Hinnebusch Brian F, Siddique Aleem, Henderson J Welles, Malo Madhu S, Zhang Wenying, Athaide Christopher P, Abedrapo Mario A, Chen Xinming, Yang Vincent W, Hodin Richard A
Deptartment of Surgery, Massachusetts General Hospital/Harvard Medical School, Boston, MA 02114, USA.
Am J Physiol Gastrointest Liver Physiol. 2004 Jan;286(1):G23-30. doi: 10.1152/ajpgi.00203.2003. Epub 2003 Aug 14.
We have examined the role that the transcription factor gut-enriched Krüppel-like factor (KLF4 or GKLF) plays in activating the enterocyte differentiation marker gene intestinal alkaline phosphatase (IAP). A yeast one-hybrid screen was used to identify proteins interacting with a previously identified cis-element (IF-III) located within the human IAP gene promoter. DNA-protein interactions were determined by using EMSA. Northern blot analysis was used to study RNA expression in human colon cancer RKO cells engineered to overexpress KLF4. Transient transfections with IAP-luciferase reporter constructs were used to characterize the mechanisms by which KLF4 activates IAP transcription. The yeast one-hybrid screen and EMSA identified KLF4 as binding to IF-III. RKO cells induced to overexpress KLF4 demonstrated a corresponding dose-dependent increase in IAP expression, and EMSA with nuclear extract from these cells confirmed that KLF4 binds to the IF-III element. Transient transfections revealed that KLF4 transactivated the IAP gene largely via a critical segment in the IAP promoter that includes the IF-III cis-element. Mutant KLF4 constructs failed to fully activate IAP. We have identified the enterocyte differentiation marker IAP as a KLF4 target gene. IAP transactivation by KLF4 is likely mediated through a critical region located within the proximal IAP promoter region.
我们研究了转录因子肠道富集型Krüppel样因子（KLF4或GKLF）在激活肠上皮细胞分化标志物基因肠碱性磷酸酶（IAP）中所起的作用。利用酵母单杂交筛选来鉴定与先前确定的位于人IAP基因启动子内的顺式元件（IF-III）相互作用的蛋白质。通过电泳迁移率变动分析（EMSA）确定DNA-蛋白质相互作用。利用Northern印迹分析研究在经基因工程改造以过表达KLF4的人结肠癌RKO细胞中的RNA表达。用IAP-荧光素酶报告构建体进行瞬时转染，以表征KLF4激活IAP转录的机制。酵母单杂交筛选和EMSA鉴定出KLF4与IF-III结合。诱导过表达KLF4的RKO细胞显示IAP表达相应地呈剂量依赖性增加，并且对这些细胞的核提取物进行EMSA证实KLF4与IF-III元件结合。瞬时转染显示KLF4主要通过IAP启动子中包含IF-III顺式元件的关键区段反式激活IAP基因。突变型KLF4构建体未能完全激活IAP。我们已将肠上皮细胞分化标志物IAP鉴定为KLF4靶基因。KLF4对IAP的反式激活可能是通过位于IAP启动子近端区域内的关键区域介导的。
