REGULATION OF GENE EXPRESSION IN THE SMALL INTESTINE

Washington University, Saint Louis MO

Investigators

Linked publications & trials

Abstract

This project will continue to use several homologous fatty acid binding protein (Fabp) genes and their protein products as model systems to address two biological questions: (i) how does the mouse intestinal epithelium establish and maintain regional differences in the differentiation programs of its 4 principal lineages in the face of its rapid and perpetual renewal? and (ii) how are proteins able to bind fatty acids in noncovalent fashion? We will extend our mapping studies of cis- acting elements that regulate the enterocyte-specific and region-specific (along both the crypt to villus and duodenal to colonic axes) patterns of Fabp expression by linking portions of their 5' nontranscribed domains to a variety of reporters and using the resulting fusion genes to generate pedigrees of transgenic mice. Cellular patterns of reporter expression will be assessed using RNA hybridization analyses and single, as well as multilabel, immunocytochemical methods. Fabp/reporter transgenes will also be used as marker systems to describe the biological properties of the multipotent crypt stem cell and the lineage relationships of its descendants. Transcriptional regulatory elements already identified in an Fabp gene will be used to direct expression of C/EBP and SV40 TAg in intestinal crypts to assess the mechanisms that regulate lineage allocation, cellular differentiation programs, and the relationship between the cell cycle and these processes. Embryonic stem cells will we transfected with Fabp/reporter fusion genes and then introduced into blastocysts in order to generate transgenic chimeras. These mice should allow us to (a) assess the biological effects of reporters on gut epithelial cell populations by comparing normal blastocyst intestinal epithelium and adjacent ES-derived epithelium; and (b) accelerate our promoter mapping studies. The structure of E coli-derived rat intestinal fatty acid binding protein (I-FABP) has been determined to 1.2A without bound ligand, to 2.0A with bound palmitate and to 1.75A with bound oleate. These x-ray studies have indicated that (i) the protein undergoes little conformation change when it acquires its fatty acid and (ii) its fatty acid ligand is bound through a series of van der Waals and hydrogen bond interactions that involve ordered waters. We will explore the nature of these interactions and a proposed binding mechanism by site-directed mutagenesis of I-FABP and by constructing a model system of the protein's fatty acid binding site based on calix[6]arene. NMR studies of the six fold symmetric calix[6]arene receptor system complexed with a fatty acid should allow us to address basic questions concerning the strength of the interactions observed between I-FABP and its bound long chain fatty acid.