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Functional analysis of the human N‐acetyltransferase 1 (NAT1) major promoter: Quantitation of tissue expression and identification of critical sequence elements
Author(s) -
Husain Anwar,
Barker David F,
Zhang Xiaoyan,
Doll Mark A,
States J. Christopher,
Hein David W.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a195-b
Subject(s) - electrophoretic mobility shift assay , promoter , biology , transcription factor , microbiology and biotechnology , reporter gene , dna binding site , binding site , oligonucleotide , transcription (linguistics) , gene , sp1 transcription factor , consensus sequence , conserved sequence , luciferase , genetics , gene expression , peptide sequence , transfection , linguistics , philosophy
N‐acetyltransferases (NAT1 & NAT2) metabolize drugs and xenobiotics A specific and quantitative RT‐PCR assay showed that the NAT1 major promoter P1 was similarly active in 29 different human tissues. Alignment of NAT1 P1 with the NAT2 promoter and the promoters of mouse, rat and cow Nat genes revealed two regions of relatively high conservation. One conserved region overlaps with a 16 bp perfect palindrome. A 213 bp DNA segment with P1 promoter function was identified by deletion analysis of luciferase expression constructs in MCF‐7 and HepG2 cell lines. Luciferase reporter constructs were prepared with larger deletions or specific site‐directed mutations in candidate transcription factor binding sites. Two sites important for basic promoter function were defined, including an Sp1 site indicated by both conservation and transcription factor database analysis. Binding of Sp1 to this site was experimentally confirmed by electrophoretic mobility shift assay (EMSA), including competition with an Sp1 consensus oligonucleotide and supershift analysis with a specific anti‐Sp1 antibody. An EMSA shift was also detected with an oligonucleotide corresponding to the conserved/palindrome sequence. This shift was not competed by consensus Sp1 or AP‐2 oligonucleotides, and may represent binding of a transcription factor common to Nat genes across species. Supported by USPHS grants CA34627 & ES12557.