CHROMATIN ACCEPTOR SITES FOR PROGESTERONE

这笔赠款的长期目标是确定地点， 核受体部位的组成和生物功能(即， 禽输卵管孕酮(PG)受体的核结合部位 (公关)。类固醇受体复合体(SR)已被报道在体外结合 并在体内与多种核基质中的特定受体部位结合 目标细胞的数量。在过去这笔赠款的资助期内，我们发现 禽染色质受体和核基质的这些部位 输卵管公关要合而为一。此外，我们提纯了一种核基质 禽类PR的“受体蛋白”，称为受体结合因子1(RBF-1)。 1)基于其生成特定的、高亲和力的PR结合的能力 禽类基因组DNA，表明它是一种独特的10kD蛋白，具有一些 与其他核蛋白同源。我们还报告了一枚核弹 类禽RBF-1在多种禽类和大鼠组织中的定位 免疫组织化学分析。RBF-1和PR在SELECT中的共定位 在禽类输卵管、大鼠卵巢和子宫中也发现了不同类型的细胞。 西南印迹分析表明，rbf与特定的dna结合。 原癌基因c-myc启动子区域的结合元件 PG可使其mRNA水平迅速降低(约15分钟)。此元素 最近发现了一个类似MAR(富含AT)的BP结构域，其两侧有GC- 丰富的序列。Rbf-1的全长cDNA已被分离并用于 鉴定一个在禽类输卵管中表达受调控的0.7kb的mRNA 在体内使用类固醇。RBF-1的基因组序列已被分离和 显示包含4个外显子，以及推测的SR和热休克反应 5‘侧翼区域中的分子。初步研究表明， RBF在人MCF-7细胞中的过表达抑制c-myc基因 启动子活性和这一活性被类固醇进一步抑制。 研究正在进行中，以继续1)类固醇和热量的分析 休克对RBF基因表达的调节；2)分析 红细胞及其DNA结合元件的生物学功能(S) 确定a)从启动子中删除该元件的效果；b) 增加，以及c)降低(使用反义寡核苷酸)RBF PG受体的核结合及稳态表达 类固醇对内源性c-myc基因表达和c-myc的调节 启动子活性；3)RBF-1-DNA元件的结构特征 4)在人中鉴定出一个同源的RBF-1mRNA/蛋白 细胞。这种RBF核基质结构可以解释类固醇是如何抑制 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.