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1 Department of Medical Biochemistry and Genetics, University of Copenhagen, Copenhagen, Denmark
* To whom correspondence should be addressed. E-mail: jolsen{at}imbg.ku.dk.
The intestinal alkaline phosphatase gene (ALPI) encodes a digestive brush border enzyme, which is highly up-regulated during small intestinal epithelial cell differentiation. To identify new putative promoter motifs responsible for the regulation of ALPI expression during differentiation of the enterocytes, we have conducted a computer-assisted cis-element search of the proximal human ALPI promoter sequence. A putative recognition site for the transcription factor HNF-4 was predicted at the positions from -94 to -82 in relation to the translational start site. The ability of HNF-4 alpha to stimulate the expression from the ALPI promoter was investigated in the non-intestinal Hela cell line. Co-transfection with an HNF-4 alpha expression vector demonstrated a direct activation of the ALPI promoter through this -94 to -82 element. Electrophoretic mobility shift assay (EMSA) showed that HNF-4 alpha from nuclear extracts of differentiated intestinal epithelial cells (Caco-2) bound with high affinity to the predicted HNF-4 binding site. A 521 bp promoter fragment containing the HNF-4 binding site demonstrated a differentiation-dependent increase in promoter activity in Caco-2 cells. The presence of the HNF-4 binding site was necessary for this increase to occur.
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