The activated mammary gland specific nuclear factor (MGF) enhances in vitro transcription of the beta-casein gene promoter.
The hormonal induction of the beta-casein gene in mammary epithelial cells is dependent on the action of peptide and steroid hormones. Epidermal growth factor, insulin, glucocorticoids and prolactin act in a sequential manner to regulate the transcription of the gene. We have studied the hormonal requirements as well as the nuclear proteins which are involved in the induction process. In vitro transcription in cell free nuclear extracts has been used to demonstrate the central role of the mammary gland specific nuclear factor, MGF, in the mediation of the hormonal signals to the transcription machinery. A gene construct comprising 344 nucleotides of wild type beta-casein promoter sequence and a G-free cassette of 220 nucleotides was used to test the activity of nuclear extracts in the in vitro transcription experiments. A construct in which the proximal MGF binding site in the beta-casein promoter region has been inactivated by mutation and a construct regulated by the adenovirus major late promoter served as controls. Nuclear extracts were prepared from Sf insect cells, HeLa cells and mammary epithelial cells of lactating rats. Strong transcription of the wild type beta-casein promoter construct was observed in the mammary cell extract, weak transcription in the extracts of Sf insect cells and the HeLa cells. The mutation of the MGF binding site drastically reduced the in vitro transcription in the mammary gland cell extract. Beta-casein promoter activity was also compared in nuclear extracts from uninduced and lactogenic hormone induced HC11 mammary epithelial cells. Extracts from induced cells are more efficient in the support of beta-casein gene transcription.
Happ
B
B
Friedrich Miescher Institute, Basel, Switzerland.
Groner
B
B
D000818:Animals; D001483:Base Sequence; D002364:Caseins / Q000235:genetics*; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000494:pharmacology*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004848:Epithelium / Q000378:metabolism; D005260:Female; D005938:Glucocorticoids / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D007774:Lactation; D008321:Mammary Glands, Animal / Q000378:metabolism*; D008894:Milk Proteins; D008969:Molecular Sequence Data; D009036:Moths; D016296:Mutagenesis; D011388:Prolactin / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D051381:Rats; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D014158:Transcription, Genetic / Q000187:drug effects*
D002364:Caseins; D004268:DNA-Binding Proteins; D005938:Glucocorticoids; D007328:Insulin; D008894:Milk Proteins; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D004815:Epidermal Growth Factor; D006160:Guanosine Triphosphate; D011388:Prolactin