Chen Fangfang, Zhang Chunxiao, Wu Haonan, Ma Yue, Luo Xiaomin, Gong Xinqi, Jiang Fan, Gui Yaoting, Zhang Hui, Lu Fei
Laboratory of Chemical Genomics, Peking University Shenzhen Graduate School, Shenzhen, Guangdong 518055, China; Guangdong and Shenzhen Key Laboratory of Male Reproductive Medicine and Genetics, Peking University Shenzhen Hospital, Shenzhen PKU-HKUST Medical Center, Shenzhen 518036, China.
Laboratory of Chemical Genomics, Peking University Shenzhen Graduate School, Shenzhen, Guangdong 518055, China; Department of Chemistry and Biochemistry, University of Nevada, Las Vegas, Nevada 89154.
J Biol Chem. 2017 Dec 8;292(49):20100-20112. doi: 10.1074/jbc.M117.815001. Epub 2017 Oct 25.
Molecular oscillators are important cellular regulators of, for example, circadian clocks, oscillations of immune regulators, and short-period (ultradian) rhythms during embryonic development. The Notch signaling factor HES1 (hairy and enhancer of split 1) is a well-known repressor of proneural genes, and HES1 ultradian oscillation is essential for keeping cells in an efficiently proliferating progenitor state. HES1 oscillation is driven by both transcriptional self-repression and ubiquitin-dependent proteolysis. However, the E3 ubiquitin ligase targeting HES1 for proteolysis remains unclear. Based on siRNA-mediated gene silencing screening, co-immunoprecipitation, and ubiquitination assays, we discovered that the E3 ubiquitin ligase SCF complex regulates HES1 ubiquitination and proteolysis. siRNA-mediated knockdown of the Cullin-RING E3 ubiquitin ligases RBX1 or CUL1 increased HES1 protein levels, prolonged its half-life, and dampened its oscillation. FBXL14, an F-box protein for SCF ubiquitin ligase, associates with HES1. FBXL14 silencing stabilized HES1, whereas FBXL14 overexpression decreased HES1 protein levels. Of note, the SCF complex promoted the ubiquitination of HES1 , and a conserved WRPW motif in HES1 was essential for HES1 binding to FBXL14 and for ubiquitin-dependent HES1 degradation. HES1 knockdown promoted neuronal differentiation, but FBXL14 silencing inhibited neuronal differentiation induced by HES1 ablation in mES and F9 cells. Our results suggest that SCF promotes neuronal differentiation by targeting HES1 for ubiquitin-dependent proteolysis and that the C-terminal WRPW motif in HES1 is required for this process.
分子振荡器是重要的细胞调节因子，例如在生物钟、免疫调节因子的振荡以及胚胎发育过程中的短周期（超日节律）节律中发挥作用。Notch信号因子HES1（毛状和分裂增强子1）是一种众所周知的神经前体基因的抑制因子，HES1的超日节律振荡对于维持细胞处于高效增殖的祖细胞状态至关重要。HES1振荡由转录自我抑制和泛素依赖性蛋白水解共同驱动。然而，靶向HES1进行蛋白水解的E3泛素连接酶仍不清楚。基于小干扰RNA（siRNA）介导的基因沉默筛选、免疫共沉淀和泛素化分析，我们发现E3泛素连接酶SCF复合物调节HES1的泛素化和蛋白水解。siRNA介导的Cullin-RING E3泛素连接酶RBX1或CUL1的敲低增加了HES1蛋白水平，延长了其半衰期，并减弱了其振荡。FBXL14是SCF泛素连接酶的一种F-box蛋白，与HES1相关联。FBXL14的沉默使HES1稳定，而FBXL14的过表达降低了HES1蛋白水平。值得注意的是，SCF复合物促进了HES1的泛素化，并且HES1中一个保守的WRPW基序对于HES1与FBXL14的结合以及泛素依赖性HES1降解至关重要。HES1的敲低促进了神经元分化，但FBXL14的沉默抑制了mES和F9细胞中HES1缺失诱导的神经元分化。我们的结果表明，SCF通过靶向HES1进行泛素依赖性蛋白水解来促进神经元分化，并且HES1中的C末端WRPW基序是这一过程所必需的。
