CCAAT/enhancer-binding protein isoforms β and δ are expressed in mammary epithelial cells and bind to multiple sites in the β-casein gene promoter

Abstract

Lactogenic hormone-dependent expression of the rat β-casein gene in mammary epithelial cells is controlled via a complex regulatory region in the promoter. The sequence between -176 and -82 is the minimal region to confer the response to glucocorticoid hormone and prolactin on a heterologous promoter. The response is further enhanced by the region between -282 and - 176. DNase I footprinting experiments and electromobility shift assays revealed the presence of four binding sites for CCAAT/enhancer-binding protein (C/EBP) isoforms in the hormone response region between -220 and - 132. In nuclear extracts from mammary epithelial cells, the prevalent C/EBP isoform binding to these sites is β (C/EBP-β). C/EBP-δ is also present in mammary epithelial cells, whereas C/EBP-α is not detectable. The C/EBP sites are located in close proximity to the previously characterized binding sites for the prolactin-inducible mammary gland factor/signal transducer and activator of transcription-5, the nuclear factor YY1, and the glucocorticoid 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 β-casein gene promoter chloramphenicol acetyltransferase construct. The results implicate C/EBPs as important regulators of β-casein gene expression in the mammary epithelium.