We have developed a method using novel latex beads for rapid identification of drug receptors using affinity purification. Composed of a glycidylmethacrylate (GMA) and styrene copolymer core with a GMA polymer surface, the beads minimize nonspecific protein binding and maximize purification efficiency. We demonstrated their performance by efficiently purifying FK506-binding protein using FK506-conjugated beads, and found that the amount of material needed was significantly reduced compared with previous methods. Using the latex beads, we identified a redox-related factor, Ref-1, as a target protein of an anti-NF-kappaB drug, E3330, demonstrating the existence of a new class of receptors of anti-NF-kappaB drugs. Our results suggest that the latex beads could provide a tool for the identification and analysis of drug receptors and should therefore be useful in drug development.

Muscle cell differentiation, or myogenesis, is a well-characterized process and involves the expression of specific sets of genes in an orderly manner. A prerequisite for myogenesis is the exit from the cell cycle, which is associated with the up-regulation of the tumor suppressor Rb. In this study, we set to investigate the regulatory mechanism of the Rb promoter that allows adequate up-regulation in differentiating myoblasts. We report that Rb expression is regulated by the transcription factors GABP, HCF-1 and YY1. Before induction of differentiation, Rb is expressed at a low level and GABP and YY1 are both present on the promoter. YY1, which exerts an inhibitory effect on Rb expression, is removed from the promoter as cells advance through myogenesis and translocates from the nucleus to the cytoplasm. On the other hand, upon induction of differentiation, the GABP cofactor HCF-1 is recruited to and coactivates the promoter with GABP. RNAi-mediated knock-down of HCF-1 results in inhibition of Rb up-regulation as well as myotube formation. These results indicate that the Rb promoter is subject to regulation by positive and negative factors and that this intricate activation mechanism is critical to allow the accurate Rb gene up-regulation observed during myogenesis.

The transcription factor hGABP/E4TF1 is a heterotetrameric complex composed of two DNA-binding subunits (hGABP␣/E4TF1-60) and two transactivating subunits (hGABP␤/E4TF1-53). In order to understand the molecular mechanism of transcriptional regulation by hGABP, we searched for proteins that interact with the non-DNA-binding subunit, hGABP␤, using yeast two-hybrid screening. We identified a human cDNA encoding a protein related to YAF-2 (YY1-associated factor 2), which was previously isolated as an interacting partner of the Ying-Yang-1 (YY1) transcription factor. Reflecting this similarity, both YAF-2 and this novel protein (named YEAF1 for YY1-and E4TF1/hGABP-associated factor-1) interacted with hGABP␤ and YY1 in vitro and in vivo, indicating that YEAF1 and YAF-2 constitute a cofactor family for these two structurally distinct transcription factors. By using yeast three-hybrid assay, we demonstrated that hGABP␤ and YY1 formed a complex only in the presence of YEAF1, indicating that YEAF1 is a bridging factor of these two transcription factors. These cofactors are functionally different in that YAF-2 positively regulates the transcriptional activity of hGABP but YEAF1 negatively regulates this activity. Also, YAF-2 mRNA is highly expressed in skeletal muscle, whereas YEAF1 mRNA is highly expressed in placenta. We speculate that the transcriptional activity of hGABP is in part regulated by the expression levels of these tissue-specific cofactors. These results provide a novel mechanism of transcriptional regulation by functionally distinct cofactor family members.