Spatiotemporal Regulation of Intestinal Gene Expression

肠道基因表达的时空调控

• 批准号: 7122296
• 负责人: ERIC SIBLEY
• 金额: $ 1.29万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7122296
• 关键词: DNA binding protein; age difference; bioluminescence; developmental genetics; enzyme activity; gastrointestinal function; gene deletion mutation; genetic regulatory element; genetic transcription; genetically modified animals; growth /development; intestines; isozymes; laboratory mouse; lactase; protein localization; reporter genes; tissue /cell culture; transcription factor

DESCRIPTION (provided by applicant): The broad objective of this proposal is to define the mechanisms mediating spatiotemporal pattern formation during gut development and maturation. The research plan will focus on the molecular mechanisms regulating the spatiotemporal restriction of intestinal lactase gene transcription. Intestinal lactase is the enterocyte disaccharidase responsible for digestion of lactose, the primary carbohydrate in milk. Lactase activity is maximal prior to weaning and then declines significantly during maturation. Decreased lactase activity combined with excessive milk consumption results in symptoms of carbohydrate malabsorption in most mature mammals, including humans. We have characterized several DNA regulatory elements and transcription factors (GATA-4/5/6, Cdx-2, HNF-1 and Pdx-1) involved in regulating lactase gene transcription in intestinal cells. In addition, we have determined that a 2.0-kilobase fragment of the lactase promoter directs appropriate spatial and temporal expression in the small intestine of transgenic mice. We present a global hypothesis that describes the likelihood of deciphering enteric codes for combinatorial interaction between DNA and protein modules mediating spatiotemporal gut gene expression. We will focus on a potential role for Pdx-1 in mediating anterior spatial restriction. Research objectives, therefore, are aimed at characterization of lactase cis regulatory elements and transcription factors. In Aim 1, we will characterize lactase DNA regulatory elements by analyzing expression of genomic deletions linked to a reporter gene in intestinal cell culture and in transgenic mice. This analysis will be facilitated by methods to allow for in vivo bioluminescent detection of reporter gene expression. In Aim 2, we will characterize the spatiotemporal expression and DNA binding patterns for known lactase transcription factors during gut development and maturation. In Aim 3, we will functionally investigate the role of the homeodomain protein Px-1 in mediating spatial restriction of the lactase gene by altering the Pdx-1-lactase promoter interaction in cell culture and in transgenic mice and assaying for transcriptional activity. The combination of ex vivo and in vivo approaches are designed to characterize the mechanisms involved in regulating the spatiotemporal restriction of lactase gene transcription.

描述（由申请人提供）：本提案的广泛目标是定义肠道发育和成熟过程中介导时空模式形成的机制。研究计划将集中在调节肠道乳糖酶基因转录时空限制的分子机制。肠乳糖酶是负责消化乳糖（牛奶中主要的碳水化合物）的肠上皮细胞二磷酸脱氢酶。乳糖酶活性在断奶前最高，然后在成熟过程中显著下降。乳糖酶活性降低与过量牛奶摄入相结合，导致大多数成熟哺乳动物（包括人类）出现碳水化合物吸收不良的症状。我们已经确定了几个DNA调控元件和转录因子（加塔-4/5/6，Cdx-2，HNF-1和Pdx-1）参与调节乳糖酶基因在肠细胞中的转录。此外，我们已经确定，乳糖酶启动子的2.0-脱氢酶片段在转基因小鼠的小肠中指导适当的空间和时间表达。我们提出了一个全球性的假设，描述了破译肠道密码的可能性，DNA和蛋白质模块之间的组合相互作用介导的时空肠道基因表达。我们将重点关注Pdx-1在介导前空间限制中的潜在作用。因此，研究目标是乳糖酶顺式调控元件和转录因子的特性。在目标1中，我们将通过分析肠细胞培养物和转基因小鼠中与报告基因相关的基因组缺失的表达来表征乳糖酶DNA调控元件。该分析将通过允许报告基因表达的体内生物发光检测的方法来促进。在目标2中，我们将描述已知乳糖酶转录因子在肠道发育和成熟过程中的时空表达和DNA结合模式。在目标3中，我们将功能性地研究同源结构域蛋白Px-1在介导乳糖酶基因的空间限制中的作用，通过改变细胞培养物和转基因小鼠中的Pdx-1-乳糖酶启动子相互作用并测定转录活性。体外和体内方法的组合被设计为表征参与调节乳糖酶基因转录的时空限制的机制。