Enhancer function and novel DNA binding protein activity in the near upstream betaAPP gene promoter.
Enhancer function and novel DNA binding protein activity in the near upstream betaAPP gene promoter.
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DOI：
10.1016/s0378-1119(99)00091-8
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发表时间：
1999-05
期刊：
影响因子：
3.5
通讯作者：
H. Querfurth;J. Jiang;W. Xia;D. Selkoe
中科院分区：
文献类型：
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作者：
H. Querfurth;J. Jiang;W. Xia;D. Selkoe
βAPP 基因转录和启动子调控在改变淀粉样蛋白 β-肽 (Aβ) 水平中的作用尚不清楚。在家族性阿尔茨海默病受试者中，当存在突变的早老素或 βAPP 等位基因时，成纤维细胞 Aβ 的产生增加或 Aβ42/Aβ40 比率发生变化。 21 三体中 βAPP mRNA 和 Aβ 水平均增加。APP 基因启动子属于一类管家基因，包含两个用于转录因子 AP1 结合的推定共有位点。使用人成纤维细胞和 HeLa 核提取物进行电泳迁移率变动 (EMSA) 和 DNase 保护测定，鉴定出具有新颖 Sp1 样特性的特定蛋白与 βAPP 启动子的近上游和下游结构域结合。上游结合活性定位于假定的 AP1 共有位点及其直接 5' 相邻的富含 GC 的元件。然而，c-Jun 抗体和竞争实验对该结构域的结合没有影响。 HeLa 和成纤维细胞中一系列 5' 缺失的 βAPP 启动子-报告基因转染表明，包含结构域的区域 n.t. -383 至 -348，对基础 pβAPP 报告基因转录产生 2.9 倍的激活影响。当进行亚克隆以测试增强子功能时，5'-GC 元件/“AP1 位点”串联构建体的活性比单独任一元件高四倍，比 3' 位置更靠前的 HSE 共有序列高两倍。佛波酯治疗在这些报告基因检测中没有效果。该元件与紧邻下游 E-box/USF 元件 5' 的结构域具有同源性和结合特性。提出了一种涉及两个域和相邻 DNA 循环的相互作用模型。我们的结论是，这种新描述的结合蛋白-增强子复合物是 βAPP 启动子完全激活所必需的。
The role of βAPP gene transcription and promoter regulation in modifying amyloid β-peptide (Aβ) levels is not well understood. Increased production of Aβ or changes in Aβ42/Aβ40ratio by fibroblasts occurs in the presence of mutant presenilin or βAPP alleles in familial Alzheimer's disease subjects. Both βAPP mRNA and Aβ levels are increased in trisomy 21. The APP gene promoter is in a class of housekeeping genes and contains two putative consensus sites for the binding of transcription factor AP1. Electrophoretic mobility shift (EMSA) and DNase protection assays using human fibroblast and HeLa nuclear extract identified specific protein binding with novel Sp1-like properties to both a near-upstream and a downstream domain of the βAPP promoter. The upstream binding activity was localized to a putative AP1 consensus site and its immediate 5′-adjacent GC-rich element. However, c-Jun antibody and competition experiments had no effect on binding to this domain. A series of 5′-deleted βAPP promoter–reporter gene transfections in HeLa and fibroblast cells showed that the domain-containing region, n.t. −383 to −348, exerts a 2.9-fold activating influence on basal pβAPP-reporter transcription. When subcloned to test enhancer function, the 5′-GC element/‘AP1 site’ tandem construct conferred four-fold greater activity than either element alone and two-fold greater than the more 3′-situated HSE consensus sequence. Phorbol ester treatment had no effect in these reporter assays. This element shares homology and binding properties with a domain immediately 5′ to the downstream E-box/USF element. An interaction model involving both domains and looping of interjacent DNA is proposed. We conclude that this newly described binding protein–enhancer complex is required for full βAPP promoter activation.