Isolation and characterization of a differentiation-dependent gene in the human colonic cell line HT29-18

The human colonic epithelial cell line HT29, and its clonal derivatives HT29-18 and HT-29-18-C1, differentiate in vitro. Differential screening of a subtraction cDNA library enriched for sequences unique to HT29-18-C1, a highly differentiated subclone of HT29-18, resulted in the isolation of a differentiation-dependent cDNA clone, A4. A full-length clone encoding A4 was obtained and sequenced to its entirety. It is 945 bp in length and contains an open reading frame (ORF) of 456 bp. The amino acid sequence deduced from the ORF reveals a polypeptide of 152 amino acids with a predicted molecular mass of 17,000 Da, a size confirmed by coupled in vitro transcription and translation directed by the full-length A4 cDNA. This polypeptide contains four potential membrane-spanning domains and consensus sequences for N-linked glycosylation as well as phosphorylation sites for protein kinase C and casein kinase II. Comparison of A4 to published DNA and protein sequences revealed no significant homology. Genomic Southern blot analysis suggests that the gene is present in a single copy within the human genome and is conserved in the rat. Northern blot analysis of RNA obtained from various rat tissues shows that the expression of the A4 gene is tissue-selective and is enriched in colonic mucosa. In situ hybridization using human intestinal tissues indicates that the expression of A4 follows a gradient along the crypt-to-villus axis with the most abundant message occurring in the lower half of the crypt. Furthermore, nuclear run-on assays suggest that the induction of the A4 gene during differentiation of HT29-18 is regulated at a transcriptional level. A clone was isolated from a human genomic library and found to contain all five exons of A4. S1 nuclease analysis localized the start site of transcription to an adenosine residue 91 nucleotides upstream from the ATG translation initiation codon. Examination of the immediate sequence 5' to the mRNA start site reveals no TATA box and multiple known enhancer sequences. A4 is also noted to share certain features with the gene encoding the cystic fibrosis transmembrane conductance regulator protein. They include a similar vertical distribution of expression along the intestinal epithelium, enhanced transcription upon differentiation of HT29-18, and multiple shared putative regulatory sequences in the promoter regions. Further characterization of the mechanisms regulating expression of the A4 gene could contribute to the understanding of mammalian intestinal differentiation.