DNA-binding proteins and their cis-acting sites controlling hormonal induction of a mouse β-casein::CAT fusion protein in mammary epithelial cells

Transcription of the mouse β-casein (βCAS)-encoding gene (casB) is regulated by the synergistic actions of insulin, glucocorticoid and prolactin in the mammary gland (MG). To delineate its regulatory sequence(s), we examined the hormonal inducibility of various chimeric constructs containing the promoter sequence of casB and the cat reporter gene in primary MG epithelial cells. A DNA fragment from bp - 258 to + 7 of casB was sufficient for the hormonal induction. Using a series of 5'- and internal deletions of the casB promoter region, at least three DNA elements were found to be necessary for full induction. These were located at bp positions -258 to -180, -154 to -136, and -98 to -62. DNase I footprinting analysis with a partially purified extract from lactating MG cells detected at least seven protected sequences, I(-242 to -219), II(-213 to -202), III(-151 to -139), IV(-125 to -110), V(-98 to -90), VI(-79 to -70) and VII(-59 to -45). Regions I/II, III and V/VI were included in the three DNA elements required for the hormonal induction, and the IV region corresponded to the MG consensus sequences of several milk protein-encoding genes. Competition gel retardation assays using nuclear extracts of lactating mouse MG revealed the presence of specific binding proteins for regions I, II, IV and VI, as well as specific protein(s) binding to both regions III and V. The binding activities of these proteins, except that associated with region IV, were increased from the virgin to lactating periods. The activity of protein binding to region IV existed in all stages, but its pattern in gel retardation assays changed during the development of MG from the virgin to lactating periods. These results suggest that multiple DNA elements and their binding proteins are involved in the hormonal induction of mouse casB expression during MG development.