CHROMATIN ACCEPTOR SITES FOR PROGESTERONE

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It has been the long range goal of this grant 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). Steroid receptor complexes (SR) have been reported to bind in vitro and in vivo to specific acceptor sites in the nuclear matrix of a variety of target cells. During the past funding period of this grant, we found these chromatin acceptor sites and nuclear matrix sites for the avian oviduct PR to be one and the same. Further, we 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 and showed it was a unique 10 kD protein with some homology to other nuclear proteins. We also reported a nuclear localization of the avian-like RBF-1 in many avian and rat tissues using immunohistochemical assays. Co-localizations of RBF-1 and PR in selected cell types in the avian oviduct and rat ovary and uterus were also found. Southwestern blot analyses demonstrated that RBF binds to a specific DNA binding element in the promoter region of the c-myc nuclear proto-oncogenes whose mRNA levels are rapidly (about 15 min) reduced by Pg. This element was recently identified as a MAR-like (AT-rich) 64 bp domain flanked by GC- rich sequences. The full length cDNA to RBF-1 has been isolated and used to identify a 0.7 kb mRNA whose levels in the avian oviduct are regulated in vivo by steroids. Genomic sequences of RBF-1 have been isolated and shown to contain 4 exons, as well as putative SR- and heat shock-response elements in the 5' flanking region. Preliminary studies indicate that the over-expression of the RBF in human MCF-7 cells inhibits the c-myc gene promoter activity and that this activity is further inhibited by steroids. Studies are underway to continue 1) the analysis of the steroid and heat shock regulation of the RBF gene expression; 2) the analyses of the biological function(s) of the RBF and its DNA binding element by determining the effects of a) deleting the element from the promoter; b) increasing, and c) decreasing (using antisense oligonucleotides) RBF expression on the Pg receptor nuclear binding, as well as the steady-state and steroid regulation of endogenous c-myc gene expression and the c-myc promoter activity; 3) structurally characterizing the RBF-1-DNA element complex, and 4) identifying an homologous RBF-1 mRNA/protein in human cells. This RBF nuclear matrix structure may explain how steroids inhibit the transcription of the c-myc and other nuclear proto-oncogenes.