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

Nature Chemistry volume 15, pages 803–814 (2023)Cite this article

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Abstract

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.

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Fig. 1: Schematic representation of the current covalent DNA-centric approaches and the design of the LIKE-XL covalent capture strategy.
Fig. 2: Design and synthesis of DNAo-NBA conjugate, and the conjugation of ssDNAo-NBA with primary-amine-containing molecules.
Fig. 3: LIKE-XL strategy for covalent capture of DNA–histone and specific DNA–TF interactions.
Fig. 4: Profiling of specific DNA–TF interactions by o-NBA-probe-based competitive crosslinking pull-down.
Fig. 5: Comparison of o-NBA probe in LIKE-XL pull-down with other photo-crosslinker-based probes for global profiling.
Fig. 6: Time-resolved profiling of dynamic alterations of o-NBA-probe-enriched TFs from SAHA-treated K562 cells.

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Data availability

All data in support of the findings of this study are available within the article and in the Supplementary Information. All relevant MS data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD039229. Source data are provided with this paper.

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Acknowledgements

We thank Y. Liu from Fudan University for assistance with the CUT&Tag sequencing data analysis. This study was supported by the National Science Foundation of China (92053106 to X.-H.C., 22225702 to M.T., 92153302 to M.T., 22177120 to X.-H.C., 81821005 to M.T. and 21907100 to H.H.); the National Key Science & Technology Program of China (2020YFE0202200 to M.T.); the Program of Shanghai Academic Research Leader (22XD1420900 to M.T.); the Innovative Research Team of High-Level Local Universities in Shanghai (SHSMU-ZDCX20212700 to M.T.); Youth Innovation Promotion Association of the Chinese Academy of Sciences (H.H.); and the Guangdong High-Level Innovative Research Institute (2021B0909050003 to M.T.).

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Author notes

  1. These authors contributed equally: An-Di Guo, Ke-Nian Yan, Hao Hu.

Authors and Affiliations

  1. State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

    An-Di Guo, Ke-Nian Yan, Hao Hu, Linhui Zhai, Haixia Su, Yijia Chi, Yechun Xu, Dongxin Zhao, Xiaojie Lu, Minjia Tan & Xiao-Hua Chen

  2. University of Chinese Academy of Sciences, Beijing, China

    An-Di Guo, Ke-Nian Yan, Yijia Chi, Dongxin Zhao, Minjia Tan & Xiao-Hua Chen

  3. School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, China

    Teng-Fei Hu & Xiao-Hua Chen

  4. State Key Laboratory of Medical Genomics, National Research Center for Translational Medicine at Shanghai, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Jinyin Zha & Jian Zhang

  5. School of Food Science and Technology, Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province, Jiangnan University, Wuxi, China

    Yong-Jiang Xu

  6. Zhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zhongshan, China

    Minjia Tan

  7. College of Pharmacy, Jiangsu Ocean University, Lianyungang, China

    Minjia Tan

  8. School of Pharmaceutical Science and Technology, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, China

    Xiao-Hua Chen

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Contributions

A.-D.G. performed the photo-crosslinking of DNA probes with TAMRA-NH2, peptides and recombinant TFs; crosslinking pull-down of probe-interacting proteins from nuclear extracts; western blots; and preparation of the RNA-seq sample and cell culture. K.-N.Y. modified the DNA probes, synthesized TAMRA-NH2, performed the photo-crosslinking of model reactions and DNA with histones and performed and analysed conjugation products. H.H. performed the proteomics sample preparation, LC-MS/MS experiments, MS data analysis and bioinformatics analysis. L.Z. assisted in the LC-MS/MS experiments and data analysis. T.-F.H. assisted in the photo-crosslinking of model reactions and analysed the conjugation products. H.S. and Y.X. performed the ITC assays. Y.C. and D.Z. performed the analysis of the CUT&Tag data. X.L. assisted in the MS analysis of the DNA probes. J. Zha and J. Zhang performed molecular docking. Y.-J.X. assisted in the data analysis. X.-H.C. and M.T. directed and supervised the project and analysed the data. X.-H.C., A.-D.G., K.-N.Y., H.H. and M.T. wrote the manuscript with collaboration from the other authors.

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Correspondence to Minjia Tan or Xiao-Hua Chen.

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Extended data

Extended Data Fig. 1 Design of LIKE-XL strategy for temporal and global profiling of DNA-protein interactions.

a, Features of lysine side chain at protein-nucleic acid interfaces. b, Schematic of the LIKE-XL workflow for temporal and global profiling of DNA-protein interactions.

Extended Data Fig. 2 Comparison of o-NBA probe based covalent LIKE-XL pulldown and non-covalent affinity pulldown.

a, The schematics of pulldown workflow. Nuclear extracts were incubated with o-NBA probe (crosslinking pulldown and control 1) or control probe (control 2) for 30 min at 4 °C. Then, crosslinking pulldown and control 2 were treated with UV-365 irradiation for 15 min at 4 °C, and control 1 did not undergo light-treatment. All samples were incubated at 25 °C. for another 30 min, and subsequently for SDS-PAGE analysis or on-bead digestion followed by LC-MS/MS analysis. b, Silver staining. c, Overlap of identified proteins. d, MS signal intensity plots of identified proteins for each samples in o-NBA probe related experiments. e, The structure and sequence of ETS-TF o-NBA probe. f, Silver staining. g, Overlap of identified proteins. h, MS signal intensity plots of identified proteins for each samples in ETS-TF o-NBA probe related experiments. In d and h, Y-axis: log-transformed MS intensities, X-axis: protein number, and each dot represents a protein. The proteins were numbered according to intensity ranks, for example the one with the highest MS intensity is number 1#. Compared with crosslinking pulldown groups (P), there were much less bands (in silver staining, b, f) and proteins (from LC-MS/MS analysis, c, d, g, h) in two controls (C1 and C2). In addition, LIKE-XL pulldown performed at even much lower probe concentration (for example 5 μM) not only successfully retrieved the majority of proteins identified in non-covalent affinity pulldown (performed at 50 μM of probe), but also identified much more additional proteins.

Source data

Extended Data Fig. 3 Screening for target proteins of o-NBA probe.

Single-shot LC-MS/MS analysis identified 1,200–1,600 in each replicate. Venn diagrams show comparison of proteins identified from pulldown groups and competition groups for replicate 1 (a) and replicate 2 (c). The log2(pulldown/competition) of proteins commonly identified in both groups are shown in scatter plot (b and d). Proteins uniquely identified in pulldown groups (brown) or with log2(pulldown/competition) > 3 (red) are considered as ‘enriched proteins’ of o-NBA probe (e). That is, there are 229 and 223 proteins enriched by o-NBA probe in replicate 1 and 2, respectively. Overlap of the enriched proteins from replicate 1 and 2 are then considered as potential targets (n = 129) of o-NBA probe (see main text Fig. 4d).

Extended Data Fig. 4 Global profiling of specific DNA-TF interactions from HCT116 cells by o-NBA probe-based competitive crosslinking pulldown.

The experiments were performed in the same condition as in Fig. 4 in the main text. a, Silver staining of the pulldown and competition samples, with the red arrows indicating protein captured. b, Overlap of proteins enriched (for criterion, see Extended Data Fig. 3) in each duplicate. Proteins in the yellow-highlighted area (n = 112) are considered as target binders of the o-NBA probe. c, Fraction of target TFs of different types. d, PWM-energy based scores (a proxy of binding affinity) of target bHLH-TFs and HD- TFs.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–25, Tables 1 and 2, experimental details, synthesis and characterization data, and unprocessed scans of blots and gels for supplementary figures.

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Supplementary Data

Supplementary Data 1. The o-NBA-probe-enriched proteins from the competitive crosslinking pull-down and TFs identified by RNA-seq (HeLa cells). Supplementary Data 2. The o-NBA-probe-enriched proteins from the competitive crosslinking pull-down (HCT116 cells). Supplementary Data 3. The Diz-probe-enriched proteins from the competitive crosslinking pull-down. Supplementary Data 4. The Azi-probe-enriched proteins from the competitive crosslinking pull-down. Supplementary Data 5. Comparison of specifically binding TFs by the o-NBA probe, Diz probe and Azi probe. Supplementary Data 6. Enriched TFs from SAHA-treated K562 cells. Supplementary Data 7. Enriched TFs from CDK9-degrader-treated MV-4-11 cells.

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Source Data Fig. 2

Unprocessed gels.

Source Data Fig. 3

Unprocessed gels and statistical source data.

Source Data Fig. 4

Unprocessed gels.

Source Data Fig. 6

Unprocessed western blots.

Source Data Extended Data Fig. 2

Unprocessed gels.

Source Data Extended Data Fig. 4

Unprocessed gels.

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Guo, AD., Yan, KN., Hu, H. et al. Spatiotemporal and global profiling of DNA–protein interactions enables discovery of low-affinity transcription factors. Nat. Chem. 15, 803–814 (2023). https://doi.org/10.1038/s41557-023-01196-z

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