YY1 REPRESSES BETA-CASEIN GENE-EXPRESSION BY PREVENTING THE FORMATIONOF A LACTATION-ASSOCIATED COMPLEX

Site-specific mutagenesis of the highly conserved milk box (-140 to -1 10) region suggested that beta-casein expression is regulated by a hor mone-mediated relief of repression (M. Schmitt-Ney, W. Doppler, R. K. Ball, and B. Groner, Mol. Cell. Biol. 11:3745-3755, 1991). However,,wh en this sequence was placed upstream of a heterologous thymidine kinas e promoter, it activated reporter gene expression. This apparent parad ox was resolved when the trans-acting factor YY1, capable of acting as both a positive and negative regulator, was shown to interact with th e milk box region, using bacterially expressed YY1 and specific oligon ucleotide and antibody competition experiments. Second, it was demonst rated that extracts prepared from several cell types contained a prote in(s) interacting with the mammary gland-specific factor (MGF) binding site, previously shown to be required far beta-casein promoter activi ty (Schmitt-Ney et al., Mol. Cell. Biol. 11:3745-3755, 1991). Sequence analysis of this site revealed similarity to the gamma interferon-act ivated sequence, suggesting that MGF may be related to the stat91 sign aling protein. Finally, using an oligonucleotide encompassing both the WI and MGF sites, we detected a slow-mobility complex only in extract s from mammary glands at late pregnancy and lactation (lactation-assoc iated complex [LAC]). Site-specific mutation of the YY1 binding site l ed to an enhancement in LAC DNA binding activity, while mutation of th e MGF site decreased detectable LAC. These results support a model in which lactogenic stimuli lead to a decrease in YY1 binding, and subseq uent increased formation of LAC at a nearby binding site, to stimulate beta-casein transcription.