Assembly requirements of PU.1-Pip (IRF-4) activator complexes:: inhibiting function in vivo using fused dimers
Assembly requirements of PU.1-Pip (IRF-4) activator complexes:: inhibiting function in vivo using fused dimers
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DOI：
10.1093/emboj/18.4.977
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发表时间：
1999-02-15
期刊：
影响因子：
11.4
通讯作者：
Singh, H
中科院分区：
文献类型：
--
作者：
Brass, AL;Zhu, AQ;Singh, H
高等真核生物中的基因表达似乎受到与调节序列结合的转录因子的特定组合的调节，Ets 因子 PU.1 和 IRF 蛋白 Pip (IRF-4) 代表一对参与调节 B 细胞特异性基因表达的相互作用的转录因子。 Pip 被磷酸化的 PU.1 招募到 DNA 上的结合位点。 PU.1-Pip 相互作用被证明是模板导向的，涉及两个不同的蛋白质-蛋白质相互作用表面：(i) ets 和 IRF DNA 结合结构域； (ii) PU.1 的磷酸化 PEST 区域和 Pip 中需要赖氨酸的推定 α 螺旋。因此，一组协调的蛋白质-蛋白质和蛋白质-DNA 接触对于 PU.1-Pip 三元复合物的组装至关重要。为了分析这些因子在体内的功能，我们设计了嵌合阻遏物，其中包含由灵活的 POU 结构域接头连接的 ets 和 IRF DNA 结合结构域。当稳定表达时，野生型融合二聚体强烈抑制重排的免疫球蛋白 lambda 基因的表达，从而确立了 PU.1-Pip 复合物在 B 细胞基因表达中的功能重要性。对野生型二聚体与一系列突变二聚体的比较分析将受 PU.1 和 Pip 调节的基因与仅受 PU.1 调节的基因区分开来。该策略在分析体内相互作用的转录因子的功能以及识别受此类复合物调节的新基因方面应该普遍有用。
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.