A nuclear matrix acceptor site for the progesterone receptor in the avian c-myc gene promoter.

It has been the goal of this project to determine the location, composition, and biological function of the nuclear acceptor sites (i.e., the nuclear binding sites) for the avian oviduct progesterone (Pg) receptor (PR). Many laboratories have demonstrated a native-(in vivo) like cell-free binding of steroid receptor complexes to specific acceptor sites in nuclei/chromatin in a variety of target tissue systems. These sites appear to involve protein-DNA complexes and some of these have been shown to reside in the nuclear matrix, including the chromatin acceptor sites for the avian oviduct PR. We have purified a nuclear matrix "acceptor protein" for the avian PR. termed receptor binding factor-1 (RBF-1), based on its ability to generate specific, high-affinity PR binding on avian genomic DNA. This 10 kD nuclear matrix protein was found to be unique with minimal homology to a couple of other proteins. Using immunohistochemical techniques and antibodies against the purified RBF-1, the RBF-1 was localized to the nuclei of many avian and rat tissues. Co-localizations of RBF-1 and PR in select cell types in the avian oviduct and rat reproductive organs were also reported. A tissue specificity was found with regard to RBF-1 concentrations. The full length cDNA to RBF-1 has been isolated and used to identify a 0.7 kb mRNA whose levels in various avian tissues reflect the protein levels. Genomic sequences of RBF-1 have been isolated and characterized. Preliminary studies indicate that the over-expression of the RBF in human MCF-7 cells results in an inhibition of the c-myc gene promoter activity which is further inhibited by steroid hormone treatments of the cells. Past studies in our laboratory demonstrated that the c-myc mRNA steady state levels are rapidly (approximately 15 min) reduced by Pg and glucocorticoids in the avian oviduct. Further, partially purified fractions of RBF-1 were shown to generate specific PR binding sites only on the genomic DNAs of certain animal species and on the c-myc gene, but not ovalbumin gene. Using Southwestern blot analyses, the purified RBF-1 was shown to bind specifically to sequences in the 5' end of c-myc, c-jun proto-oncogenes but not to genomic sequences of the ovalbumin gene. A specific DNA binding element in the promoter region of the c-myc proto-oncogene has been identified as AT-rich domain of homopurine/pyrimidine stretches flanked by GC-rich sequences. Southern blot analyses using 200 bp matrix DNA fragments protected by the nuclear matrix structure indicate that the matrix is attached on either side of the RBF-1 binding element. A model is described for a nuclear matrix acceptor site attached to the c-myc promoter which may mediate the Pg-induced down-regulation of the c-myc gene expression.