CCAAT ENHANCER-BINDING PROTEIN ISOFORM-BETA AND ISOFORM-DELTA ARE EXPRESSED IN MAMMARY EPITHELIAL-CELLS AND BIND TO MULTIPLE SITES IN THE BETA-CASEIN GENE PROMOTER/

Lactogenic hormone-dependent expression of the rat beta-casein gene in mammary epithelial cells is controlled via a complex regulatory regio n in the promoter. The sequence between -176 and -82 is the minimal re gion to confer the response to glucocorticoid hormone and prolactin on a heterologous promoter. The response is further enhanced by the regi on between -282 and -176. DNase I footprinting experiments and electro mobility shift assays revealed the presence of four binding sites for CCAAT/enhancer-binding protein (C/EBP) isoforms in the hormone respons e region between -220 and -132. In nuclear extracts from mammary epith elial cells, the prevalent C/EBP isoform binding to these sites is bet a (C/EBP-beta). C/EBP-delta is also present in mammary epithelial cell s, whereas C/EBP-alpha is not detectable. The C/EBP sites are located in close proximity to the previously characterized binding sites for t he prolactin-inducible mammary gland factor/signal transducer and acti vator of transcription-5, the nuclear factor YY1, and the glucocortico id receptor. The importance of the two proximal C/EBP binding sites at the 5' border of the minimal region was tested by mutational analysis . Mutations of each site were found to inhibit strongly both the basal and the lactogenic hormone-induced transcription of a beta-casein gen e promoter chloramphenicol acetyltransferase construct. The results im plicate C/EBPs as important regulators of beta-casein gene expression in the mammary epithelium.