MOLECULAR MECHANISMS OF INTESTINAL DEVELOPMENT

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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.