CHROMATIN ACCEPTOR SITES FOR PROGESTERONE

确定地点是这项赠款的长期目标， 组成和核受体位点的生物学功能（即， 鸟输卵管孕酮（Pg）受体的核结合位点 （PR）. 类固醇受体复合物（SR）已被报道在体外结合 并在体内结合到多种细胞核基质中的特异性受体位点 的靶细胞。 在过去的资助期内，我们发现 这些鸟类的染色质受体位点和核基质位点 输卵管PR是同一个。 此外，我们纯化了一个核基质 禽PR的“受体蛋白”，称为受体结合因子-1（RBF-B）。 1），基于其产生特异性、高亲和力PR结合的能力， 结果表明，它是一个独特的10 kD蛋白， 与其他核蛋白同源。 我们还报告了一个核 使用免疫组织化学技术在许多禽类和大鼠组织中定位禽类样RBF-1， 免疫组织化学测定。 RBF-1和PR在选择的细胞中的共定位 在鸟类输卵管和大鼠卵巢和子宫中也发现了细胞类型。 Southwestern印迹分析表明，RBF与特定的DNA结合 c-myc核原癌基因启动子区结合元件 其mRNA水平被Pg迅速降低（约15分钟）。 最近被鉴定为MAR样（AT丰富）64 bp结构域，侧翼为GC- 丰富的序列。 RBF-1的全长cDNA已被分离和使用 为了鉴定一个0.7 kb的mRNA，其在禽类输卵管中的水平受到调节， 在体内通过类固醇。 RBF-1的基因组序列已被分离， 显示含有4个外显子，以及推定的SR和热休克反应 在5'侧翼区的元件。 初步研究表明， RBF在人MCF-7细胞中的过表达抑制c-myc基因 启动子活性，并且该活性进一步被类固醇抑制。 研究正在进行中，以继续1）类固醇和热量的分析 RBF基因表达的冲击调控; 2）对RBF基因表达的分析。 RBF及其DNA结合元件的生物学功能， 确定a）从启动子中删除所述元件; B） 增加，和c）降低（使用反义寡核苷酸）RBF 表达对Pg受体核结合，以及稳态 内源性c-myc基因表达的类固醇调节和c-myc 启动子活性; 3）结构表征RBF-1-DNA元件 复合物，以及4）鉴定人中的同源RBF-1 mRNA/蛋白质 细胞 这种RBF核基质结构可以解释类固醇如何抑制 c-myc和其他核原癌基因的转录。

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.