Cloning and characterization of the rat lysyl oxidase gene promoter - Identification of core promoter elements and functional nuclear factor I-binding sites
Cloning and characterization of the rat lysyl oxidase gene promoter - Identification of core promoter elements and functional nuclear factor I-binding sites
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DOI：
10.1074/jbc.m610108200
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发表时间：
2007-08-31
影响因子：
4.8
通讯作者：
Li, Wande
中科院分区：
文献类型：
--
作者：
Gao, Song;Zhao, Yinzhi;Li, Wande
赖氨酰氧化酶（LO）通过交联胶原蛋白和弹性蛋白来稳定细胞外基质。为了评估LO的转录调控，我们克隆了大鼠LO基因的5 '侧翼区，长度为3，979 bp。LO转录起始于聚集在ATG上游-78至-51区域的多个位点。下游核心启动子元件功能上独立于启动子主要激活TATA-少LO基因。5'端缺失分析表明ATG上游804 bp的序列足以引发最大启动子活性，而-709/-598区域对瞬时转染的RFL 6细胞中报告基因表达表现出强烈的增强作用。DNA酶I足迹分析显示，在片段-612/-580中存在受保护的模式，其在-594/-580区域含有核因子I（NFI）结合位点，电泳迁移率超移分析证实了这一点。该作用位点上的突变降低了凝胶迁移试验中的NFI结合亲和力和用NFI结合位点-SV 40启动子嵌合构建体转染的细胞中SV 40启动子活性的刺激。此外，在LO启动子区-804/-1中鉴定了至少两个功能性NFI结合位点，包括另一个位于-147/-133的位点。只有NFI-A和NFI-B在大鼠肺成纤维细胞中表达，并且它们与LO基因的相互作用被外源刺激如香烟烟雾冷凝物敏感地调节。总之，分离的大鼠LO基因启动子包含功能独立的启动子和下游核心启动子元件，保守的NFI结合位点在LO基因激活中起着关键作用。
Lysyl oxidase (LO) stabilizes the extracellular matrix by crosslinking collagen and elastin. To assess the transcriptional regulation of LO, we cloned the 5'-flanking region with 3,979 bp of the rat LO gene. LO transcription started at multiple sites clustered at the region from -78 to -51 upstream of ATG. The downstream core promoter element functionally independent of the initiator predominantly activated the TATA-less LO gene. 5' Deletion assays illustrated a sequence of 804 bp upstream of ATG sufficient for eliciting the maximal promoter activity and the region -709/-598 exhibiting strongly enhancing effects on the reporter gene expression in transiently transfected RFL6 cells. DNase I footprinting assays showed a protected pattern existing in the fragment -612/-580, which contains a nuclear factor I ( NFI)-binding site at the region -594/-580 confirmed by electrophoretic mobility supershift assays. Mutations on this acting site decreased both NFI binding affinity in gel shift assays and stimulation of SV40 promoter activities in cells transfected with the NFI-binding site-SV40 promoter chimeric construct. Furthermore, at least two functional NFI-binding sites, including another one located at -147/-133, were identified in the LO promoter region -804/-1. Only NFI-A and NFI-B were expressed in rat lung fibroblasts, and their interaction with the LO gene was sensitively modulated by exogenous stimuli such as cigarette smoke condensate. In conclusion, the isolated rat LO gene promoter contains functionally independent initiator and downstream core promoter elements, and the conserved NFI-binding sites play a critical role in the LO gene activation.