Pirot Pierre, van Grunsven Leo A, Marine Jean-Christophe, Huylebroeck Danny, Bellefroid Eric J
Laboratoire d'Embryologie Moléculaire, Université Libre de Bruxelles, Institut de Biologie et de Médecine Moléculaires, rue des Profs. Jeener et Brachet 12, B-6041 Gosselies, Belgium.
Biochem Biophys Res Commun. 2004 Sep 17;322(2):526-34. doi: 10.1016/j.bbrc.2004.07.157.
Nrarp encodes for an evolutionarily conserved small ankyrin repeat-containing protein that functions as a negative regulator of Notch signaling. Interestingly, increased Nrarp transcription was observed following induction of Notch signaling, suggesting the existence of a negative feedback loop. We show here that both mouse and human promoter regions of Nrarp share two conserved regions located approximately 2 and approximately 3 kb upstream of the transcription start site each containing a perfect putative binding site for the Notch-dependent transcription factor Su(H). A 4.4 kb genomic fragment of the mouse Nrarp locus containing those conserved regions and fused to a luciferase reporter gene showed basal promoter activity in 293T cells and this activity was strongly increased by the intracellular domain of Notch, NICD. NICD-dependent stimulation was attenuated by a dominant negative mutant of Su(H), Su(H)DBM, and was not observed in Su(H)-deficient cells (OT-11). Promoter bashing and gel shift assays revealed that the most distal putative Su(H) binding site located within the -3 kb conserved element plays a crucial role in this induction. Collectively, these results provide definitive support for direct regulation of the Nrarp gene by the Notch pathway.
Nrarp编码一种进化上保守的含小锚蛋白重复序列的蛋白质，该蛋白质作为Notch信号通路的负调节因子发挥作用。有趣的是，在Notch信号通路激活后观察到Nrarp转录增加，这表明存在负反馈回路。我们在此表明，Nrarp的小鼠和人类启动子区域在转录起始位点上游约2 kb和约3 kb处共有两个保守区域，每个区域都含有一个与Notch依赖性转录因子Su(H)完美匹配的假定结合位点。包含这些保守区域并与荧光素酶报告基因融合的小鼠Nrarp基因座的4.4 kb基因组片段在293T细胞中显示出基础启动子活性，并且这种活性被Notch的细胞内结构域NICD强烈增强。Su(H)的显性负突变体Su(H)DBM减弱了NICD依赖性刺激，并且在缺乏Su(H)的细胞（OT-11）中未观察到这种刺激。启动子敲除和凝胶迁移试验表明，位于-3 kb保守元件内最远端的假定Su(H)结合位点在这种诱导中起关键作用。总体而言，这些结果为Notch信号通路对Nrarp基因的直接调控提供了确凿的支持。
