Abstract
Transcriptional regulation involves complex and dynamic protein–DNA interactions, which alter chromatin states and, consequently, regulate gene expression. In plants, current technologies face challenges in efficiently capturing dynamically DNA-binding proteins, especially transcription factors. Here, by leveraging the binding ability of dead Cas9 to specific DNA fragments and the labelling capacity of the TurboID protein for adjacent proteins, we have developed a CRISPR-based sequence proximity binding protein labelling system (CSPL) to detect promoter-binding proteins. Using this approach, we identified both known and novel upstream binding proteins on the PIF4 promoter in Arabidopsis, cabbage and rice. This demonstrates the powerful capabilities and broad potential applications of CSPL for detecting promoter-binding proteins in plants.
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Data availability
The LC–MS/MS data have been deposited to Integrated Proteome Resources with the data identifier IPX0013570001. The sequence data for PIF4 (At2G43010), TCP14 (AT3G47620), GTL2 (AT1G33240), FHY3 (AT3G22170), LSH3 (AT2G31160) and OsPIF4 (LOC127767328) are available via NCBI GeneBank, while those for BolPIF4 (BolC03g026110.2J), BolGTL2 (BolC08g010870.2J), BolTGA6 (BolC05g054340.2J) and BolC3H (BolC05g063020.2J) are available via BRAD (http://brassicadb.cn/#/). Source data are provided with this paper. All other supporting data for this study are available in the Supplementary Information.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (NSFC grant nos 32302570 to L.Z., 32402574 to C.C. and 31972411 to F.C.), the Agricultural Science and Technology Innovation Program (ASTIP grant nos JCKJ2025-CG-03 to F.C. and Y2024QC05 to K.Z.), and the Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture and Rural Affairs, China. We thank the Key Facility Center of ICS, CAAS, for LC–MS/MS measurement.
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Contributions
F.C. conceived and designed the study. L.Z. performed the experiments. C.C. conducted the bioinformatics analyses. Q.C. and X.T. prepared and collected the samples. K.Z. and S.C. helped with the data interpretation. L.Z. and C.C. mainly wrote the manuscript. F.C., L.Z. and C.C. revised the manuscript.
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Nature Plants thanks Bo Sun, Lun Zhao and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 Verification of PIF4 promoter-binding proteins in Arabidopsis thaliana.
(a) Diagram of the primers position for LUC/REN assay and Y1H assay in the PIF4 promoter. (b) Diagram to show the probes for EMSA assay and cis-elements of PIF4 promoter-binding candidate proteins. (c) Diagrams of constructs used for the LUC/REN assay. The PIF4 promoter was used as the reporter and FHY3 or LSH3 as the effectors. Meanwhile, the empty vector pGreen II 62-SK was used as mock control. (d) The results of the LUC/REN assay showed that both FHY3 and LSH3 inhibit the PIF4 expression (n = 12). Unpaired t-test (two tailed) was used to evaluate the statistical significance. The data are reported as the mean ± SD. Boxes represent the interquartile range of data, whiskers mean the 5-95 pecentile, and shading indicates the density profile. (e) The Y1H assay validated the regulatory abilities of FHY3 or LSH3 to the PIF4 promoter. AbA: Aureobasidin A. The empty vector pGAD-T7 was set as the ‘Mock’ control. SD-UL+ represents the SD-UL medium supplied with certain amount of AbA. (f, g) EMSA assessing the binding ability of FHY3 or LSH3 to the PIF4 promoter. ‘Competitor’ represents unlabeled probes and ‘Mut’ represents the mutated probes. (h) ChIP-qPCR assessing the binding abilities of TCP14-GFP, GTL-GFP, FHY3-GFP, and LSH3-GFP to the PIF4 promoter in Arabidopsis protoplast (set as “Treated”). The empty GFP was set as “Control”. Relative fold enrichment was calculated as Treated/Control or Control/Control (n = 4). Unpaired t-test (two tailed) was used to evaluate the statistical significance. The data are reported as the mean ± SD. Figure created with BioRender.com.
Extended Data Fig. 2 Identification and verification of BolPIF4 promoter-binding proteins in Brassica oleracea (cabbage).
(a) Diagram of the target site in the BolPIF4 promoter. (b) Schematic of cabbage leaf treatment. (c) Venn analysis of proteins captured in different treatments using LC-MS/MS. BolT0 and BolT1 represent the non-target and BolT1 target of the BolPIF4 promoter, while 0B and 200B represent the 0 μM and 200 μM biotin treatments, respectively. The red number indicates the proteins selected for further analysis. (d) Identification of nuclear-localized proteins by DeepLoc 2.1 and transcription factors (TFs) by PlantTFDB. Previously reported and newly identified TFs in this study are shown below the circle. (e) Diagrams of constructs used for the LUC/REN assay. The BolPIF4 promoter was used as the reporter and BolGTL2, BolTGA6 or BolC3H as the effectors. Meanwhile, the empty vector pGreen II 62-SK was used as mock control. (f) The results of the LUC/REN assay showed that BolGTL2, BolTGA6, and BolC3H activate the BolPIF4 expression (n = 13-15). Unpaired t-test (two tailed) was used to evaluate the statistical significance. The data are reported as the mean ± SD. Boxes represent the interquartile range of data, whiskers mean the 5-95 pecentile, and shading indicates the density profile. (g) The Y1H assay validated the regulatory abilities of BolGTL2, BolTGA6, and BolC3H to the BolPIF4 promoter. AbA: Aureobasidin A. SD-UL+ represents the SD-UL medium supplied with certain amount of AbA. (h–j) EMSA assessing the binding ability of BolGTL2, BolTGA6, and BolC3H to the BolPIF4 promoter. ‘Competitor’ represents unlabeled probes and ‘Mut’ represents the mutated probes. The experiment was repeated two times independently with similar results. Figure created with BioRender.com.
Extended Data Fig. 3 Western Blot of purified biotinylated proteins in different samples.
BolT0-200B, BolT1-200B, and BolT1-0B represent purified biotinylated proteins before IP-MS. The experiment was repeated two times independently with similar results. Figure created with BioRender.com.
Extended Data Fig. 4 Venn analysis of proteins captured in BolT1-200B and AC12-200B using LC-MS/MS.
BolT1 represents the BolT1 targeting the BolPIF4 promoter, while the AC12 means an unrelated region targeting control. 200B represents the 200 μM biotin treatments. Figure created with BioRender.com.
Extended Data Fig. 5 Identification of OsPIF4 promoter-binding proteins in Oryza sativa (rice).
(a) Scheme of the CSPL vector for monocot plants ‘dCas9-TDmo’. (b) Schematic of rice leaf treatment. (c) Venn analysis of proteins captured in different treatments using LC-MS/MS. OsT0 and OsT1 represent the non-target and OsT1 of the OsPIF4 promoter, while 0B and 200B represent the 0 μM and 200 μM biotin treatments, respectively. The red number indicates the proteins selected for further analysis. (d) Identification of nuclear-localized proteins by DeepLoc 2.1 and TFs by PlantTFDB. (e) The Y1H assay validated the regulatory abilities of the four candidate TFs to the OsPIF4 promoter. AbA: Aureobasidin A. SD-UL+ represents the SD-UL medium supplied with certain amount of AbA. Figure created with BioRender.com.
Supplementary information
Supplementary Information (download PDF )
Supplementary Sequences 1 and 2, Tables 1–4 and Methods.
Source data
Source Data Fig. 2e,i and Extended Data Figs. 1d,h and 2f (download XLSX )
Statistical source data for Fig. 2e,i and Extended Data Figs. 1d,h and 2f.
Source Data Extended Data Fig. 3 (download PDF )
Unprocessed western blots.
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Zhang, L., Cai, C., Chen, Q. et al. A CRISPR-based sequence proximity binding protein labelling system for scanning upstream regulatory proteins. Nat. Plants 12, 277–283 (2026). https://doi.org/10.1038/s41477-025-02212-5
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DOI: https://doi.org/10.1038/s41477-025-02212-5
