Spodsberg N, Troelsen J T, Carlsson P, Enerbäck S, Sjöström H, Norén O
Department of Medical Biochemistry and Genetics, Biochemical Laboratory C, The Panum Institute, University of Copenhagen, Copenhagen, Denmark.
Gastroenterology. 1999 Apr;116(4):842-54. doi: 10.1016/s0016-5085(99)70067-3.
BACKGROUND & AIMS: One-kilobase sequence of the upstream fragment of the pig lactase-phlorizin hydrolase gene has been shown to control small intestinal-specific expression and postweaning decline of lactase-phlorizin hydrolase in transgenic mice. The aim of this study was to identify the regulatory DNA elements and transcription factors controlling lactase-phlorizin hydrolase expression.
The activity of different lactase-phlorizin hydrolase promoter fragments was investigated by transfection experiments using Caco-2 cells. Electrophoretic mobility shift assays and supershift analyses were used to characterize the interaction between intestinal transcription factors and the identified regulatory elements.
Functional analysis revealed three previously undescribed regulatory regions in the lactase-phlorizin hydrolase promoter: a putative enhancer between -894 and -798 binding hepatocyte nuclear factor (HNF)-1 at position -894 to -880; a repressor-binding element between -278 to -264 to which an HNF-3-like factor is able to bind; and an element between -178 to -164 that binds an activating transcription factor.
Identification of three new regulatory regions and HNF-1 and HNF-3-like transcription factor as players in the regulation of lactase-phlorizin hydrolase gene transcription has an impact on the understanding of the molecular mechanisms behind age-dependent, tissue-specific, differentiation-dependent, and regional regulation of expression in the intestine.
猪乳糖酶-根皮苷水解酶基因上游片段的1千碱基序列已被证明可控制转基因小鼠小肠特异性表达及断奶后乳糖酶-根皮苷水解酶的下降。本研究的目的是鉴定控制乳糖酶-根皮苷水解酶表达的调控DNA元件和转录因子。
通过使用Caco-2细胞的转染实验研究不同乳糖酶-根皮苷水解酶启动子片段的活性。电泳迁移率变动分析和超迁移分析用于表征肠道转录因子与已鉴定调控元件之间的相互作用。
功能分析揭示了乳糖酶-根皮苷水解酶启动子中三个先前未描述的调控区域：一个推定的增强子，位于-894至-798之间，在-894至-880位点结合肝细胞核因子（HNF）-1；一个阻遏物结合元件，位于-278至-264之间，一种HNF-3样因子能够与之结合；以及一个位于-178至-164之间的元件，可结合一种激活转录因子。
鉴定出三个新的调控区域以及HNF-1和HNF-3样转录因子作为乳糖酶-根皮苷水解酶基因转录调控的参与者，对理解肠道中年龄依赖性、组织特异性、分化依赖性和区域表达调控背后的分子机制具有重要意义。
