Promoter regulation of a differentially expressed gene in the human colonic epithelial cell lines HT29-18 and HT29-18-C1.
Gene A4 is transcriptionally activated upon enterocyte differentiation of the human colonic epithelial cell line HT29-18 and its highly differentiated subclone HT29-18-C1 [Oliva et al., Arch. Biochem. Biophys. 302 (1993) 183-192]. To characterize the mechanisms regulating the differential transcription of A4, we analyzed its immediate 5'-flanking region for regulatory elements. Promoter-linked transfection experiments of progressively deleted A4 5'-flanking sequences fused to the bacterial cat reporter gene suggest the presence of one negative and two positive DNA elements within the first 371 bp of the A4 promoter (pA4). DNase I footprint and electrophoretic mobility shift assays demonstrate that one positive element which contains the core binding sequence for the transcription factor, Sp1, mediates an equal level of transcription in the two cell types. The second positive element, localized between nucleotide positions--169 and -152, contains a sequence previously unrecognized as a transcription factor-binding site. This element mediates a twofold increase in the activity of pA4 in HT29-18-C1, as compared to HT29-18. Furthermore, nuclear extracts obtained from HT29-18-C1 contain a higher binding activity for this element than those from HT29-18. Southwestern blot analysis suggests that the protein interacting with this element has an estimated molecular mass of 50 kDa. We conclude that this protein may be involved in the differential regulation of A4 in these intestinal cell lines.
Oliva
M M
MM
Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Cortese
J F
JF
Yang
V W
VW
D001483:Base Sequence; D001665:Binding Sites; D002454:Cell Differentiation; D002460:Cell Line; D003106:Colon / Q000166:cytology / Q000502:physiology*; D004252:DNA Mutational Analysis; D004847:Epithelial Cells; D004848:Epithelium / Q000502:physiology; D005786:Gene Expression Regulation; D062792:MARVEL Domain-Containing Proteins; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011510:Proteolipids; D011994:Recombinant Proteins; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor; D014162:Transfection
D062792:MARVEL Domain-Containing Proteins; D008565:Membrane Proteins; D009687:Nuclear Proteins; C094765:PLP2 protein, human; D011510:Proteolipids; D011994:Recombinant Proteins; D016329:Sp1 Transcription Factor