Departamento de Biología Celular y Desarrollo, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México, Distrito Federal 04510, México.
J Biol Chem. 2012 Aug 3;287(32):26764-76. doi: 10.1074/jbc.M112.386599. Epub 2012 Jun 6.
The human SKI-like (SKIL) gene encodes the SMAD transcriptional corepressor SNON that antagonizes TGF-β signaling. SNON protein levels are tightly regulated by the TGF-β pathway: whereas a short stimulation with TGF-β decreases SNON levels by its degradation via the proteasome, longer TGF-β treatment increases SNON levels by inducing SKIL gene expression. Here, we investigated the molecular mechanisms involved in the self-regulation of SKIL gene expression by SNON. Bioinformatics analysis showed that the human SKIL gene proximal promoter contains a TGF-β response element (TRE) bearing four groups of SMAD-binding elements that are also conserved in mouse. Two regions of 408 and 648 bp of the human SKIL gene (∼2.4 kb upstream of the ATG initiation codon) containing the core promoter, transcription start site, and the TRE were cloned for functional analysis. Binding of SMAD and SNON proteins to the TRE region of the SKIL gene promoter after TGF-β treatment was demonstrated by ChIP and sequential ChIP assays. Interestingly, the SNON-SMAD4 complex negatively regulated basal SKIL gene expression through binding the promoter and recruiting histone deacetylases. In response to TGF-β signal, SNON is removed from the SKIL gene promoter, and then the activated SMAD complexes bind the promoter to induce SKIL gene expression. Subsequently, the up-regulated SNON protein in complex with SMAD4 represses its own expression as part of the negative feedback loop regulating the TGF-β pathway. Accordingly, when the SNON-SMAD4 complex is absent as in some cancer cells lacking SMAD4 the regulation of some TGF-β target genes is modified.
人类 SKI 样（SKIL）基因编码 SMAD 转录共抑制因子 SNON，拮抗 TGF-β信号。SNON 蛋白水平受 TGF-β 途径的严格调控：尽管 TGF-β 的短暂刺激通过蛋白酶体降解降低了 SNON 水平，但较长时间的 TGF-β 处理通过诱导 SKIL 基因表达增加了 SNON 水平。在这里，我们研究了 SNON 自我调节 SKIL 基因表达的分子机制。生物信息学分析表明，人 SKIL 基因近端启动子包含一个 TGF-β 反应元件（TRE），其中包含四个 SMAD 结合元件组，这些元件在小鼠中也保守。人 SKIL 基因的两个区域（408 和 648 bp，位于 ATG 起始密码子上游约 2.4 kb）包含核心启动子、转录起始位点和 TRE，用于功能分析。ChIP 和连续 ChIP 实验证明，TGF-β 处理后，SMAD 和 SNON 蛋白结合到 SKIL 基因启动子的 TRE 区域。有趣的是，SNON-SMAD4 复合物通过结合启动子并招募组蛋白去乙酰化酶，负调控基础 SKIL 基因表达。对 TGF-β 信号的反应，SNON 从 SKIL 基因启动子中被移除，然后激活的 SMAD 复合物结合启动子诱导 SKIL 基因表达。随后，作为调节 TGF-β 途径的负反馈循环的一部分，上调的与 SMAD4 结合的 SNON 蛋白抑制其自身表达。因此，当像某些缺乏 SMAD4 的癌细胞中不存在 SNON-SMAD4 复合物时，一些 TGF-β 靶基因的调控会发生改变。
