The intestinal epithelium is spatially segregated into a proliferating, undifferentiated compartment and a non- proliferating, differentiated compartment in both the small and large intestine. The differentiated columnar epithelium is the interface between the contents of the intestinal lumen and the internal environment of higher vertebrates and, therefore, expresses genes important for the functional activity at this interface. To date, however, little is known about the following: 1) The molecular mechanism by which genes are targeted to the colonic epithelium, 2) The processes underlying differentiation in the colonic epithelium, or 3) The mechanisms by which intestinal gene regulation is altered in pathologic disease states. Therefore, based upon the following hypothesis, we propose to study the transcriptional regulation of a gene known as DRA (Down Regulated in Adenoma): 1) The regulation of DRA gene expression may be used as a model to understand how gene expression is directed to the differentiated surface epithelium of the colon. DRA encodes a sulfate/chloride anion transporter that is expressed principally by the differentiated epithelium in the colon and whose expression is lost in colonic adenomas and adenocarcinomas. DRA has been recently identified by positional cloning as the gene which is mutated in congenital chloride diarrhea and represents the only gene yet identified that, in isolation, plays a role in the pathogenesis of diarrhea. We have demonstrated that two zinc finger transcription factors, YY1 and GATA, play an important role in the transcriptional activation of the DRA promoter in vitro. Transgenic animals demonstrate that 3.6 kb of the DRA 5'-flank directs high level reporter gene expression to the differentiated epithelium of both the small and large intestine.
Three specific aims will be pursued to understand how the DRA gene is regulated: 1) To elucidate the transcriptional mechanisms by which the DRA 5'-flank is activated using sodium butyrate induction of DRA in LS174T cells as an in vitro model system, 2) To identify potential cis-acting regions within the DRA promoter which interact which nuclear proteins isolated from murine small and large intestine, and 3) To characterize the mechanisms that regulate DRA gene expression in vivo using transgenic mice. Ultimately, once the mechanisms by which DRA gene transcription is regulated in the normal colon have been elucidated, DRA may be a useful model with which to investigate how gene expression is altered in pathobiology.
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