Spelsberg T C, Lauber A H, Sandhu N P, Subramaniam M
Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, USA.
Recent Prog Horm Res. 1996;51:63-96.
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.
本项目的目标是确定禽类输卵管孕酮（Pg）受体（PR）的核受体位点（即核结合位点）的位置、组成和生物学功能。许多实验室已证明，在多种靶组织系统中，类固醇受体复合物可在细胞核/染色质中与特定受体位点进行类似天然（体内）的无细胞结合。这些位点似乎涉及蛋白质 - DNA复合物，其中一些已被证明存在于核基质中，包括禽类输卵管PR的染色质受体位点。我们已经纯化了一种禽类PR的核基质“受体蛋白”，基于其在禽类基因组DNA上产生特异性、高亲和力PR结合的能力，将其命名为受体结合因子 - 1（RBF - 1）。这种10 kD的核基质蛋白被发现是独特的，与其他几种蛋白质的同源性极低。使用免疫组织化学技术和针对纯化的RBF - 1的抗体，RBF - 1定位于许多禽类和大鼠组织的细胞核中。还报道了RBF - 1和PR在禽类输卵管和大鼠生殖器官的特定细胞类型中的共定位。发现RBF - 1浓度具有组织特异性。已分离出RBF - 1的全长cDNA，并用于鉴定一种0.7 kb的mRNA，其在各种禽类组织中的水平反映了蛋白质水平。已分离并鉴定了RBF - 1的基因组序列。初步研究表明，在人MCF - 7细胞中过表达RBF会导致c - myc基因启动子活性受到抑制，而细胞经类固醇激素处理后这种抑制作用会进一步增强。我们实验室过去的研究表明，在禽类输卵管中，Pg和糖皮质激素可使c - myc mRNA的稳态水平迅速（约15分钟）降低。此外，部分纯化的RBF - 1组分仅在某些动物物种的基因组DNA和c - myc基因上产生特异性PR结合位点，而不在卵清蛋白基因上产生。通过蛋白质印迹分析表明，纯化的RBF - 1可特异性结合c - myc、c - jun原癌基因5'端的序列，但不结合卵清蛋白基因的基因组序列。已确定c - myc原癌基因启动子区域中的一个特异性DNA结合元件是富含AT的同型嘌呤/嘧啶延伸结构域，两侧为富含GC的序列。使用受核基质结构保护的200 bp基质DNA片段进行的Southern印迹分析表明，核基质附着在RBF - 1结合元件的两侧。本文描述了一种附着于c - myc启动子的核基质受体位点模型，该模型可能介导Pg诱导的c - myc基因表达下调。
