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Structural insights into CDF1 accumulation on the CONSTANS promoter via a plant-specific DNA-binding domain

Nature Plants volume 11, pages 836–848 (2025)Cite this article

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Abstract

DNA-binding with one-finger (Dof) proteins are a family of plant-specific transcription factors distinguished by the highly conserved Dof DNA-binding domain. Various members play crucial roles in diverse plant biological processes. However, it remains unclear how the Dof domain recognizes a restricted set of promoters for gene regulation by binding to just four nucleotides, AAAG/CTTT. Here we present the crystal structure of the Dof domain of CYCLING DOF FACTOR 1 (CDF1), a well-characterized Dof protein acting as a transcriptional repressor by binding to the CONSTANS promoter to regulate photoperiodic flowering, in complex with DNA containing two cis elements. The data reveal that the Dof domain exhibits a unique zinc ribbon fold that includes a three-stranded antiparallel β-sheet and a carboxy-terminal loop, enabling DNA recognition accompanied by directional expansion of the major groove. These features facilitate binding to contiguous target cis elements in a proper arrangement to effectively regulate gene expression.

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Fig. 1: Overall structure of the CDF1Dof–DNA complex.
Fig. 2: DNA-binding mode of CDF1Dof.
Fig. 3: Simultaneous binding ability of CDF1Dof towards the two cis elements.
Fig. 4: Effective transcriptional regulation of CDF1 through binding to contiguous cis elements.
Fig. 5: Phenotypic changes resulting from altered CDF1–DNA interactions in planta.
Fig. 6: Model for the efficiency of the transcriptional regulation of CDF1 through recruitment of TPL to different arrangements of cis elements on the CO promoter.

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Data availability

The atomic coordinates and structure factors are available in the PDB under accession number 8XUF for the CDF1Dof–dsDNA complex. The atomic coordinates of ZNF217 bound to DNA, which were used for molecular replacement, are also available in the PDB under accession number 4IS1. Source data are provided with this paper.

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Acknowledgements

We thank N. Mitsuda (AIST, Japan) for providing the pGLHNew_RLH vector and the pDEST–35SHSP vector and K. Ifuku (Kyoto University, Japan) for allowing us to use the necessary equipment to capture fluorescence images of the gels in EMSA. This work was supported by Grants-in-Aid for Scientific Research (no. JP22H04977 to S.Y. and T.M.; nos. JP22K18945, JP23H01818 and JP23H03073 to H.Y.; no. JP23K27467 to Y.I.; and no. JP24KJ1579 to A.T.) and a Grant-in-Aid for Scientific Research on Innovative Areas (no. JP19H04855 to T.M.) from the Japan Society for the Promotion of Science, the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from the Japan Agency for Medical Research and Development (AMED) under grant no. JP24ama121010 (T.S. and T.M.), the Cooperative Research Grant of the Plant Transgenic Design Initiative from Gene Research Center at the University of Tsukuba (no. 2102 to T.M. and S.N.), CiDER from the University of Osaka (H.Y.), and the Frontier Research Grant from the Japan Institute of Metals (H.Y.). We thank T. Senda, Y. Yamada and the beamline staff for the synchrotron radiation experiments performed with the AR-NE3A beamline in the Photon Factory (Tsukuba, Japan) (2023PF-B006), which were supported by BINDS from AMED under grant no. JP24ama121001 (support no. 5013).

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Author notes

  1. These authors contributed equally: Hirotake Furihata, Zhangliang Zhu, Kaisei Nishida, Yasuhito Sakuraba.

Authors and Affiliations

  1. Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan

    Hirotake Furihata, Zhangliang Zhu, Shohei Nosaki, Masaru Tanokura & Takuya Miyakawa

  2. Proteo-Science Center, Ehime University, Matsuyama, Japan

    Hirotake Furihata & Tatsuya Sawasaki

  3. Graduate School of Biostudies, Kyoto University, Kyoto, Japan

    Zhangliang Zhu, Kaisei Nishida, Ryo Tachibana, Ayumi Yamagami, Takeshi Nakano & Takuya Miyakawa

  4. Agro-Biotechnology Research Center, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan

    Yasuhito Sakuraba & Shuichi Yanagisawa

  5. Graduate School of Engineering Science, The University of Osaka, Osaka, Japan

    Akihiro Tsuji, Hayato Yamashita & Masayuki Abe

  6. Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Japan

    Shohei Nosaki

  7. Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto, Japan

    Yoshiki Ikeda

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  1. Hirotake Furihata
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Contributions

T.M., S.Y. and M.T. conceived and designed the project. T.M., S.Y., T.N. and T.S. supervised the experiments. H.F. and Z.Z. performed the protein and DNA preparation as well as the EMSA experiments, with assistance from S.N. H.F. determined the crystal structure with the aid of X-ray diffraction experiments carried out by T.M. Z.Z. collected the ITC data with support from Y.I. K.N. conducted the transient reporter assays with help from R.T. and A.Y. A.T., H.Y. and M.A. performed the HS-AFM experiments and analysed the data. Y.S. and S.Y. carried out the in planta experiments and the associated data analysis. H.F., T.M., S.Y., and M.T. wrote the paper, with important contributions from Z.Z., K.N., Y.S., A.T. and H.Y. All authors have read and approved the final paper.

Corresponding authors

Correspondence to Shuichi Yanagisawa, Masaru Tanokura or Takuya Miyakawa.

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Extended data

Extended Data Fig. 1 The sequence of the CO promoter ranging from ‒550 to ‒1.

The cis elements of the Dof domain are highlighted in yellow.

Extended Data Fig. 2 The structure around the Zn2+-binding site.

a, The Fo‒Fc omit map of the Zn2+-coordinating residues in the two CxxC motifs. The Fo‒Fc electron density is not clearly resolved between each Cys residue and Zn2+ ion due to their close proximity. b, The β-hairpin-like structure of the region containing the first CxxC motif. Dashed lines and a yellow sphere indicate hydrogen bonds and the backbone phosphate group of DNA, respectively.

Extended Data Fig. 3 Sequence alignments of the Dof domains in each subgroup.

Dof5.4 (G6) and Dof5.6 (G7) are aligned with each other, although they belong to different subgroups. A circle and an arrowhead on the sequence indicate residues involved in the interaction with the nucleobase and backbone phosphate group (except for R40) of DNA, respectively. Black lines at the bottom of each alignment indicate two CxxC motifs. Green boxes indicate variable residues in the G1 members. CDF1 corresponds to Dof5.5, which belongs to group II (G2).

Extended Data Fig. 4 The Fo‒Fc omit map of the key structures for the interaction between CDF1Dof and dsDNA.

a, A1 and A2 nucleobases recognition by N71 and N70, respectively. b, A3 and G4 nucleobases recognition by Y68. The cyan sphere represents a water molecule. c, The interaction between four residues and backbone phosphate groups of DNA. d, R95 orientation into the minor groove of DNA. The cyan sphere represents a water molecule.

Extended Data Fig. 5 Topology diagrams of zinc finger (ZF) domains.

The Dof domain observed in the structure of CDF1Dof bound to DNA is compared with the three major groups of ZF domains. The green box and blue arrow represent the α-helix and β-strand, respectively. Two translucent arrows with dashed outlines at the N-terminus of the Dof domain indicate pseudo β-strands.

Extended Data Fig. 6 Structural comparison between CDF1Dof (left) and the eight protein structures coordinating a Zn2+ ion (right).

The regions matching CDF1Dof on the protein structures found with the DALI server (Z-score of 2.0 or higher) are shown in magenta.

Extended Data Fig. 7 Comparison between the DNA-binding modes of CDF1Dof and the NrdR dimer.

a, Two DNA-binding domains of NrdR (magenta and mint) in complex with DNA. The orange sphere indicates a Zn2+ ion. b, CDF1Dof in complex with a DNA whose direction matches the NrdR (magenta)-binding region of DNA in panel a. c, Nucleobase recognition of NrdR. The consensus binding sequence for NrdR is shown below the ribbon model. Dashed lines indicate hydrogen bonds between the residues of NrdR and nucleobases. d, Nucleobase recognition pattern of CDF1Dof, which is different from that of NrdR. e, Sequence alignment of CDF1Dof with the DNA binding domain of NrdR. The secondary structures are superimposed on the top (CDF1Dof) and at the bottom (NrdR) of the alignment. A circle and an arrowhead on the sequence indicate residues involved in the interaction with the nucleobase and backbone phosphate group (except for R95 of CDF1) of DNA, respectively.

Extended Data Fig. 8 No simultaneous binding ability of CDF1Dof toward the two tandem repeats of cis elements without a linker.

The DNA concentration for EMZA experiments was 0.125 μM. The experiment was independently repeated three times with consistent results.

Source data

Extended Data Fig. 9 ITC analysis of the interaction between CDF1Dof and 40-bp DNA fragments containing two tandem repeats of cis elements connected via a 10-base linker.

The upper panel for each experiment displays the representative ITC titration curves (differential power, DP), while the lower panel shows integrated heats (enthalpy, ΔH) of injection (black squares). Each dataset was fitted using the MicroCal origin software with “one binding site” model. The dissociation constant (KD) and the binding molar ratio (N) are listed at the bottom of each set of ITC data, representing the mean ± s.d. from three independent experiments. The DNA sequence highlights the cis element (CTTT) and its mutationally dysfunctional sequence (TCGT), colored in orange and blue, respectively.

Source data

Extended Data Fig. 10 The analysis of high-speed atomic force microscopy (HS-AFM) images of interaction processes between CDF1Dof molecules and DNA fragments with no cis elements, single cis element, and four tandem repeats.

The following procedure was applied to each set of the sequential HS-AFM images. A curve was defined along the DNA fragment in each image using Kappa, a plugin of ImageJ software81 (Red dashed curves in the upper HS-AFM images). Here, an initialization curve was created and fitted to the DNA fragment in each image using an iterative minimization algorithm. For HS-AFM images of DNA with CDF1Dof molecules bound around the centre, the curve was adjusted so that it overlaps the highest position of such molecules. Then, the height profiles along the curves were extracted from each AFM image (bottom panels). For DNA fragments without molecules bound in the centre, the height at the centre of each line profile was analysed (indicated by the black arrow in the lower graphs). For DNA fragments with molecules bound around the centre, distinct peaks appear in the height profiles. In this case, the highest point of each peak in the profiles were analysed (indicated by the green, magenta and blue circles in the lower graphs). The analysed heights were plotted against time for each dataset with the lowest height set to 0 nm (Fig. 4e).

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1 and 3–6 and Figs. 1–4.

Reporting Summary

Supplementary Table 2

DALI results using the CDF1Dof structure as a search model.

Supplementary Video 1

HS-AFM video of the interaction process between CDF1Dof molecules and DNA with no cis elements. Scan size, 80 nm × 48 nm; frame rate, 1.0 s per frame. This video is played at 5× speed.

Supplementary Video 2

HS-AFM video of the interaction process between CDF1Dof molecules and DNA with a single cis element. The cis element was positioned at the centre of the DNA fragments. Scan size, 80 nm × 48 nm; frame rate, 1.5 s per frame. This video is played at 7.5× speed.

Supplementary Video 3

HS-AFM video of the interaction process between CDF1Dof molecules and DNA with four tandem repeats of cis elements connected via a three-base linker. The cis elements were positioned at the centre of the DNA fragments. Scan size, 80 nm × 40 nm; frame rate, 0.5 s per frame. This video is played at 2.5× speed.

Supplementary Data 1

Source data for Supplementary Figs. 1–4.

Source data

Source Data Figs. 1–3 and Extended Data Fig. 8

Unprocessed gels for Figs. 1–3 and Extended Data Fig. 8.

Source Data Figs. 4 and 5 and Extended Data Figs. 9 and 10

Source data for Figs. 4 and 5 and Extended Data Figs. 9 and 10.

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Furihata, H., Zhu, Z., Nishida, K. et al. Structural insights into CDF1 accumulation on the CONSTANS promoter via a plant-specific DNA-binding domain. Nat. Plants 11, 836–848 (2025). https://doi.org/10.1038/s41477-025-01946-6

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