Spatiotemporal Regulation of Intestinal Gene Expression

描述(申请人提供)：这项建议的广泛目标是确定肠道发育和成熟过程中时空模式形成的中介机制。该研究计划将重点放在调节肠道乳糖酶基因转录时空限制的分子机制上。肠道乳糖酶是一种肠道细胞二糖酶，负责消化牛奶中的主要碳水化合物乳糖。乳糖酶活性在断奶前最高，成熟期显著下降。乳糖酶活性的降低和过量的牛奶摄入会导致包括人类在内的大多数成熟哺乳动物出现碳水化合物吸收不良的症状。我们已经鉴定了几个DNA调控元件和转录因子(GATA-4/5/6、CDX-2、HNF-1和PDX-1)，它们参与调节肠道细胞中乳糖酶基因的转录。此外，我们已经确定乳糖酶启动子的2.0kb碱基片段在转基因小鼠的小肠中引导了适当的空间和时间表达。我们提出了一个全球假说，描述了破译DNA和蛋白质模块之间的组合相互作用的肠道密码的可能性，这些模块介导了肠道基因的时空表达。我们将关注PDX-1在调节前空间受限中的潜在作用。因此，研究的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，我们将通过分析与报告基因连锁的基因组缺失在肠道细胞培养和转基因小鼠中的表达来表征乳糖酶DNA调控元件。这一分析将通过允许体内生物发光检测报告基因表达的方法来促进。在目标2中，我们将描述已知的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，我们将通过改变细胞培养和转基因小鼠中PDX-1-乳糖酶启动子的相互作用并检测转录活性，从功能上研究同源结构域蛋白PX-1在介导乳糖酶基因空间限制中的作用。体外和体内相结合的方法被设计用来描述调控乳糖酶基因转录的时空限制的机制。

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.