The expression of intestinal hormone genes is limited to specific enteroendocrine cell types. The overall objective of the studies proposed in this application is to characterize the regulatory mechanisms controlling cell-specific expression of the secretin gene in cell lines and transgenic mice. This laboratory has identified a 98 bp cis-active element in the secretin gene which enhances its expression in secretin producing cell lines. In addition we have recently expressed a viral oncogene in secretin cells of transgenic mice. Analysis of enteroendocrine tumors in the transgenic mice suggests that the secretin-producing S cell may be genetically related to at least one other cell type, the enteroglucagon cell. The present investigations will focus on defining the functional sequences of the secretin enhancer and characterizing the trans-acting proteins that bind to these sequences in cell lines. The cell-specific expression of secretin and differentiation of the secretin cell will be examined in transgenic mice to complement the in vitro studies. The following five specific aims are proposed: 1) To define the functional elements of the rat secretin gene enhancer by deletional analysis and scanning substitution mutagenesis. 2) To characterize and partially purify the proteins which bind to cis-active elements using gel retardation, DNase I footprinting, methylation interference, and in vitro transcription assays. 3) To determine which sequences in the rat secretin gene are necessary for the correct tissue-specific and developmental expression of this hormone in the normal S cells of transgenic mice. These studies will examine the expression of a hybrid transgene consisting of the human growth hormone gene coding region under the control of the secretin gene 5' flanking sequences. 4) To immortalize the secretin cell progenitor in transgenic mice by expressing a viral oncogene, SV40 large T antigen, in S cells of transgenic mice. The hormonal products of T antigen expressing cells and tumors will be characterized by immunocytochemistry to identify steps in the pathway of secretin cell differentiation and further characterize the relationship of the S cell to other enteroendocrine cells. Tumors which develop in animals will be used to establish cell lines. 5) To selectively ablate secretin cells in transgenic mice by expressing a toxic gene, herpes simplex thymidine kinase, in S cells. Ablation of other enteroendocrine cell populations by a toxic gene under control of the secretin promoter will identify other members of the S cell lineage. In addition, the secretin deficient mice will be used as an animal model to examine the effects of this hormone on pancreatic growth in developing animals. By using genetic rather than morphologic criteria, the studies proposed in this application will provide new insights into how enteroendocrine cells differentiate and may provide a genetic foundation to understand the production of multiple hormones by endocrine neoplasia of the intestine and pancreas.
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