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Spatiotemporal and global profiling of DNA-protein interactions enables discovery of low-affinity transcription factors

ABSTRACT: Precise dissection of DNA-protein interactions is essential for elucidating 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 light-induced lysine (K) enabled crosslinking (LIKE-XL) strategy for spatiotemporal and global profiling of DNA-protein interactions. Harnessing the unique abilities to capture weak and transient DNA-protein interactions, we demonstrate that LIKE-XL enables discovery of low-affinity transcription factors (TFs)-DNA interactions via sequence-specific DNA baits, determining the binding sites for TFs previously unachieved. More importantly, we successfully decipher the dynamics of TF subproteome in response to drug treatment in time-resolved manner, and find new downstream target TFs from drug perturbations, providing insights into their dynamic transcriptional networks. The LIKE-XL offers a novel and complementary method to expand the DNA-protein profiling toolbox and map accurate DNA-protein interactomes previously inaccessible via noncovalent strategies, for better understanding of protein functions in health and disease.

ORGANISM(S): Homo Sapiens

SUBMITTER: Minjia Tan

PROVIDER: PXD039229 | iProX | Tue Jan 03 00:00:00 GMT 2023

REPOSITORIES: iProX

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Spatiotemporal and global profiling of DNA-protein interactions enables discovery of low-affinity transcription factors.

Guo An-Di AD Yan Ke-Nian KN Hu Hao H Zhai Linhui L Hu Teng-Fei TF Su Haixia H Chi Yijia Y Zha Jinyin J Xu Yechun Y Zhao Dongxin D Lu Xiaojie X Xu Yong-Jiang YJ Zhang Jian J Tan Minjia M Chen Xiao-Hua XH

Nature chemistry 20230427 6

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 ...[more]

PMID: 37106095

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Project description:The Nuclear Factor I (NFI) family of transcription factors (TFs) plays key roles in cellular differentiation, proliferation, and homeostasis. As such, NFI family members engage in large number of interactions with other proteins and the chromatin. However, despite their well-established significance, the NFIs interactomes, their dynamics, and their functions have not been comprehensively examined. Here, we employed complementary omics-level techniques, i.e. interactomics (affinity purification mass spectrometry (AP-MS) and proximity-dependent biotinylation (BioID)), and chromatin immunoprecipitation sequencing (ChIP-Seq), to obtain a comprehensive view of the NFI proteins and their interactions. Our analyses included all four main NFI family members, and a less studied short isoform of NFIB (NFIB4), which lacks the DNA binding domain. We observed that, despite exhibiting some redundancy, each family member had unique high-confidence interactors and target genes, highlighting distinct roles within the transcriptional regulatory networks. The study revealed that NFIs interact with a large number of other TFs to co-regulate a broad range of regulatory networks and cellular processes. Notably, time-dependent proximity-labeling unveiled a highly dynamic nature of NFI protein-protein interaction networks and hinted at temporal modulation of NFI interactions. Furthermore, gene ontology (GO) enrichment analysis of NFI interactome and targetome revealed the involvement of NFIs in transcriptional regulation, chromatin organization, and cellular signaling pathways, and pathways related with cancer. Additionally, we observed that NFIB4 engages with proteins associated with mRNA regulation, which suggests that NFIs have roles beyond traditional DNA binding and transcriptional modulation. We propose that NFIs serve as pioneering TFs, playing critical roles in regulating other TFs and influencing a wide range of cellular processes. These insights into NFI protein-protein interactions and their dynamic, context-dependent nature provide a deeper understanding of gene regulation mechanisms and hint at the role of NFIs as master regulators.

Dataset Information

Spatiotemporal and global profiling of DNA-protein interactions enables discovery of low-affinity transcription factors

Publications

Spatiotemporal and global profiling of DNA-protein interactions enables discovery of low-affinity transcription factors.

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