Plisov S Y, Poirot M E, Modarress K J, Cavanaugh A H, Edwards D P, Simons S S
Steroid Hormones Section, NIDDK/LMCB, National Institutes of Health, Bethesda, MD, USA.
J Steroid Biochem Mol Biol. 1998 Nov;67(3):251-66. doi: 10.1016/s0960-0760(98)00118-6.
We previously reported evidence for two subpopulations of several classes of steroid receptors that could be distinguished by their requirement of a low molecular weight factor (Mr=700-3000 Da) for binding to nonspecific, calf thymus DNA-cellulose [Cavanaugh, A. H. and Simons Jr., S. S., Journal of Steroid Biochemistry and Molecular Biology, 48, 433-446 (1994)]. This factor appeared to be enriched in (NH4)2SO4 precipitates of nuclear extracts. Using human progesterone receptors (PRs) and biologically active DNA sequences in a modified avidin/biotin-coupled DNA (ABCD) binding assay, we now report a factor-mediated increase in PR binding to specific DNA sites that was indistinguishable from that seen with nonspecific sites. The main advantages of this modified assay are that both kinetic and equilibrium binding of receptor-steroid complexes to DNA can be directly monitored in solution. The ability of either Sephadex G-50 chromatography or sodium arsenite to prevent that binding which is increased by added factor supported the existence of PR subpopulations that are independent of the acceptor DNA sequence. The factor was found, surprisingly, to be low concentrations (> or = 5 mM) of (NH4)2SO4, which anomalously is partially excluded from Sephadex G-10 columns, and can be mimicked by some salts but not sodium arsenite. Kinetic analyses demonstrated that the mechanism of action of salt was to accelerate the rate of binding of PR. Salt also had a much greater effect on the nonspecific binding of PR, such that the ratio of specific to nonspecific DNA binding was greatest at elevated salt concentrations (approximately 75 mM) that afforded submaximal levels of PR binding to specific DNA sites. Further analysis of the DNA-bound receptors revealed that the smaller, A-form of PR is preferentially bound to specific DNA sequences both in the presence and in the absence of various salt concentrations. Thus, the differences in DNA binding of PR +/- salt do not correlate with the preferential binding of A or B isoform. The unequal behavior of PR subpopulations and/or isoforms for binding to specific DNA sequences offers added mechanisms for selective transcriptional regulation of genes in intact cells.
我们之前报道了几类甾体受体存在两个亚群的证据，这两个亚群可通过它们与非特异性小牛胸腺DNA - 纤维素结合时对低分子量因子（分子量为700 - 3000道尔顿）的需求来区分[卡瓦诺，A. H. 和西蒙斯，S. S. 小，《甾体生物化学与分子生物学杂志》，48，433 - 446（1994）]。该因子似乎在核提取物的硫酸铵沉淀中富集。在改良的抗生物素蛋白/生物素偶联DNA（ABCD）结合试验中使用人孕酮受体（PRs）和生物活性DNA序列，我们现在报告了一种因子介导的PR与特定DNA位点结合的增加，这与在非特异性位点观察到的情况无法区分。这种改良试验的主要优点是受体 - 甾体复合物与DNA的动力学和平衡结合都可以在溶液中直接监测。葡聚糖G - 50柱层析或亚砷酸钠阻止添加因子增加的结合的能力支持了与受体DNA序列无关的PR亚群的存在。令人惊讶的是，发现该因子是低浓度（≥5 mM）的硫酸铵，硫酸铵异常地部分被葡聚糖G - 10柱排除，并且可以被一些盐模拟但不能被亚砷酸钠模拟。动力学分析表明盐的作用机制是加速PR的结合速率。盐对PR的非特异性结合也有更大的影响，使得在盐浓度升高（约75 mM）时特异性与非特异性DNA结合的比率最大，此时PR与特定DNA位点的结合水平低于最大值。对结合到DNA上的受体的进一步分析表明，无论是否存在各种盐浓度，较小的A形式的PR都优先结合到特定DNA序列上。因此，PR ± 盐时DNA结合的差异与A或B异构体的优先结合无关。PR亚群和/或异构体在结合特定DNA序列时的不同行为为完整细胞中基因的选择性转录调控提供了额外的机制。
