Zou Min-Xu, Butcher Darci T, Sadikovic Bekim, Groves Timothy C, Yee Siu-Pok, Rodenhiser David I
London Regional Cancer Centre, University of Western Ontario, London, Ontario, Canada.
Oncogene. 2004 Jan 15;23(2):330-9. doi: 10.1038/sj.onc.1207053.
An essential requirement to understand how genes contribute to genetic disease is the thorough knowledge of the transcriptional regulation of gene expression. Here, we have characterized transcription factor binding sites within the type 1 neurofibromatosis (NF1) proximal regulatory region, and addressed the molecular mechanisms that regulate NF1 transcription. Overlapping regions of the NF1 proximal promoter have been cloned and characterized for use in luciferase reporter assays. These assays have identified a 500 bp region displaying activities up to 80-fold higher than control reporter levels. Mutations at putative CRE and SP1-binding sites immediately 5' to the transcription start site have dramatic effects that lead to a 70-90% decrease in reporter activity in all cell lines tested. Gelshift assays confirm binding of CREB and SP1/KLF family members to their putative recognition sequences, and provide the first evidence identifying functional sites likely involved in regulating NF1 transcription. These assays have also revealed a putative repressor region within the NF1 promoter region corresponding to CCCTC-rich sequences between the transcription and translation start sites. This work provides new information concerning the transcriptional regulation of the NF1 gene, and is the most thorough attempt to date to map functionally relevant regions within the NF1 proximal promoter region.
了解基因如何导致遗传疾病的一个基本要求是全面掌握基因表达的转录调控。在此，我们对1型神经纤维瘤病（NF1）近端调控区域内的转录因子结合位点进行了表征，并探讨了调控NF1转录的分子机制。已克隆并表征了NF1近端启动子的重叠区域，用于荧光素酶报告基因检测。这些检测确定了一个500 bp的区域，其活性比对照报告基因水平高80倍。转录起始位点上游紧邻的假定CRE和SP1结合位点处的突变具有显著影响，导致所有测试细胞系中的报告基因活性降低70 - 90%。凝胶迁移实验证实了CREB和SP1/KLF家族成员与其假定识别序列的结合，并提供了首个证据，确定了可能参与调控NF1转录的功能位点。这些检测还揭示了NF1启动子区域内一个假定的抑制区域，对应于转录起始位点和翻译起始位点之间富含CCCTC的序列。这项工作提供了有关NF1基因转录调控的新信息，是迄今为止对NF1近端启动子区域内功能相关区域进行图谱绘制的最全面尝试。
