Nucleo-cytoplasmic shuttling of Drosophila Hairless/Su(H) heterodimer as a means of regulating Notch dependent transcription.
Nucleo-cytoplasmic shuttling of Drosophila Hairless/Su(H) heterodimer as a means of regulating Notch dependent transcription.
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DOI：
10.1016/j.bbamcr.2019.07.008
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发表时间：
2019-10
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通讯作者：
Dorina B. Wolf;Thomas K. Smylla;Jan Reichmuth;Philipp Hoffmeister;L. Kober;M. Zimmermann;A. Turkiewicz;T. Borggrefe;A. Nagel;F. Oswald;A. Preiss;D. Maier
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作者：
Dorina B. Wolf;Thomas K. Smylla;Jan Reichmuth;Philipp Hoffmeister;L. Kober;M. Zimmermann;A. Turkiewicz;T. Borggrefe;A. Nagel;F. Oswald;A. Preiss;D. Maier
Notch靶基因的激活和抑制是由转录因子CSL介导的，在果蝇中被称为无毛抑制因子（Suppressor of Hairless，Su（H）），在人类中被称为CBF 1或RBPJ。CSL与共激活因子Notch或共抑制因子如DrosophilaHairless相关。在哺乳动物和果蝇中，转录因子CSL的核转位依赖于辅因子的结合。果蝇CSL同源基因Su（H）需要Hairless来形成阻遏物复合物。基于其在转录沉默中的作用，H蛋白被认为是严格的核蛋白。但H蛋白也是胞浆性的，这可能与其在Su（H）蛋白的稳定和核转位中的作用有关。本研究利用GFP融合蛋白、报告基因分析和Hairless野生型和穿梭缺陷型突变体的体内分析，研究了预测的Hairless辅阻遏物核定位信号（NLS 1-3）和单个核输出信号（内斯）的功能。我们确定NLS 3和内斯对无毛功能至关重要。事实上，H NLS 3突变体果蝇与H无效突变体相匹配，而H NLS 3 NES双突变体显示较弱的表型，这与内斯在H输出中的关键作用一致。正如对转录抑制子的预期，Notch靶基因在HNNLS 3突变细胞中被解除调控，证明其活性需要细胞核。重要的是，我们揭示了Su（H）蛋白严格遵循Hairless蛋白定位。总之，我们提出，在核质区室之间穿梭提供了通过平衡用于Notch激活的Su（H）蛋白可用性来微调Notch靶基因表达的调节的可能性。
Activation and repression of Notch target genes is mediated by transcription factor CSL, known as Suppressor of Hairless (Su(H)) inDrosophilaand CBF1 or RBPJ in human. CSL associates either with co-activator Notch or with co-repressors such asDrosophilaHairless. The nuclear translocation of transcription factor CSL relies on co-factor association, both in mammals and inDrosophila. TheDrosophilaCSL orthologue Su(H) requires Hairless for repressor complex formation. Based on its role in transcriptional silencing, H protein would be expected to be strictly nuclear. However, H protein is also cytosolic, which may relate to its role in the stabilization and nuclear translocation of Su(H) protein. Here, we investigate the function of the predicted nuclear localization signals (NLS 1–3) and single nuclear export signal (NES) of co-repressor Hairless using GFP-fusion proteins, reporter assays andin vivoanalyses using Hairless wild type and shuttling-defective Hairless mutants. We identify NLS3 and NES to be critical for Hairless function. In fact,H⁎NLS3mutant flies match H null mutants, whereasH⁎NLS3⁎NESdouble mutants display weaker phenotypes in agreement with a crucial role for NES in H export. As expected for a transcriptional repressor, Notch target genes are deregulated inH⁎NLS3mutant cells, demonstrating nuclear requirement for its activity. Importantly, we reveal that Su(H) protein strictly follows Hairless protein localization. Together, we propose that shuttling between the nucleo-cytoplasmic compartments provides the possibility to fine tune the regulation of Notch target gene expression by balancing of Su(H) protein availability for Notch activation.