Ai Walden, Zheng Hai, Yang Xiangdong, Liu Ying, Wang Timothy C
Division of Digestive and Liver Diseases, Department of Medicine, Columbia University Medical Center, New York, NY 10032, USA.
Nucleic Acids Res. 2007;35(18):6137-49. doi: 10.1093/nar/gkm656. Epub 2007 Sep 7.
KLF4 is a transcription factor that is highly expressed in the gastrointestinal tract. Previously we have demonstrated that KLF4 represses HDC promoter activity in a gastric cell line through both an upstream Sp1 binding GC box and downstream gastrin responsive elements. However, the mechanism by which KLF4 inhibits HDC promoter is not well defined. In the current study, by using yeast two-hybrid screening, Tip60 was identified as a KLF4 interacting protein. Further coimmunoprecipitation and functional reporter assays support the interaction between these two proteins. In addition, Tip60 and HDAC7, previously shown to interact with each other and repress transcription, inhibited HDC promoter activity in a dose-dependent fashion. Consistently, knock down of Tip60 or HDAC7 gene expression by specific shRNA increased endogenous HDC mRNA level. Co-immunoprecipitation assays showed that HDAC7 was pulled down by KLF4 and Tip60, suggesting that these three proteins form a repressive complex. Further chromatin immuno-precipitation indicated that all three proteins associated with HDC promoter. Two-hour gastrin treatment, known to activate HDC gene expression, significantly decreased the association of KLF4, Tip60 and HDAC7 with HDC promoter, suggesting that gastrin activates HDC gene expression at least partly by decreasing the formation of KLF4/Tip60/HDAC7 repressive complexes at the HDC promoter.
KLF4是一种在胃肠道中高度表达的转录因子。此前我们已经证明，KLF4通过上游的Sp1结合GC盒和下游的胃泌素反应元件抑制胃细胞系中HDC启动子的活性。然而，KLF4抑制HDC启动子的机制尚不清楚。在当前的研究中，通过酵母双杂交筛选，Tip60被鉴定为一种与KLF4相互作用的蛋白。进一步的免疫共沉淀和功能报告基因检测支持了这两种蛋白之间的相互作用。此外，Tip60和HDAC7（此前已证明它们相互作用并抑制转录）以剂量依赖的方式抑制HDC启动子的活性。同样，通过特异性shRNA敲低Tip60或HDAC7基因表达可提高内源性HDC mRNA水平。免疫共沉淀检测表明，HDAC7被KLF4和Tip60拉下，提示这三种蛋白形成一个抑制复合物。进一步的染色质免疫沉淀表明，这三种蛋白均与HDC启动子相关。已知能激活HDC基因表达的胃泌素处理两小时后，显著降低了KLF4、Tip60和HDAC7与HDC启动子的结合，提示胃泌素至少部分通过减少HDC启动子处KLF4/Tip60/HDAC7抑制复合物的形成来激活HDC基因表达。
