Functional analysis of the human N-acetyltransferase 1 major promoter:: Quantitation of tissue expression and identification of critical sequence elements
Functional analysis of the human N-acetyltransferase 1 major promoter:: Quantitation of tissue expression and identification of critical sequence elements
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DOI：
10.1124/dmd.107.016485
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发表时间：
2007-09-01
影响因子：
3.9
通讯作者：
Hein, David W.
中科院分区：
文献类型：
--
作者：
Husain, Anwar;Zhang, Xiaoyan;Hein, David W.
芳胺 N-乙酰转移酶 1 (NAT1) 在异生物质的生物转化中发挥着重要作用，遗传变异与癌症和出生缺陷的易感性有关。对主要 NAT1 启动子转录进行的特异性定量逆转录聚合酶链反应测定检测到人体组织中高表达且变异性有限。通过将荧光素酶报告基因构建体转染至 MCF-7 和 HepG2 细胞系中，鉴定出 213 个碱基对 (bp) 的最小启动子。 213 bp 区域与旁系同源和直系同源启动子的比对揭示了两个保守区域片段，其中一个与 16 bp 完美回文重叠。最小启动子中具有人工突变的荧光素酶构建体的转染定义了对启动子功能重要的两个位点。其中一个位点与 Sp1 转录因子结合共有序列紧密匹配。电泳迁移率变动分析 (EMSA)，随后进行竞争性和超迁移分析，证实了 Sp1 结合。 16 bp 回文结构的高度保守部分的突变使启动子活性降低了 3 倍以上，并且使用跨越该片段的寡核苷酸 200L29 检测到 EMSA 位移。 200L29 EMSA 转变无法通过共有 Sp1 或 AP-2 寡核苷酸竞争，并且可能代表人类和其他物种中 N-乙酰转移酶基因常见的转录因子的结合。
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.