Identification of Transactivation, Repression, and Protein-Protein Interaction Domains Using GAL4-Fusion Proteins

The Epstein-Barr viral nuclear antigen 2 (EBNA2) latency protein is a transcriptional activator that plays a critical role in regulation of Epstein-Barr virus (EBV) latency gene expression and in EBV-induced B-cell immortalization. GAL4-fusion constructions have been instrumental in elucidating the mechanism of action of EBNA2 (1 –4 ). Regulatory proteins are modular in structure with separate domains for functions such as promoter targeting, transcriptional activation, transcriptional repression, and protein-protein interactions. Functional chimeras can be generated by fusing domains from different proteins. In the case of transcriptional activation, the transactivator protein must be targeted to the responsive promoter and contain an activation domain that either directly or indirectly interacts with the core cellular transcriptional machinery to increase transcriptional efficiency. Early work on transcriptional transactivators demonstrated the modular nature of these proteins by showing that the DNA binding domain of the yeast transcriptional activator GAL4, GAL4 (aa1-147) could be fused to a variety of yeast, cellular, bacterial, and viral sequences to reconstitute functional transcriptional activator proteins (5 ,6 ). Subsequently, GAL4-fusions became the standard experimental approach for identifying activation domains.