Mitchelmore C, Troelsen J T, Spodsberg N, Sjöström H, Norén O
Department of Medical Biochemistry and Genetics, Biochemical Laboratory C, The Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
Biochem J. 2000 Mar 1;346 Pt 2(Pt 2):529-35.
Lactase-phlorizin hydrolase is a brush-border enzyme which is specifically expressed in the small intestine where it hydrolyses lactose, the main carbohydrate found in milk. We have previously demonstrated in transgenic mice that the tissue-specific and developmental expression of lactase is controlled by a 1 kb upstream region of the pig lactase gene. Two homeodomain transcription factors, caudal-related homeodomain protein (Cdx2) and hepatic nuclear factor 1alpha (HNF1alpha), are known to bind to regulatory cis elements in the promoters for several intestine-specific genes, including lactase, and are present in mammalian intestinal epithelia from an early stage in development. In the present study, we examined whether Cdx2 and HNF1alpha physically interact and co-operatively activate transcription from the lactase-phlorizin hydrolase promoter. We show that the presence of both factors leads to a much higher level of transcription than the sum of the activation by either factor alone. The N-terminal activation domain of Cdx2 is required for maximal synergy with HNF1alpha. With the use of pull-down assays, we demonstrate a direct protein-protein interaction between Cdx2 and HNF1alpha. The interaction domain includes the homeodomain region of both proteins. This is the first demonstration of a functional interaction between two transcription factors involved in the activation of a number of intestine-specific genes. Synergistic interaction between tissue-restricted factors is likely to be an important mechanism for reinforcing developmental and tissue-specific gene expression within the intestine.
乳糖酶-根皮苷水解酶是一种刷状缘酶，在小肠中特异性表达，在小肠中它可水解乳糖，乳糖是牛奶中主要的碳水化合物。我们之前在转基因小鼠中证明，乳糖酶的组织特异性和发育表达受猪乳糖酶基因上游1 kb区域的控制。已知两种同源结构域转录因子，尾相关同源结构域蛋白（Cdx2）和肝细胞核因子1α（HNF1α），可结合包括乳糖酶在内的几种肠道特异性基因启动子中的调控顺式元件，并且在发育早期就存在于哺乳动物肠道上皮中。在本研究中，我们检测了Cdx2和HNF1α是否发生物理相互作用并协同激活乳糖酶-根皮苷水解酶启动子的转录。我们发现，两种因子同时存在时导致的转录水平比单独一种因子激活时的总和要高得多。Cdx2的N端激活结构域是与HNF1α实现最大协同作用所必需的。通过下拉实验，我们证明了Cdx2和HNF1α之间存在直接的蛋白质-蛋白质相互作用。相互作用结构域包括两种蛋白质的同源结构域区域。这是首次证明参与激活多种肠道特异性基因的两种转录因子之间存在功能相互作用。组织限制性因子之间的协同相互作用可能是加强肠道内发育和组织特异性基因表达的重要机制。
