A pregnancy-specific mammary nuclear factor involved in the repression of the mouse beta-casein gene transcription by progesterone.

The sequence-specific binding pattern of mammary nuclear proteins to the 545-base pair (bp) 5'-flanking region of the mouse beta-casein gene was compared between pregnant and lactating mice by a gel mobility shift assay. By using appropriate probes, two complexes were detected only during pregnancy, whereas an additional four complexes were detected during both pregnancy and lactation. The two pregnancy-specific complexes showed identical electrophoretic mobility and were cross-competed with two unlabeled DNA fragments used to generate the probes. Methylation interference experiments indicated that the two binding regions involved guanosine residues at nucleotides -350 and -8, and the sequences around each guanosine residue have a common palindromic sequence, 5'-TGAT/ATCA-3'. This binding factor is termed pregnancy-specific mammary nuclear factor. In gene transfection experiments, expression of the chimeric beta-casein-CAT gene linked to the 545 bp of mouse beta-casein gene promoter region was maximally induced when transfected mammary epithelial cells were cultured with the lactogenic hormones prolactin, hydrocortisone, and insulin, whereas it was very low when insulin alone was added. The addition of progesterone together with the lactogenic hormones inhibited the hormonal induction of the chimeric gene, whereas co-transfection with an oligonucleotide containing the pregnancy-specific mammary nuclear factor binding site substantially overcome the progesterone-mediated repression of transcription. Furthermore, mutation of the pregnancy-specific mammary nuclear factor binding sites of the chimeric beta-casein-CAT gene resulted in attenuation of the inhibitory effect of progesterone without blocking the stimulatory effect of the lactogenic hormones. These results indicate the presence of a pregnancy-specific mammary nuclear factor(s) that bind to two separate sites of the beta-casein gene promoter and suggest that it may serve as a repressor that mediates the inhibitory action of progesterone on beta-casein gene transcription.