May 3, 2007.
Revised June 14, 2007.
Accepted for publication June 21, 2007.

Functional Analysis of the Human N-Acetyltransferase 1 Major Promoter: Quantitation of Tissue Expression and Identification of Critical Sequence Elements

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 RT-PCR assay for transcription from the major NAT1 promoter detected high expression with limited variability in human tissues. A 213 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 regions segment, 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 (EMSA), 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.

Key words: acetyltransferases, bioactivation, carcinogen metabolism, genetic polymorphism, inactivation, pharmacogenetics, phase II drug metabolism