MOLECULAR MECHANISMS OF INTESTINAL DEVELOPMENT

肠道的上皮细胞是由复杂的 组织发生和基因表达的发育模式。之甚少 已知的分子机制，指导这些发展 程序.蔗糖酶-异麦芽糖酶（SI）将被用作模型基因来检测 这些机制，因为它有一个时间表达的模式， 反映了许多重要的发展转型。SI已被用于 作为肠道发育的标志， 分化和我们实验室最近的研究提供了 为研究其调控的分子机制奠定基础。 具体目标1：问题：是什么调节了 小鼠小肠中的肠基因表达， 从哺乳到断奶的发育过渡期呢假设：The 断奶时SI基因表达的诱导是转录的， 通过位于SI的5 '侧翼区的DNA调控元件 基因目的：鉴定DNA调控元件及其同源序列 结合蛋白，其调节小肠中SI基因的表达 在哺乳-断奶的发育过程中， 过渡初步数据表明， 利用转基因小鼠研究顺式作用调节的可行性 SI的正确发育调节所需的元素 基因这些调节元件的精确定位将是 使用转基因小鼠的组合实现，体外分析 DNA酶I超敏位点，稳定转染的肠细胞的应用 线，和DNA-蛋白质相互作用。最后，结合这些蛋白的蛋白质 调控元件将被表征和克隆。 具体目标2：问题：限制 肠内特定细胞系的基因表达 上皮？具体来说，是什么机制限制了SI 基因转录到肠上皮细胞？假设：SI基因表达是 限制在肠上皮细胞系的转录沉默， 其他上皮细胞类型。瞄准为了鉴定DNA调控元件， 在肠上皮细胞谱系中沉默SI基因转录 除了肠细胞。转基因小鼠和细胞中的初步数据 线表明肠上皮细胞的表达限制受到控制， 在转录抑制的水平上。稳定转染的细胞系 将利用微基因构建体来鉴定转录因子 沉默元素转基因小鼠将被用作体内报告者 系统，以确定是否DNA调控元件中确定的 转染分析如预期在体内起作用。

The epithelial lining of the intestinal tract results from complex developmental patterns of histogenesis and gene expression. Little is known about the molecular mechanisms that direct these developmental programs. Sucrase-isomaltase (SI) will be used as a model gene to examine these mechanisms since it has a pattern of temporal expression that mirrors many important developmental transitions. SI has been utilized for over three decades as a marker for intestinal development and differentiation and recent studies from our laboratory have provided the foundation to examine the molecular mechanisms of its regulation. SPECIFIC AIM 1: Question: What regulates the dramatic increase in intestinal gene expression in the small intestine of mice as they progress through the suckling-weaning developmental transition? Hypothesis: The induction of SI gene expression at weaning is transcriptional and occurs via DNA regulatory elements located in the 5'-flanking region of the SI gene. Aim: To identify the DNA regulatory elements, and their cognate DNA binding proteins, which regulate SI gene expression in the small intestine of mice as they progress through the suckling-weaning developmental transition. Preliminary data are presented which demonstrate the feasibility of using transgenic mice to examine the cis-acting regulatory elements that are required for correct developmental regulation of the SI gene. The precise localization of these regulatory elements will be achieved using a combination of transgenic mice, in vitro analysis of DNase I hypersensitive sites, use of stably transfected intestinal cell lines, and DNA-protein interactions. Finally, the proteins that bind these regulatory elements will be characterized and cloned. SPECIFIC AIM 2: Question: What are the mechanisms that restrict the expression of genes to specific cell lineages in the intestinal epithelium? And specifically, what are the mechanisms that restrict SI gene transcription to enterocytes? Hypothesis: SI gene expression is restricted to the enterocyte lineage by the silencing of transcription in other epithelial cell types. Aim. To identify the DNA regulatory elements that silence SI gene transcription in intestinal epithelial cell lineages other than enterocytes. Preliminary data in both transgenic mice and cell lines suggest that restriction of expression to enterocytes is controlled at the level of repression of transcription. Cell lines stably transfected with minigene constructs will be utilized to identify transcriptionaI silencing elements. Transgenic mice will be used as an in vivo reporter system to determine if the DNA regulatory elements identified in the transfection analysis functions as expected in vivo.