CIS-ACTING ELEMENTS AND TRANSCRIPTION FACTORS INVOLVED IN THE INTESTINAL SPECIFIC EXPRESSION OF THE RAT CALBINDIN-D9K GENE - BINDING OF THEINTESTINE-SPECIFIC TRANSCRIPTION FACTOR CDX-2 TO THE TATA BOX

The calbindin-D9k (CaBP9k) gene is mainly expressed in differentiated duodenal epithelial cells and is used as a model for studying the mole cular mechanisms of intestine-specific transcription. The gene has bee n cloned, two major DNase-I-hypersensitive sites in the duodenum have been described, and a vitamin-D-response element has been identified. We have now analysed the transcription factors and regulatory sequence s involved in the transcription of the CaBP9k gene in the intestine in ex vivo and in vitro experiments. Transfection experiments in intesti nal (CaCo-2) and non-intestinal (HeLa) cell lines defined two regions in the 5'-flanking sequences of the rat CaBP9k gene, A minimal proxima l region (-117 to +20) promoted transcription in both intestinal expre ssing and non-expressing cell lines. Tissue specificity was conferred by the sequences situated further upstream, which are responsible for complete repression in the non-intestinal cells. Intestinal transcript ion was specified by the proximal region, containing a specialized TAT A box, and a distal region, which contains a previously described inte stinal DNase-I-hypersensitive site. In vitro DNase I footprinting, ele ctrophoretic mobility shift assays and antibody supershift assays were used to examine the factors bound to the proximal promoter region (-8 00 to +80 bp). Rat duodenal nuclear extracts protected 12 sites. Some of them appear to be binding sites for ubiquitous (nuclear factor 1) o r hepatic-enriched sites (hepatocyte nuclear factors 1 and 4, enhancer binding protein alpha and beta) factors. DNA binding studies and tran sfection experiments indicated that an intestine-specific transcriptio n factor, caudal homeobox-2, binds to the TATA box of the rat CaBP9k g ene. These data contribute to our understanding of the control of the intestinal transcription of the CaBP9k gene and demonstrate that sever al traits-acting factors, other than the vitamin D receptor, may be fa ctors for intestine-specific CaBP9k gene expression.