Gene expression in higher eukaryotes appears to be regulated by specific combinations of transcription factors binding to regulatory sequences. The Ets factor PU. 1 and the IRF protein Pip (IRF-4) represent a pair of interacting transcription factors implicated in regulating B cell-specific gene expression. Pip is recruited to its binding site on DNA by phosphorylated PU. 1. PU. 1-Pip interaction is shown to be template directed and involves two distinct protein-protein interaction surfaces: (i) the ets and IRF DNA-binding domains; and (ii) the phosphorylated PEST region of PU. 1 and a lysine-requiring putative alpha-helix in Pip. Thus, a coordinated set of protein-protein and protein-DNA contacts are essential for PU. 1-Pip ternary complex assembly. To analyze the function of these factors in vivo, we engineered chimeric repressors containing the ets and IRF DNA-binding domains connected by a flexible POU domain linker. When stably expressed, the wild-type fused dimer strongly repressed the expression of a rearranged immunoglobulin lambda gene, thereby establishing the functional importance of PU VSports手机版. 1-Pip complexes in B cell gene expression. Comparative analysis of the wild-type dimer with a series of mutant dimers distinguished a gene regulated by PU. 1 and Pip from one regulated by PU. 1 alone. This strategy should prove generally useful in analyzing the function of interacting transcription factors in vivo, and for identifying novel genes regulated by such complexes. .