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Brassinosteroid-induced gene repression requires specific and tight promoter binding of BIL1/BZR1 via DNA shape readout

Nature Plants volume 8, pages 1440–1452 (2022)Cite this article

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Abstract

BRZ-INSENSITIVE-LONG 1 (BIL1)/BRASSINAZOLE-RESISTANT 1 (BZR1) and its homologues are plant-specific transcription factors that convert the signalling of the phytohormones brassinosteroids (BRs) to transcriptional responses, thus controlling various physiological processes in plants. Although BIL1/BZR1 upregulates some BR-responsive genes and downregulates others, the molecular mechanism underlying the dual roles of BIL1/BZR1 is still poorly understood. Here we show that BR-responsive transcriptional repression by BIL1/BZR1 requires the tight binding of BIL1/BZR1 alone to the 10 bp elements of DNA fragments containing the known 6 bp core-binding motifs at the centre. Furthermore, biochemical and structural evidence demonstrates that the selectivity for two nucleobases flanking the core motifs is realized by the DNA shape readout of BIL1/BZR1 without direct recognition of the nucleobases. These results elucidate the molecular and structural basis of transcriptional repression by BIL1/BZR1 and contribute to further understanding of the dual roles of BIL1/BZR1 in BR-responsive gene regulation.

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Fig. 1: Characteristics of DNA binding and transcriptional regulatory patterns of the plant-specific BIL1/BZR1 TFs.
Fig. 2: Impact of two nucleobase pairs flanking the binding motif of BIL1/BZR1.
Fig. 3: The presence of two nucleobases flanking the core-binding motifs is critical for transcriptional repression by BIL1/BZR1.
Fig. 4: Structural and mutational analyses provide insight into the specificity of BIL1/BZR1 for nucleobases flanking the core-binding motif.
Fig. 5: Schematic model for BR-induced transcriptional regulation via BIL1/BZR1.

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Data availability

The atomic coordinates and structural factors of MBP-fused BIL1/BZR1 in complex with several DNA fragments have been deposited in the Protein Data Bank (PDB) under accession codes 7VN2–8. Microarray data have been deposited in NCBI GEO under accession number GSE181871. The bioinformatics data and EMSA data generated in this study are provided in the Source data files. Source data are provided with this paper.

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Acknowledgements

This work was supported by Grants-in-aid for Scientific Research on Innovative Areas (Nos. 17H05835 and 19H04855 to T.M.) and Grants-in-Aid for Scientific Research (Nos. 18H02140 to T.N. and N.M., 19K23658 to S.N. and 21H02114 to T.M., T.N., N.M. and S.N.) from the Japan Society for the Promotion of Science (JSPS), by 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 number JP20am0101077 (T.M., support number 2013) and JP21am0101107 (T.T., support number 1083), by the Cooperative Research Grant of the Plant Transgenic Design Initiative by Gene Research Center, University of Tsukuba (Nos. 2102 and 2121), and in part by grants from the NARO Bio-oriented Technology Research Advancement Institution (BRAIN) to T.N. We thank the Beamline staff for supporting the synchrotron radiation experiments performed with beamline AR-NE3A at the Photon Factory with the approval of the High Energy Accelerator Research Organization (Proposal No. 2016G648 and 2018G618). We also thank T. Asami (The University of Tokyo) for his gift of the compound Brz and Y. Takiguchi (AIST) for technical support with the microarray experiment.

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Author notes

  1. These authors contributed equally: Shohei Nosaki, Nobutaka Mitsuda.

Authors and Affiliations

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

    Shohei Nosaki, Kazuki Kusubayashi, Yuqun Xu, Thi Bao Chau Bui, Masaru Tanokura & Takuya Miyakawa

  2. Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan

    Shohei Nosaki & Kenji Miura

  3. Tsukuba Plant-Innovation Research Center (T-PIRC), University of Tsukuba, Tsukuba, Ibaraki, Japan

    Shohei Nosaki & Kenji Miura

  4. Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan

    Nobutaka Mitsuda & Shingo Sakamoto

  5. Global Zero Emission Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan

    Nobutaka Mitsuda & Shingo Sakamoto

  6. Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto, Japan

    Ayumi Yamagami, Takeshi Nakano & Takuya Miyakawa

  7. Gene Discovery Research Group, RIKEN CSRS, Wako, Saitama, Japan

    Ayumi Yamagami & Takeshi Nakano

  8. Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

    Tohru Terada

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  1. Shohei Nosaki
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Contributions

T.M. and M.T. supervised the research. S.N., N.M, T.N., M.T. and T.M. conceived and designed the research. S.N., N.M., S.S., Y.X., K.M., T.N. and T.M. constructed plasmids for the experiments. S.N., K.K., Y.X., T.B.C.B and T.M. performed the biochemical experiments. S.N. prepared crystals. S.N. and T.M. collected X-ray diffraction data. S.N. processed and analysed the data with support from T.M. and T.T. A.Y. and T.N. prepared plants for microarray analysis. N.M. performed the microarray analysis. N.M. performed the bioinformatics analysis. S.N., N.M. and S.S. performed the transient assay. S.N., N.M., T.N., M.T. and T.M. wrote the paper. All authors reviewed the article.

Corresponding authors

Correspondence to Masaru Tanokura or Takuya Miyakawa.

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Nature Plants thanks Nitzan Shabek, Yanhai Yin 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 The domain structure of BZR TFs with amino acid sequence alignment of the conserved DNA binding domains (DBDs).

Amino acids sequences of paralogues in Arabidopsis BZR (AtBIL1/BZR1, AtBES1, AtBEH1–4) and orthologues in other plants, including Nicotiana tabacum (NtLOC107792445), Solanum lycopersicum (Sl101246895), Glycine max (Gm100803869), Oryza sativa (Os07g0580500) and Zea mays (Zm00014a_030121), were aligned. The blue triangles point to the three residues Glu37, Arg52 and Asp64 of AtBIL1/BZR1, which are suggested to generate the DNA-binding specificity toward nucleobases flanking the core-binding motif.

Extended Data Fig. 2 The BIL1/BZR1 DAP-seq peaks are concentrated at proximal upstream of transcription start sites.

a, DAP-seq peak distribution over promoters. The position 0 corresponds to the transcriptional start sites. b, Venn diagrams showing the overlap between genes differentially expressed in bil1-1D/bzr1-1D with Brz/BL vs. Col-0 with Brz/mock (left: down, right: up) and genes with BIL1/BZR1 DAP-seq signals from –500 to –1. Each P value by Fisher’s exact test (one sided) and odds ratio are shown below the Venn diagram. c, Distributions of the natural logarithm fold change Ln(FC) in expression levels of genes with and without BIL1/BZR1 DAP-seq signals in the region from –500 to –1. The genes differentially expressed in bil1-1D/bzr1-1D/Brz/BL vs. Col-0/Brz/mock are coloured blue to indicate downregulation and red to denote upregulation. d, Distribution map of the Ln(FC) in the expression levels of genes with BIL1/BZR1 DAP-seq signals in the region from –500 to –1.

Source data

Extended Data Fig. 3 EMSA experiments of the BZR TFs towards DNA fragments derived from BR-responsive gene promoters.

a, Coomassie-stained SDS-PAGE analysis of the purified MBP-fused DBDs of the six members of Arabidopsis BZR TFs, and the BIL1/BZR1 E37A D64A double mutant. This experiment was repeated independently with similar results at least twice. b, Electrophoretic patterns in the EMSA experiments of MBP-fused BIL1/BZR1 DBD towards 0.25 μM carboxyfluorescein (FAM)-labelled DNA fragments derived from promoters of BR-regulated BIL1/BZR1 target genes. Asterisks and arrowheads indicate the positions of free DNA and the BIL1/BZR1-DNA complex, respectively. This experiment was repeated independently with similar results in triplicate. The data were used to calculate the fluorescence densitometric profile shown in Fig. 2d. c, EMSA experiments of MBP-fused Arabidopsis BZR DBDs towards 0.25 μM FAM-labelled DNA fragments. Molar protein-to-DNA ratios are shown at the top of each gel. Asterisks and arrowheads indicate the positions of free DNA and the BZR TF-DNA complex, respectively. This experiment was repeated independently with similar results at least twice.

Extended Data Fig. 4 Interactions of AtPIF4 and AtFD with several DNA fragments containing different binding motifs.

EMSA experiments of 4 μM MBP-fused AtPIF4 DBD (a, b) and 4 μM AtFD DBD (T282E) in complex with 4 μM AtGRF6/GF14l (c, d) towards 0.25 μM FAM-labelled DNA fragments derived from promoters of BR-regulated genes. The electrophoretic patterns are shown with asterisks and arrowheads indicating the positions of free DNA and the TF-DNA complex, respectively (a, c). These experiments were repeated independently with similar results four or three times for AtPIF4 and AtFD, respectively. Bar graph showing the fluorescence densitometric profile (b, d). Data are presented as mean ± s.e.m. (n = 4 and 3 independent experiments for AtPIF4 and AtFD, respectively). Dots denote individual data points.

Source data

Extended Data Fig. 5 ITC analysis of the interaction between BIL1/BZR1 and the different G-box-containing DNA fragments.

The ITC assays were performed by adding 100 μM MBP-fused BIL1/BZR1 (21–104) to 5 μM DNA fragments containing CA|G-box|TG or AT|G-box|AT. The upper panel for each experiment illustrates the representative ITC titration curves, and the bottom panel shows integrated heats of injection (black squares). Data were fitted using the MicroCal origin software with ‘one binding site’ model. b, Thermodynamic parameters determined by ITC. The values of thermodynamic parameters are expressed as mean ± s.d. for three independent experiments. The s.d. values are in parenthesis. ND indicates not detected.

Source data

Extended Data Fig. 6 Structures of BIL1/BZR1 and target DNA fragments.

a, Mutant MBP (mMBP)-fused BIL1/BZR1 (21–90) via one alanine linker. b, Palindromic 14-bp G-box-containing DNA fragments with one-nucleotide overhangs at the 3’-ends. c, Electron density maps of the dinucleotides flanking the G-box of BIL1/BZR1-bound DNA are shown as Fo–Fc omit map contoured at 3σ. d, e, EMSA competition assay in the presence of 5 mM maltose using 0.5 μM mMBP-Ala-BIL1/BZR1 (21–90) and 0.25 mM FAM-labelled pSAUR36-derived DNA mixed with 50-fold excess competitors as used for crystallization. The electrophoretic patterns are shown with asterisks and arrowheads indicating the positions of the free FAM-labelled DNA probe and BIL1/BZR1-DNA complex, respectively (d). This experiment was repeated independently with similar results in triplicate. The data were used to calculate the fluorescence densitometric profile shown in Fig. 4d, e. Bar graph showing the fluorescence densitometric profile (e). Data are presented as mean ± s.e.m. (n = 3 independent experiments). Dots denote individual data points.

Source data

Extended Data Fig. 7 The direct G-box recognition mode by BIL1/BZR1 with seven different DNA fragments.

a, The C1A2 bases recognition by the Glu37 residues. b, The G4 base recognition by the Arg41 residues. Hydrogen bonds are shown as black dashed lines, and the distances (Å) are shown as blue letters. ‘PN’ and a red sphere represent a phosphate group at position N and a water molecule mediating the hydrogen bonding network, respectively. For comparison, each structure is superposed on a translucent model of the complex of BIL1/BZR1 and DNA containing CA|G-box|TG.

Extended Data Fig. 8 The recognition mode of DNA phosphate groups by BIL1/BZR1 with seven different DNA fragments.

The phosphate groups recognition by the Arg30 and Arg34 residues (a) and the Lys61 and His62 residues (b). Hydrogen bonds are shown as black dashed lines, and the distances (Å) are shown as blue letters. ‘PN’ represents a phosphate group at position N. For comparison, each structure is superposed on a translucent model of the complex of BIL1/BZR1 and DNA containing CA|G-box|TG.

Extended Data Fig. 9 Comparison of the interaction network among BIL1/BZR1 and the backbones of seven DNA fragments with different nucleobases flanking the G-box.

a, Hydrogen bonds and salt bridges among amino acid residues and phosphate groups are shown as black dashed lines, and the distances (Å) are shown as blue letters. DNA and two chains of BIL1/BZR1 are shown in different colours. ‘PN’ and a red sphere represent a phosphate group at position N and a water molecule mediating the hydrogen bonding network, respectively. For comparison, each structure is superposed on a translucent model of the complex of BIL1/BZR1 and DNA containing CA|G-box|TG. b, Electron density maps of the amino acid residues and phosphate groups forming the interaction network in the BIL1/BZR1-DNA complexes are shown as 2Fo–Fc map contoured at 1.5σ.

Extended Data Fig. 10 Local base step parameters of seven DNA fragments with different dinucleotides flanking the G-box in complex with BIL1/BZR1.

Base step parameters are shift (Å), slide (Å), rise (Å), tilt (°), roll (°) and twist (°) calculated with 3DNA41, which describe the stacking geometry of a dinucleotide step from a local perspective. The reference values of the regular B-form DNA are as follows: shift, 0 Å; slide, 0 Å; rise, 3.34 Å; tilt, 0, roll, 0°; and twist 36°. The sequence of DNA fragment with CA|TG at the position of dinucleotides flanking the G-box motif (red dashed lines) is shown to explain the dinucleotide step.

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Nosaki, S., Mitsuda, N., Sakamoto, S. et al. Brassinosteroid-induced gene repression requires specific and tight promoter binding of BIL1/BZR1 via DNA shape readout. Nat. Plants 8, 1440–1452 (2022). https://doi.org/10.1038/s41477-022-01289-6

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