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首页> 外文期刊>Molecular and Cellular Endocrinology >Transcriptional regulation of the human chromogranin A gene by its 5' distal regulatory element: novel effects of orientation, structure, flanking sequences, and position on expression.
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Transcriptional regulation of the human chromogranin A gene by its 5' distal regulatory element: novel effects of orientation, structure, flanking sequences, and position on expression.

机译：人类嗜铬粒蛋白A基因的5'末端调控元件对转录的调控：方向，结构，侧翼序列和位置对表达的新影响。

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In previous studies of human chromogranin A (hCgA) gene expression, we had identified a 27 bp distal regulatory element (DRE) located between -576 and -550 bp that interacts with a CRE-containing promoter to enhance gene transcription specifically in neuroendocrine (NE) cells. To characterize the DRE we have now mutated its various parts and assessed the effects on protein binding by electrophoretic mobility shift assays (EMSAs) and hCgA transcription within BEN cells. We found that the sequence TGACTAA, an AP-1 binding site that we refer to as DRE-AP-1, was necessary but not sufficient to produce the DRE's enhancer effect. Moreover, while AP-1 (Jun/Fos) bound this site, binding was not correlated with transcriptional effects. Protein binding by the DRE-AP-1 could be attenuated by mutations of its flanking sequences, and transcriptional enhancement by the DRE was dependent on its orientation and spatial relationship to the hCgA proximal promoter. Mutation of the DRE-AP-1 to a consensus AP-1 did not produce greater transcriptional activity, even though it increased binding of nuclear factors. Co-transfection with c-jun and/or c-fos expression plasmids showed that the DRE was unresponsive to the over-expressed AP-1 proteins. Co-transfection with wild-type DRE oligonucleotides competitively inhibited DRE-mediated transcription, while co-transfection with mutant DRE oligonucleotides had a lesser effect. Our studies indicate that transcriptional enhancement of hCgA by the DRE is dependent on a unique NE-specific DRE-binding factor, which we refer to as DBF, that specifically and directionally binds the DRE to assemble and synergize a functional transcription complex.
机译：在以前的人类嗜铬粒蛋白A（hCgA）基因表达研究中，我们发现了一个位于-576至-550 bp之间的27 bp远端调控元件（DRE），它与含CRE的启动子相互作用，以增强神经内分泌（NE）中的基因转录） 细胞。为了表征DRE，我们现在已经对其各个部分进行了突变，并通过电泳迁移率迁移分析（EMSA）和BEN细胞内的hCgA转录评估了对蛋白质结合的影响。我们发现序列TGACTAA（AP-1结合位点，我们称为DRE-AP-1）是必需的，但不足以产生DRE的增强子作用。此外，虽然AP-1（Jun / Fos）结合了该位点，但结合与转录作用无关。 DRE-AP-1的蛋白质结合可通过其侧翼序列的突变而减弱，而DRE的转录增强取决于其与hCgA近端启动子的方向和空间关系。 DRE-AP-1突变为共识AP-1不会产生更大的转录活性，即使它增加了核因子的结合。用c-jun和/或c-fos表达质粒共转染显示DRE对过表达的AP-1蛋白无反应。与野生型DRE寡核苷酸的共转染竞争性抑制DRE介导的转录，而与突变DRE寡核苷酸的共转染的效果较小。我们的研究表明，DRE对hCgA的转录增强依赖于独特的NE特异性DRE结合因子，我们将其称为DBF，该因子特异性和方向性地结合DRE组装并协同功能性转录复合物。

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