Liu Chao, Zhang Dijuan, Shen Yuxian, Tao Xiaofang, Liu Lihua, Zhong Yongwang, Fang Shengyun
Department of Histology and Embryology, Institute of Stem Cell and Tissue Engineering, School of Basic Medical Sciences, Anhui Medical University, Hefei, Anhui 230032 China; Center for Biomedical Engineering and Technology (BioMET), University of Maryland, Baltimore, MD 21201 USA.
Department of Histology and Embryology, Institute of Stem Cell and Tissue Engineering, School of Basic Medical Sciences, Anhui Medical University, Hefei, Anhui 230032 China.
Biochim Biophys Acta. 2015 Dec;1853(12):3279-93. doi: 10.1016/j.bbamcr.2015.09.029. Epub 2015 Sep 28.
The amount of transcription factor OCT4 is strictly regulated. A tight regulation of OCT4 levels is crucial for mammalian embryonic development and oncogenesis. However, the mechanisms underlying regulation of OCT4 protein expression and nuclear distribution are largely unknown. Here, we report that DPF2, a plant homeodomain (PHD) finger protein, is upregulated during H9 cell differentiation induced by retinoic acid. Endogenous interaction between DPF2 and OCT4 in P19 cells was revealed by an immunoprecipitation assay. GST-pull down assay proved that OCT4 protein in H9 cells and recombinant OCT4 can precipitate with DPF2 in vitro. In vitro ubiquitination assay demonstrated DPF2 might serve as an E3 ligase. Knock down of dpf2 using siRNA increased OCT4 protein level and stability in P19 cells. DPF2 siRNAs also up-regulates OCT4 but not NANOG in H9 cells. However, RA fails to downregulates OCT4 protein level in cells infected by lenitviruses containing DPF2 siRNA. Moreover, overexpression of both DPF2 and OCT4 in 293 cells proved the DPF2-OCT4 interaction. DPF2 but not PHD2 mutant DPF2 enhanced ubiquitination and degradation of OCT4 in 293 cells co-expressed DPF2 and OCT4. Both wild type DPF2 and PHD2 mutant DPF2 redistributes nuclear OCT4 without affecting DPF2-OCT4 interaction. Further analysis indicated that DPF2 decreases monomeric and mono-ubiquitinated OCT4, assembles poly-ubiquitin chains on OCT4 mainly through Ub-K48 linkage. These findings contribute to an understanding of how OCT4 protein level and nuclear distribution is regulated by its associated protein.
转录因子OCT4的量受到严格调控。OCT4水平的严格调控对哺乳动物胚胎发育和肿瘤发生至关重要。然而，OCT4蛋白表达和核分布的调控机制在很大程度上尚不清楚。在此，我们报告DPF2，一种植物同源结构域（PHD）指蛋白，在视黄酸诱导的H9细胞分化过程中上调。免疫沉淀试验揭示了P19细胞中DPF2与OCT4之间的内源性相互作用。GST下拉试验证明H9细胞中的OCT4蛋白和重组OCT4在体外可与DPF2沉淀。体外泛素化试验表明DPF2可能作为一种E3连接酶。使用siRNA敲低dpf2可增加P19细胞中OCT4蛋白水平和稳定性。DPF2 siRNAs也上调H9细胞中的OCT4但不上调NANOG。然而，视黄酸未能下调含有DPF2 siRNA的慢病毒感染细胞中的OCT4蛋白水平。此外，在293细胞中过表达DPF2和OCT4证明了DPF2 - OCT4相互作用。在共表达DPF2和OCT4的293细胞中，DPF2而非PHD2突变体DPF2增强了OCT4的泛素化和降解。野生型DPF2和PHD2突变体DPF2均使核OCT4重新分布，而不影响DPF2 - OCT4相互作用。进一步分析表明，DPF2减少单体和单泛素化的OCT4，主要通过Ub - K48连接在OCT4上组装多聚泛素链。这些发现有助于理解OCT4蛋白水平和核分布是如何由其相关蛋白调控的。
