MacDonald G H, Itoh-Lindstrom Y, Ting J P
Department of Microbiology-Immunology, University of North Carolina at Chapel Hill 27599-7295.
J Biol Chem. 1995 Feb 24;270(8):3527-33. doi: 10.1074/jbc.270.8.3527.
YB-1 is a member of a newly defined family of DNA- and RNA-binding proteins, the Y box factors. These proteins have been shown to affect gene expression at both the transcriptional and translational levels. Recently, we showed that YB-1 represses interferon-gamma-induced transcription of class II human major histocompatibility (MHC) genes (1). Studies in this report characterize the DNA binding properties of purified, recombinant YB-1 on the MHC class II DRA promoter. The generation of YB-1-specific antibodies further permitted an analysis of the DNA binding properties of endogenous YB-1. YB-1 specifically binds single-stranded templates of the DRA promoter with greater affinity than double-stranded templates. The single-stranded DNA binding sites of YB-1 were mapped to the X box, whereas the double-stranded binding sites were mapped to the Y box of the DRA promoter, by methylation interference analysis. Most significantly, YB-1 can induce or stabilize single-stranded regions in the X and Y elements of the DRA promoter, as revealed by mung bean nuclease analysis. In concert with the findings that YB-1 represses DRA transcription, this study of YB-1 binding properties suggests a model of repression in which YB-1 binding results in single-stranded regions within the promoter, thus preventing loading and/or function of other DRA-specific transactivating factors.
YB-1是新定义的DNA和RNA结合蛋白家族Y盒因子的成员之一。这些蛋白已被证明在转录和翻译水平上都会影响基因表达。最近，我们发现YB-1可抑制干扰素γ诱导的人类主要组织相容性复合体（MHC）II类基因的转录（1）。本报告中的研究对纯化的重组YB-1在MHC II类DRA启动子上的DNA结合特性进行了表征。YB-1特异性抗体的产生进一步使得对内源性YB-1的DNA结合特性进行分析成为可能。YB-1与双链模板相比，以更高的亲和力特异性结合DRA启动子的单链模板。通过甲基化干扰分析，YB-1的单链DNA结合位点被定位到X盒，而双链结合位点被定位到DRA启动子的Y盒。最为重要的是，绿豆核酸酶分析表明，YB-1可诱导或稳定DRA启动子X和Y元件中的单链区域。结合YB-1抑制DRA转录这一发现，对YB-1结合特性的这项研究提出了一种抑制模型，即YB-1的结合会导致启动子内出现单链区域，从而阻止其他DRA特异性反式激活因子的加载和/或功能发挥。
