Enterocyte differentiation marker intestinal alkaline phosphatase is a target gene of the gut-enriched Kruppel-like factor
Enterocyte differentiation marker intestinal alkaline phosphatase is a target gene of the gut-enriched Kruppel-like factor
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DOI：
10.1152/ajpgi.00203.2003
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发表时间：
2004-01-01
影响因子：
4.5
通讯作者：
Hodin, RA
中科院分区：
文献类型：
--
作者：
Hinnebusch, BF;Siddique, A;Hodin, RA
我们研究了转录因子肠道富集型Kruppel样因子(KLF4或GKLF)在激活肠细胞分化标志基因肠碱性磷酸酶(IAP)中的作用。酵母单杂交筛选用于鉴定与位于人IAP基因启动子内的顺式元件(IF-III)相互作用的蛋白质。用EMSA测定DNA与蛋白质的相互作用。用Northern印迹分析研究了人结肠癌RKO细胞中过表达KLF4的RNA表达。瞬时转染IAP-荧光素酶报告载体用于研究KLF4激活IAP转录的机制。酵母单杂交筛选和EMSA鉴定KLF4与IF-III结合。被诱导过表达KLF4的RKO细胞表现出相应的IAP表达的剂量依赖性增加，EMSA证实KLF4与IF-III元件结合。瞬时转染法显示，KLF4主要通过IAP启动子中包含IF-III顺式元件的关键片段反式激活IAP基因。突变的KLF4构建体未能完全激活IAP。我们已经确定肠细胞分化标记IAP是KLF4的靶基因。KLF4对IAP的反式激活可能是通过位于IAP近端启动子区域的一个关键区来实现的。
We have examined the role that the transcription factor gut-enriched Kruppel-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.