Tuthill Matthew C, Wada Randal K, Arimoto Jason M, Sugino Christine N, Kanemaru Kelli K, Takeuchi Kenneth K, Sidell Neil
Molecular Carcinogenesis Section, Cancer Etiology Program, Cancer Research Center of Hawaii, The University of Hawaii at Manoa, 1236 Lauhala Street, Honolulu, HI 96813-2424, USA.
Mol Genet Metab. 2003 Sep-Oct;80(1-2):272-80. doi: 10.1016/s1096-7192(03)00133-1.
Regulation of N-myc oncogene expression is an important determinant of the biological behavior of neuroblastoma. The N-myc promoter contains several potential binding sites for transcription factors of the Sp1 family. Mutation of a CT-box motif contained within a 26 bp region required for N-myc downregulation by retinoic acid decreased basal transcriptional activity and altered DNA-protein interactions of the promoter, while mutations flanking this motif did neither. On super-shift, this region was shown to recruit Sp1 and Sp3 transcription factor proteins, while a functionally significant CT-box mutation resulted in their replacement by NF-1 transcription factor. Lysates from Drosophila S2 cells expressing exogenous Sp1, Sp3, and NF-1 proteins were able to partially mimic gel shift complexes seen with neuroblastoma nuclear extract and either wild type or mutant probes. Transient transfections of S2 cells showed that both individually and together, Sp1 and Sp3 were able to trans-activate a wild type CT-box-driven luciferase reporter construct in a dose-dependent manner. Transfection of the wild type but not mutant CT-box oligonucleotide was able to decrease endogenous N-myc expression in neuroblastoma cells. Together these results suggest that the CT-box element serves a critically functional role, and in the basal state, allows for N-myc trans-activation by Sp1 and Sp3. Moreover when mutated, the CT-box may still function as a binding motif for alternate transcription factors such as NF-1 that can allow persistent N-myc expression.
N - myc癌基因表达的调控是神经母细胞瘤生物学行为的重要决定因素。N - myc启动子包含几个Sp1家族转录因子的潜在结合位点。维甲酸下调N - myc所需的26 bp区域内的CT - box基序发生突变，会降低基础转录活性并改变启动子的DNA - 蛋白质相互作用，而该基序侧翼的突变则不会。在超迁移实验中，该区域显示可募集Sp1和Sp3转录因子蛋白，而功能上重要的CT - box突变导致它们被NF - 1转录因子取代。表达外源性Sp1、Sp3和NF - 1蛋白的果蝇S2细胞裂解物能够部分模拟神经母细胞瘤核提取物以及野生型或突变型探针所观察到的凝胶迁移复合物。S2细胞的瞬时转染表明，Sp1和Sp3单独或共同作用时，都能够以剂量依赖的方式反式激活野生型CT - box驱动的荧光素酶报告基因构建体。野生型而非突变型CT - box寡核苷酸的转染能够降低神经母细胞瘤细胞中内源性N - myc的表达。这些结果共同表明，CT - box元件起着关键的功能作用，在基础状态下，允许Sp1和Sp3对N - myc进行反式激活。此外，当发生突变时，CT - box仍可能作为诸如NF - 1等替代转录因子的结合基序，从而允许N - myc持续表达。
