Maturational Regulation of Lactase Gene Transcription

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DESCRIPTION (provided by applicant): The broad objective of this proposal is to enhance the transition of the investigator to an independent and productive status in the area of molecular and developmental biology of the gastrointestinal tract. The research goal of this proposal is to define in greater detail the molecular mechanisms regulating the maturational decline in 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 and Cdx-2) 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 spatio-temporal expression in the small intestine of transgenic mice. Our hypothesis, based on this data, is that the maturational decline in lactase expression is mediated by differential interaction between this 2.0 kb promoter region DNA and specific nuclear transcription factors. Specific research objectives, therefore, are aimed at characterization of cis regulatory elements and identification of nuclear proteins interacting with those cis elements. We aim to characterize the lactase DNA regulatory elements by analyzing expression of additional genomic deletions linked to a reporter gene and expressed in transgenic mice. This analysis will be facilitated by the application of methods to allow for in vivo photonic detection of reporter gene expression. We will identify specific nuclear proteins interacting with lactase gene fragments using DNase I footprint and gel shift assays. We aim to functionally characterize the proteins by altering their expression in cell culture and in transgenic mice and by assaying for transcriptional activity. The continued research experience, supplemented with mentored advice and seminars in the fields of molecular and developmental biology, will facilitate the investigator's development as an independent and productive scientist performing research on the molecular biology of intestinal development.