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
