Characterization of the APP proximal promoter and 5′-untranslated regions:: identification of cell-type specific domains and implications in APP gene expression and Alzheimer's disease
Characterization of the APP proximal promoter and 5′-untranslated regions:: identification of cell-type specific domains and implications in APP gene expression and Alzheimer's disease
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DOI：
10.1096/fj.04-2900fje
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发表时间：
2005-02-01
期刊：
影响因子：
4.8
通讯作者：
Maloney, B
中科院分区：
文献类型：
--
作者：
Lahiri, DK;Ge, YW;Maloney, B
阿尔茨海默病的特点是由A β前体蛋白（APP）产生的有毒淀粉样β肽（A β）在脑内沉积。APP基因的表达通过近端启动子区（PPR，人类序列- 46/- 1，+ 1转录起始）和5'-非翻译区（5'- utr, + 1/+ 147）调控。我们最近发现了一个独特的CAGA序列，“淀粉样蛋白”（+83/+86），仅存在于淀粉样斑块形成物种的APP基因中，不存在于所有APP样蛋白（APLP1和APLP2）基因中。为了测定含有或缺乏“淀粉样蛋白”盒子的PPR + UTR和5'- UTR区域的功能活性，我们在瞬时转染研究中测试了9种构建体。我们观察到- 46/144、- 46/100、- 46/54和54/144构建体的报告基因活性显著提高。54/100片段包含转化生长因子- β /“淀粉样蛋白”/白介素-1急性盒盒，根据细胞类型显示不同的活性。电泳迁移位移分析（EMSA）显示，所有片段中dna -核蛋白相互作用明显，不同细胞类型和特定片段之间存在差异。报告基因的表达与dna -蛋白结合模式一致。为了直接检查“淀粉样蛋白”盒子，我们为CAGA突变体或突变的邻近核苷酸生成了低聚物。EMSA结果表明，改变“淀粉样蛋白”或邻近序列会以突变和细胞类型特异性的方式改变特异性dna -核蛋白相互作用。报告基因试验揭示突变特异性表达效应。因此，- 46/54区域似乎对APP基因的基础表达至关重要，54/100和100/144区域可能具有组织特异性活性，“淀粉样蛋白”CAGA盒在APP基因调控中发挥作用。
Alzheimer's disease is characterized by brain deposition of toxic amyloid beta-peptide (A beta), generated from the A beta precursor protein (APP). APP gene expression is regulated via the proximal promoter region (PPR; - 46/- 1 in the human sequence; + 1 transcription start) and the 5'- untranslated region (5'-UTR; + 1/+ 147). We have recently identified a unique CAGA sequence, "amyloid," (+83/+86) present only in the APP gene from amyloid plaque-forming species, absent in all APP-like-proteins' (APLP1 and APLP2) genes. To assay functional activity of PPR + UTR and 5'- UTR regions that either contain or lack the " amyloid" box, we tested nine constructs in transient transfection studies. We observed significantly high reporter gene activity with - 46/144, - 46/100, - 46/54, and 54/144 constructs. The 54/100 fragment, which contains a transforming growth factor-beta/"amyloid"/interleukin-1 acute box cassette, showed different activity depending on cell type. Electrophoretic mobility shift assay ( EMSA) showed distinct DNA-nuclear protein interaction in all fragments, differing among both cell types and specific fragment. Reporter gene expression corroborates with the DNA-protein binding pattern. To directly examine the " amyloid" box, we generated oligomers for CAGA mutants or mutated adjacent nucleotides. EMSA results showed that altering " amyloid" or adjacent sequence alters specific DNA-nuclear protein interaction in both mutation- and cell-type-specific manners. Reporter gene assay reveals mutant-specific expression effects. Therefore, the - 46/54 region appears to be essential for basal expression of the APP gene, the 54/100 and 100/144 regions may have tissue-specific activity, and the " amyloid" CAGA box plays a role in APP gene regulation.