Molecular basis of neuroendocrine cell type-specific expression of the chromogranin B gene: crucial role of the transcription factors CREB, AP-2, Egr-1 and Sp1
Molecular basis of neuroendocrine cell type-specific expression of the chromogranin B gene: crucial role of the transcription factors CREB, AP-2, Egr-1 and Sp1
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DOI：
10.1111/j.1471-4159.2006.04128.x
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发表时间：
2006-10-01
影响因子：
4.7
通讯作者：
Mahata, Sushil K.
中科院分区：
文献类型：
--
作者：
Mahapatra, Nitish R.;Mahata, Manjula;Mahata, Sushil K.
嗜铬粒蛋白B基因（Chgb）神经内分泌特异性表达的分子基础仍不清楚。利用野生型和突变型Chgb启动子/荧光素酶报告基因构建体，本研究确定了cAMP反应元件（CRE）盒在内分泌[大鼠嗜铬细胞瘤]中的关键作用，（嗜铬）细胞系（PC 12）和大鼠垂体生长激素细胞系（GC）]和神经元[大鼠背根神经节/小鼠神经母细胞瘤杂交细胞系（F-11），皮质和海马初级神经元]细胞。此外，在-134/-127，-125/-117和-115/-110 bp处的G/C富集结构域对Chgb基因的内分泌特异性表达起特别重要的作用。CREB、激活蛋白-2（转录因子）（AP-2）、早期生长反应蛋白（转录因子）（Egr-1）或特异性蛋白1（转录因子）（Sp1）的表达质粒与Chgb启动子的共转染构建了Chgb基因的反式激活表达。来自PC 12或F-11细胞的核提取物与Chgb（-110/-87 bp）（CRE）寡核苷酸形成特异性复合物，其被抗CREB抗体超移位或破坏。此外，PC 12核提取物还与含有三个富含G/C的区域的Chgb（-140/-104-bp）寡核苷酸形成特异性复合物，其被抗AP-2、抗Egr-1或抗Sp1抗体剂量依赖性地破坏;实际上，这三种抗体中的任何一种完全消除了复合物，表明所有三种因子同时结合该区域，至少在体外是这样。染色质免疫沉淀试验记录了在PC 12细胞中，在染色质背景下，转录因子CREB、AP-2、Egr-1和Sp1与染色体Chgb基因启动子的体内结合。我们的结论是，Chgb的神经内分泌特异性表达的CRE和G/C盒的顺式和转录因子CREB，AP-2，Egr-1和Sp1的反式介导。
The molecular basis of neuroendocrine-specific expression of chromogranin B gene (Chgb) has remained elusive. Utilizing wild-type and mutant Chgb promoter/luciferase reporter constructs, this study established a crucial role for the cAMP response element (CRE) box at -102/-95 bp in endocrine [rat pheochromocytoma (chromaffin) cell line (PC12) and rat pituitary somatotrope cell line (GC)] and neuronal [rat dorsal root ganglion/mouse neuroblastoma hybrid cell line (F-11), cortical and hippocampal primary neurons] cells. Additionally, G/C-rich domains at -134/-127, -125/-117 and -115/-110 bp played especially important roles for endocrine-specific expression of the Chgb gene. Co-transfection of expression plasmids for CREB, activator protein-2 (transcription factor) (AP-2), early growth response protein (transcription factor) (Egr-1) or specificity protein 1 (transcription factor) (Sp1) with the Chgb promoter constructs trans-activated expression of the Chgb gene. Nuclear extracts from either PC12 or F-11 cells formed specific complexes with the Chgb (-110/-87 bp) (CRE) oligonucleotide, which were either supershifted or disrupted by anti-CREB antibodies. In addition PC12 nuclear extracts also formed a specific complex with a Chgb (-140/-104-bp) oligonucleotide containing three G/C-rich regions, which was dose-dependently disrupted by anti-AP-2, anti-Egr-1 or anti-Sp1 antibodies; indeed, any one of these three antibodies completely abolished the complex, suggesting that all three factors bind the region simultaneously, at least in vitro. Chromatin immunoprecipitation assays documented the binding of the transcription factors CREB, AP-2, Egr-1 and Sp1 to the chromosomal Chgb gene promoter in vivo in PC12 cells within the context of chromatin. We conclude that the neuroendocrine-specific expression of Chgb is mediated by the CRE and G/C boxes in cis and the transcription factors CREB, AP-2, Egr-1 and Sp1 in trans.