Department of Brain and Cognitive Sciences, Seoul National University School of Biological Sciences, Seoul, Korea.
Department of Legal Medicine, Korea University College of Medicine, Seoul, Korea.
Endocrinol Metab (Seoul). 2015 Sep;30(3):381-8. doi: 10.3803/EnM.2015.30.3.381. Epub 2015 May 18.
Hes6 is a transcriptional regulator that induces transcriptional activation by binding to transcription repressor Hes1 and suppressing its activity. Hes6 is controlled by the ubiquitin-proteosome-mediated degradation system. Here we investigated the sumoylation of Hes6 and its functional role in its rhythmic expression.
Hes6, SUMO, and ubiquitin were transfected into HeLa cells and the expression pattern was observed by Western blot and immunoprecipitation. To confirm the effect of sumoylation on the rhythmic expression of Hes6, we generated mouse Hes6 promoter-driven GFP-Hes6 fusion constructs and expressed these constructs in NIH 3T3 cells.
Overexpression of SUMO led to sumoylation of Hes6 at both lysine 27 and 30. Protein stability of Hes6 was decreased by sumoylation. Moreover, expression of a Hes6 sumoylation-defective mutant, the 2KR (K27/30R) mutant, or co-expression of SUMO protease SUSP1 with native Hes6, strongly reduced ubiquitination. In addition, sumoylation was associated with both the rhythmic expression and transcriptional regulation of Hes6. Wild type Hes6 showed oscillatory expression with about 2-hour periodicity, whereas the 2KR mutant displayed a longer period. Furthermore, sumoylation of Hes6 derepressed Hes1-induced transcriptional repression.
Hes6 sumoylation plays an important role in the regulation of its stability and Hes1-mediated transcription. These results suggest that sumoylation may be crucial for rhythmic expression of Hes6 and downstream target genes.
Hes6 是一种转录调节因子，通过与转录抑制因子 Hes1 结合并抑制其活性来诱导转录激活。Hes6 受泛素-蛋白酶体介导的降解系统调控。本研究探讨了 Hes6 的 SUMO 化及其在其节律性表达中的功能作用。
将 Hes6、SUMO 和泛素转染入 HeLa 细胞，通过 Western blot 和免疫沉淀观察其表达模式。为了证实 SUMO 化对 Hes6 节律性表达的影响，我们构建了由小鼠 Hes6 启动子驱动的 GFP-Hes6 融合构建体，并在 NIH 3T3 细胞中表达这些构建体。
SUMO 的过表达导致 Hes6 在赖氨酸 27 和 30 处发生 SUMO 化。Hes6 的蛋白稳定性因 SUMO 化而降低。此外，表达 Hes6 SUMO 突变体（2KR，K27/30R）或与天然 Hes6 共表达 SUMO 蛋白酶 SUSP1，强烈降低了泛素化。此外，SUMO 化与 Hes6 的节律性表达和转录调控有关。野生型 Hes6 表现出约 2 小时周期性的振荡表达，而 2KR 突变体则显示出更长的周期。此外，Hes6 的 SUMO 化解除了 Hes1 诱导的转录抑制。
Hes6 的 SUMO 化在其稳定性和 Hes1 介导的转录调控中发挥重要作用。这些结果表明，SUMO 化可能对 Hes6 和下游靶基因的节律性表达至关重要。
