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.
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