Spatiotemporal and global profiling of DNA-protein interactions and substrates of lysine-modifying enzymes in living cells

Precise dissection of DNA-protein and enzyme-substrate interactions is essential for elucidating the recognition basis, dynamics, signaling transduction and gene regulation mechanism. However, global profiling of weak and dynamic DNA-protein and enzyme-substrate interactions remains a long-standing challenge. Here, we establish a 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. Using a similar light-induced chemical crosslinking tool, we further develop a strategy to directly capture substrates of lysine-modifying enzymes via PTM-acceptor residue crosslinking in living cells, enabling global profiling of substrates of PTM-enzymes and validation of PTM-sites in a straightforward manner. By integrating enzymatic PTM-mechanisms, and genetically encoding residue-selective photo-crosslinker into PTM-enzymes, our strategy expands the substrate profiles of both bacterial and mammalian lysine acylation enzymes, including bacterial lysine acylases PatZ, YiaC, LplA, TmcA, and YjaB, as well as mammalian acyltransferases GCN5 and Tip60, leading to discovery of distinct yet functionally important substrates and acylation sites. Together, our strategies offer complementary approaches to expand the DNA-protein profiling and substrates of PTM-enzyme identification that were previously inaccessible via non-covalent strategies.

References:

Hu H, et al. Spatiotemporal and direct capturing global substrates of lysine-modifying enzymes in living cells. Nat Commun. 2024, 15:1465.Guo A, et al. Spatiotemporal and global profiling of DNA-protein interactions enables discovery of low-affinity transcription factors. Nat Chem 2023, 15:803-814

Keywords: light-induced crosslinking; DNA-protein interactions; transcription factors; lysine-modifying enzyme substrates; proteomics analysis