[HTML][HTML] Assembly requirements of PU. 1–Pip (IRF‐4) activator complexes: inhibiting function in vivo using fused dimers

AL Brass, AQ Zhu, H Singh - The EMBO journal, 1999 - embopress.org
AL Brass, AQ Zhu, H Singh
The EMBO journal, 1999embopress.org
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; …
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 α‐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 λ 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.
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