PROMOTER REGULATION OF A DIFFERENTIALLY EXPRESSED GENE IN THE HUMAN COLONIC EPITHELIAL-CELL LINES HT29-18 AND HT29-18-C-1

Gene A4 is transcriptionally activated upon enterocyte differentiation of the human colonic epithelial cell line HT29-18 and its highly diff erentiated subclone HT29-18-C-1 [Oliva et al., Arch. Biochem. Biophys. 302 (1993) 183-192]. To characterize the mechanisms regulating the di fferential transcription of A4, we analyzed its immediate 5'-flanking region for regulatory elements. Promoter-linked transfection experimen ts of progressively deleted A4 5'-flanking sequences fused to the bact erial cat reporter gene suggest the presence of one negative and two p ositive DNA elements within the first 371 bp of the A4 promoter (pA4). DNase I footprint and electrophoretic mobility shift assays demonstra te that one positive element which contains the core binding sequence for the transcription factor, Spl, mediates an equal level of transcri ption in the two cell types. The second positive element, localized be tween nucleotide positions -169 and -152, contains a sequence previous ly unrecognized as a transcription factor-binding site. This element m ediates a twofold increase in the activity of pA4 in HT29-18-C-1, as c ompared to HT29-18. Furthermore, nuclear extracts obtained from HT29-1 8-C-1 contain a higher binding activity for this element than those fr om HT29-18. Southwestern blot analysis suggests that the protein inter acting with this element has an estimated molecular mass of 50 kDa. We conclude that this protein may be involved in the differential regula tion of A4 in these intestinal cell lines.