Fitzgerald K, Bazar L, Avigan M I
Department of Pathology, Georgetown University School of Medicine, Washington, District of Columbia 20007, USA.
Am J Physiol. 1998 Feb;274(2):G314-24. doi: 10.1152/ajpgi.1998.274.2.G314.
Lactase-phlorizin hydrolase (LPH) synthesis is restricted to differentiated small intestinal enterocytes and is highly regulated during development. Analysis of expression of LPH promoter segments fused with luciferase transfected in Caco-2 cells, a line that uniquely expresses LPH mRNA, mapped an 18-base pair (bp) segment 100 bp upstream of the transcription start site that is required for transactivation. Remarkably, the LPH upstream element (LUE) has no stimulatory activity in both human intestinal and nonintestinal lines in which LPH mRNA is absent. Electrophoretic analysis of sequence-specific DNA-nuclear protein complexes demonstrated the presence of a Caco-2 cell-specific protein(s) (CCP), which is uniformly absent in LPH nonproducer cell lines. Mutational analysis of the LUE demonstrated that bases contained within a GATA consensus motif are critical for both CCP binding and transcription from the LPH promoter. Caco-2 cells express high levels of GATA-6 mRNA in a cell line-specific manner, suggesting that GATA-6 is a CCP that complexes with the LUE. When expressed by a plasmid, GATA-6 transactivated the LPH promoter. The stimulation was abrogated with mutations in the GATA consensus motif as well as mutations in a flanking downstream element. These studies are consistent with an important role of an intestinal GATA binding protein in cell type-specific transactivation of the LPH promoter.
乳糖酶 - 根皮苷水解酶（LPH）的合成仅限于分化的小肠肠上皮细胞，并且在发育过程中受到高度调控。对与荧光素酶融合的LPH启动子片段在Caco - 2细胞（一种独特表达LPH mRNA的细胞系）中进行转染后的表达分析，确定了转录起始位点上游100 bp处一个18个碱基对（bp）的片段，该片段是反式激活所必需的。值得注意的是，LPH上游元件（LUE）在缺乏LPH mRNA的人肠道和非肠道细胞系中均无刺激活性。对序列特异性DNA - 核蛋白复合物的电泳分析表明存在一种Caco - 2细胞特异性蛋白（CCP），而在不产生LPH的细胞系中该蛋白均不存在。对LUE的突变分析表明，GATA共有基序内的碱基对于CCP结合和LPH启动子的转录均至关重要。Caco - 2细胞以细胞系特异性方式高水平表达GATA - 6 mRNA，这表明GATA - 6是一种与LUE结合的CCP。当通过质粒表达时，GATA - 6可反式激活LPH启动子。GATA共有基序以及侧翼下游元件中的突变均消除了这种刺激作用。这些研究表明肠道GATA结合蛋白在LPH启动子的细胞类型特异性反式激活中起重要作用。
