Abstract
The molecular mechanisms that regulate intestine-specific gene expression and the transition from proliferating, undifferentiated crypt cells to nonproliferating, differentiated villus cells are unknown. Sucrase-isomaltase is an apical membrane disaccharidase that is found exclusively in enterocytes of adult intestine and is expressed in a complex pattern along the intestinal crypt-villus axis. To investigate the regulation of sucrase-isomaltase, we have cloned and sequenced 3.6 kilobases of the 5'-flanking region of the human sucrase-isomaltase gene. The transcriptional start site was mapped in human small intestine and in a colonic adenocarcinoma cell line (Caco-2) using an anchored polymerase chain reaction, primer extension, and RNase protection assays. The 5'-flanking DNA of the gene was linked to either chloramphenicol acetyltransferase or luciferase reporter genes and used for transfection into Caco-2, HeLa, and HepG2 cells. This analysis demonstrated that intestine-specific transcription of the sucrase-isomaltase gene involves both proximal and distal regulatory elements. Use of sucrase-isomaltase as a model gene will allow investigation of the mechanisms that regulate transcription of enterocyte-specific genes, developmental gene expression in the small intestine and colon, and the process of differentiation as epithelial cells migrate from intestinal crypts onto the villus in adult intestine.
Highlights
The molecular mechanisms that regulate intestine- cells are enterocytes, constituting 90% of cells in crypts and specific gene expressionand the transition from pro- greater than 95% of cells on the villi [3]
We have previously shown by in situ hybridization that tion of the mechanisms that regulate transcription of the pattern of SI mRNA expression along the crypt-villus enterocyte-specific genes, developmental gene expresa-xis was similar in both rat [8]and human intestine[9].Little sion in the small intestine and colon, and the process or no SI mRNA is detectable in crypt cells, whereas there is of differentiation as epithelial cells migrate from in- aprominent appearance of mRNA in cells located at the testinal crypts onto thveillus in adult intestine
SI mRNA is abundant in enterocytes from the base of villi to the mid-villus region and decreases as enterocytes move from mid-villus toward the tip, resulting
Summary
Vol 267, No 11, Issue of April 15, pp. 7863-7870, 1992 Printed in U.S.A. Isolation and Characterization of the Human Sucrase-Isomaltase Gene and Demonstration of Intestine-specific Transcriptional Elements*. In addition to being ferase or luciferase reporter geneasnd used for trans- intestine-specific, SI represents a useful model for investigafectionintoCaco-2, HeLaa, ndHepG2 cells This tion of enterocyte-specific gene expression, because it is the analysis demonstratedthatintestine-specifictranproduct of a single gene [8,9],is expressed in intestinal tumor scription of the sucrase-isomaltase gene involves both cell lines, and has a complex pattern of expression during proximal and distal regulatory elements. We have previously shown by in situ hybridization that tion of the mechanisms that regulate transcription of the pattern of SI mRNA expression along the crypt-villus enterocyte-specific genes, developmental gene expresa-xis was similar in both rat [8]and human intestine[9].Little sion in the small intestine and colon, and the process or no SI mRNA is detectable in crypt cells, whereas there is of differentiation as epithelial cells migrate from in- aprominent appearance of mRNA in cells located at the testinal crypts onto thveillus in adult intestine. The diminished expression of SI mRNA in villus tip cells maybe a manifestation of senescence prior tothe extrusion of these cells into the intestinal lumen
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