PDF(Pubmed)
Abstract：
Transcription factors (TFs) regulate gene expression by recognizing specific target enhancers in the genome. The DNA-binding and regulatory activity of TFs depend on the presence of additional protein partners, leading to the formation of versatile and dynamic multimeric protein complexes. Visualizing these protein-protein interactions (PPIs) in the nucleus is key for decrypting the molecular cues underlying TF specificity in vivo. Over the last few years, Bimolecular Fluorescence Complementation (BiFC) has been developed in several model systems and applied in the analysis of different types of PPIs. In particular, BiFC has been applied when analyzing PPIs with hundreds of TFs in the nucleus of live Drosophila embryos. However, the visualization of PPIs at the level of specific target enhancers or genomic regions of interest awaits the advent of DNA-labelling methods that can be coupled with BiFC. Here, we present a novel experimental strategy that we have called BiFOR and that is based on the coupling of BiFC with the bacterial ANCHOR DNA-labelling system. We demonstrate that BiFOR enables the precise quantification of the enrichment of specific dimeric protein complexes on target enhancers in Drosophila salivary gland nuclei. Given its versatility and sensitivity, BiFOR could be applied more widely to other tissues during Drosophila development. Our work sets up the experimental basis for future applications of this strategy.
摘要：
转录因子(TF)通过识别基因组中的特定靶增强子来调节基因表达。TFs的DNA结合和调节活性取决于其他蛋白质伴侣的存在，导致形成多功能和动态多聚体蛋白复合物。可视化细胞核中的这些蛋白质-蛋白质相互作用（PPIs）是解密体内TF特异性的分子线索的关键。在过去的几年里，双分子荧光互补（BiFC）已在多个模型系统中开发并应用于不同类型的PPI的分析。特别是,在分析活果蝇胚胎细胞核中具有数百个TF的PPI时，已应用BiFC。然而,PPIs在特定靶增强子或感兴趣的基因组区域水平的可视化等待着可以与BiFC结合的DNA标记方法的出现.这里,我们提出了一种称为BiFOR的新实验策略，该策略基于BiFC与细菌锚定DNA标记系统的偶联。我们证明BiFOR能够精确定量果蝇唾液腺核中目标增强剂上特定二聚体蛋白质复合物的富集。鉴于其多功能性和灵敏度，在果蝇发育过程中，BiFOR可以更广泛地应用于其他组织。我们的工作为该策略的未来应用奠定了实验基础。
