Department of Chemistry, University of Cambridge, Cambridge, UK.
Cancer Research UK Cambridge Institute, Li Ka Shing Centre, University of Cambridge, Cambridge, UK.
Nat Chem. 2021 Jul;13(7):626-633. doi: 10.1038/s41557-021-00736-9. Epub 2021 Jun 28.
DNA-protein interactions regulate critical biological processes. Identifying proteins that bind to specific, functional genomic loci is essential to understand the underlying regulatory mechanisms on a molecular level. Here we describe a co-binding-mediated protein profiling (CMPP) strategy to investigate the interactome of DNA G-quadruplexes (G4s) in native chromatin. CMPP involves cell-permeable, functionalized G4-ligand probes that bind endogenous G4s and subsequently crosslink to co-binding G4-interacting proteins in situ. We first showed the robustness of CMPP by proximity labelling of a G4 binding protein in vitro. Employing this approach in live cells, we then identified hundreds of putative G4-interacting proteins from various functional classes. Next, we confirmed a high G4-binding affinity and selectivity for several newly discovered G4 interactors in vitro, and we validated direct G4 interactions for a functionally important candidate in cellular chromatin using an independent approach. Our studies provide a chemical strategy to map protein interactions of specific nucleic acid features in living cells.
DNA-蛋白质相互作用调节关键的生物过程。鉴定与特定功能基因组位置结合的蛋白质对于在分子水平上理解潜在的调控机制至关重要。在这里，我们描述了一种共结合介导的蛋白质谱分析（CMPP）策略，用于研究天然染色质中 DNA 四链体（G4s）的互作组。CMPP 涉及可渗透细胞的、功能化的 G4 配体探针，该探针可与内源性 G4 结合，并随后在原位交联到共结合的 G4 相互作用蛋白上。我们首先通过体外接近标记 G4 结合蛋白来证明 CMPP 的稳健性。然后，我们在活细胞中使用这种方法，从各种功能类别中鉴定了数百种可能的 G4 相互作用蛋白。接下来，我们在体外证实了几种新发现的 G4 相互作用蛋白具有高的 G4 结合亲和力和选择性，并且我们使用独立的方法验证了细胞染色质中一个功能重要的候选物的直接 G4 相互作用。我们的研究提供了一种在活细胞中绘制特定核酸特征的蛋白质相互作用图谱的化学策略。
