The objective of our current research is to elucidate the molecular mechanism(s) of tissue- and stage-specific, multi-hormone regulated gene expression using the beta-casein gene as a tool. Beta-Casein is the major milk protein synthesized by the mammary gland during lactation. The expression of the mouse beta-casein gene is regulated both positively and negatively by the interaction of various peptide and steroid hormones during mammary gland development. Insulin (I), glucocorticoid (H) and prolactin (PRL) are essential for the induction of casein gene transcription and progesterone inhibits the induction process. To study the molecular mechanism of casein gene expression, we have established a transient expression system. Primary mouse mammary epithelial cells were transfected with a beta-casein promoter-CAT (chloramphenicol acetyltransferase) reporter plasmid and its expression was examined in the presence of appropriate hormones. Using this system, we found that approximately 500 nucleotides upstream from the transcription initiation site are sufficient to confer hormone responsiveness to the CAT reporter gene. CAT activity was induced by I, H and PRL and was inhibited by progesterone. Additional studies with a series of 5'- and internal deletions of the promoter indicated that at least three regions, located within bp positions -258/+7, contain DNA elements required for hormonal induction. In order to define the transcription factors mediating the hormonal regulation of beta-casein gene transcription, we have analyzed the binding of mammary gland nuclear proteins to the casein gene promoter using gel mobility shift assays, DNase I footprinting and methylation interference experiments. We identified at least six DNA elements (I - VI) that bind nuclear proteins in the promoter region required for hormonal induction. The binding of nuclear proteins to the six elements increased in the mammary gland during transition from the virgin to lactating periods. Elements III and V are probably recognized by a mammary gland-specific factor whose appearance is induced by PRL. Element IV corresponds to the consensus sequence for milk protein genes. Transfection analysis showed that this element is important for enhancement of beta-casein gene transcription. The binding protein to element IV existed in two forms, one dominant in virgin gland and the other in lactating gland. The appearance of the latter form is induced by I. Our results suggest that the multi-hormonal regulation of beta- casein gene transcription involves the interplay of multiple transcription factors whose appearance is controlled by various hormones.
