Regulatory sequences on the human villin gene trigger the expression of a reporter gene in a differentiating HT29 intestinal cell line.
To develop a molecular tool for tissue-specific targeting of gene expression in immature and differentiated epithelial cells of the small and large intestinal mucosa, we have isolated the 2-kb 5'-flanking region of the human villin gene. This region contains numerous short sequences that are conserved among other tissue-specific promoters of genes expressed in differentiated enterocytes. This DNA fragment promotes the transcription and expression of the luciferase reporter gene in villin-positive intestinal, renal, and hepatoma cell lines but not in a villin-negative keratinocyte cell line. The pattern of expression corresponds that of the endogenous gene, indicating that this sequence can direct intestine-specific transcription. In the differentiating HT29 intestinal cell line, expression of the reporter gene is already detectable in undifferentiated cells, and dramatically increases when terminal differentiation is induced. Thus, as previously reported for the endogenous gene the isolated 5'-flanking region of the villin gene responds positively to conditions known to stimulate terminal differentiation of these cultured epithelial intestinal cells. The reported results indicate that this genomic fragment contains sufficient regulatory elements to recapitulate the expression pattern of the villin promoter during intestinal differentiation.
Institut Pasteur, Département de Biologie Moléculaire, URA-Centre National de la Recherche Scientifique 1149, Paris, France.
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Robine
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Sahuquillo-Merino
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Louvard
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Pringault
D000230:Adenocarcinoma; D000818:Animals; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000235:genetics; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics*; D002454:Cell Differentiation; D002460:Cell Line; D003001:Cloning, Molecular; D003110:Colonic Neoplasms; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007413:Intestinal Mucosa / Q000502:physiology; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D008840:Microfilament Proteins / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection; D014407:Tumor Cells, Cultured
D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D008840:Microfilament Proteins; D009838:Oligodeoxyribonucleotides; C026615:villin; D008156:Luciferases