Assembly requirements of PU.1-Pip (IRF-4) activator complexes: inhibiting function in vivo using fused dimers.
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.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.
Mesh Terms:
Amino Acid Sequence, Animals, B-Lymphocytes, DNA-Binding Proteins, Dimerization, Enhancer Elements, Genetic, Gene Expression Regulation, Immunoglobulin lambda-Chains, Interferon Regulatory Factors, Mice, Molecular Sequence Data, Mutation, Protein Engineering, Proto-Oncogene Proteins, Recombinant Fusion Proteins, Repressor Proteins, Sequence Alignment, Trans-Activators, Transcription Factors, Transcriptional Activation, Transfection, Tumor Cells, Cultured
Amino Acid Sequence, Animals, B-Lymphocytes, DNA-Binding Proteins, Dimerization, Enhancer Elements, Genetic, Gene Expression Regulation, Immunoglobulin lambda-Chains, Interferon Regulatory Factors, Mice, Molecular Sequence Data, Mutation, Protein Engineering, Proto-Oncogene Proteins, Recombinant Fusion Proteins, Repressor Proteins, Sequence Alignment, Trans-Activators, Transcription Factors, Transcriptional Activation, Transfection, Tumor Cells, Cultured
EMBO J.
Date: Feb. 15, 1999
PubMed ID: 10022840
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