Precise dissection of DNA–protein interactions is essential for elucidating the recognition basis, dynamics and gene regulation mechanism. However, global profiling of weak and dynamic DNA–protein interactions remains a long-standing challenge. Here, we establish the light-induced lysine (K) enabled crosslinking (LIKE-XL) strategy for spatiotemporal and global profiling of DNA–protein interactions. Harnessing unique abilities to capture weak and transient DNA–protein interactions, we demonstrate that LIKE-XL enables the discovery of low-affinity transcription-factor/DNA interactions via sequence-specific DNA baits, determining the binding sites for transcription factors that have been previously unknown. More importantly, we successfully decipher the dynamics of the transcription factor subproteome in response to drug treatment in a time-resolved manner, and find downstream target transcription factors from drug perturbations, providing insight into their dynamic transcriptional networks. The LIKE-XL strategy offers a complementary method to expand the DNA–protein profiling toolbox and map accurate DNA–protein interactomes that were previously inaccessible via non-covalent strategies, for better understanding of protein function in health and disease.

精确剖析 DNA-蛋白质相互作用对于阐明识别基础、动力学和基因调控机制至关重要。然而，弱动态 DNA-蛋白质相互作用的全局分析仍然是一个长期存在的挑战。在这里，我们建立了光诱导赖氨酸 (K) 启用的交联 (LIKE-XL) 策略，用于 DNA-蛋白质相互作用的时空和全局分析。利用独特的能力捕捉微弱和瞬时的 DNA-蛋白质相互作用，我们证明 LIKE-XL 能够通过序列特异性 DNA 诱饵发现低亲和力的转录因子/DNA 相互作用，确定之前已经发现的转录因子的结合位点未知。更重要的是，我们以时间分辨的方式成功破译了转录因子亚蛋白质组响应药物治疗的动力学，并从药物扰动中找到下游目标转录因子，从而深入了解它们的动态转录网络。LIKE-XL 策略提供了一种补充方法来扩展 DNA-蛋白质分析工具箱并绘制准确的 DNA-蛋白质相互作用组，这些相互作用组以前无法通过非共价策略获得，从而更好地了解蛋白质在健康和疾病中的功能。