Graduate School of Frontier Biosciences, Osaka University, Suita, Japan.
PLoS One. 2011;6(10):e25694. doi: 10.1371/journal.pone.0025694. Epub 2011 Oct 3.
Sox9 is a transcription factor that is required for tissue development in mammals. In general, such transcription factors require co-regulators for precise temporal and spatial control of the activation and inactivation of the numerous genes necessary for precise development during embryogenesis. Here we identify a new Sox9 co-regulator: Using affinity chromatography with immobilized Sox9 protein, we identified exportin 4 (Exp4) as an interacting protein of Sox9 in human cultured cells. Interaction between endogenous Exp4 and Sox9 proteins was confirmed in the human osteosarcoma U2OS cells by immunoprecipitation experiments using anti-Sox9 antibody. siRNA depletion of Exp4 enhanced transcription of Sox9 target genes in U2OS cells, but did not affect nuclear localization of Sox9. These results suggest that Exp4 regulates Sox9 activity in the nucleus. Furthermore we found that the HMG box of Sox9 was responsible for binding to Exp4, and the HMG box was required for suppression of Sox9-mediated transcription. This contrasts with the known Sox9 co-regulators which bind to its transcriptional activation domain. Chromatin immunoprecipitation analyses revealed that Exp4 prevents Sox9 binding to the enhancers of its target genes. These results demonstrate that Exp4 acts as a Sox9 co-regulator that directly regulates binding of Sox9 to its target genes.
Sox9 是一种转录因子，对于哺乳动物的组织发育是必需的。一般来说，这类转录因子需要共激活因子来精确地调控其激活和失活，以确保胚胎发生过程中众多基因的精确发育。在这里，我们鉴定了一种新的 Sox9 共激活因子：利用固定化 Sox9 蛋白的亲和层析，我们在人培养细胞中鉴定出了输出蛋白 4（Exp4）是 Sox9 的一个相互作用蛋白。免疫沉淀实验使用抗 Sox9 抗体，在人骨肉瘤 U2OS 细胞中证实了内源性 Exp4 和 Sox9 蛋白之间的相互作用。Exp4 的 siRNA 耗竭增强了 U2OS 细胞中 Sox9 靶基因的转录，但不影响 Sox9 的核定位。这些结果表明 Exp4 在核内调节 Sox9 的活性。此外，我们发现 Sox9 的 HMG 盒负责与 Exp4 结合，并且 HMG 盒对于抑制 Sox9 介导的转录是必需的。这与已知的 Sox9 共激活因子形成对比，后者结合其转录激活结构域。染色质免疫沉淀分析显示 Exp4 阻止 Sox9 与靶基因的增强子结合。这些结果表明 Exp4 作为 Sox9 共激活因子，直接调节 Sox9 与其靶基因的结合。
