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EBS is a bivalent histone reader that regulates floral phase transition in Arabidopsis

Abstract

The ability of cells to perceive and translate versatile cues into differential chromatin and transcriptional states is critical for many biological processes1,2,3,4,5. In plants, timely transition to a flowering state is crucial for successful reproduction6,7,8,9. EARLY BOLTING IN SHORT DAY (EBS) is a negative transcriptional regulator that prevents premature flowering in Arabidopsis thaliana10,11. We found that EBS contains bivalent bromo-adjacent homology (BAH)–plant homeodomain (PHD) reader modules that bind H3K27me3 and H3K4me3, respectively. We observed co-enrichment of a subset of EBS-associated genes with H3K4me3, H3K27me3, and Polycomb repressor complex 2 (PRC2). Notably, EBS adopted an autoinhibition mode to mediate its switch in binding preference between H3K27me3 and H3K4me3. This binding balance was critical because disruption of either EBS–H3K27me3 or EBS–H3K4me3 interaction induced early floral transition. Our results identify a bivalent chromatin reader capable of recognizing two antagonistic histone marks, and we propose a distinct mechanism of interaction between active and repressive chromatin states.

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Fig. 1: Structural basis for recognition of H3K27me3 by BAH.
Fig. 2: Structural basis for an autoinhibition loop formed by the EBS C terminus.
Fig. 3: EBS colocalizes with H3K4me3 and H3K27me3.
Fig. 4: Functional analysis of EBS mutants defective in H3K4me3 or H3K27me3 binding and a C-terminus deletion mutant.
Fig. 5: EBS interacts with MSI4.

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Acknowledgements

We thank the staff at the Shanghai Synchrotron Radiation Facility for data collection, the staff at the UW–Madison Biotechnology Center for high-throughput sequencing, J. Jiang and J. Chen (UW–Madison) for MSI4 constructs, Z. Shen for help with the split-LUC assay, M. Piñeiro (Centro de Biotecnología y Genómica de Plantas, Spain) for ebs seeds, Y. He (Shanghai Center for Plant Stress Biology) for discussion, and R. Amasino and D. Patel for manuscript comments. This study was supported by the National Key R&D Program (2016YFA0503200), the National Science Foundation of China (31622032 and 31770782), and the Chinese Academy of Sciences to J.D.; the Alexander von Humboldt Foundation and NSF CAREER (MCB-1552455) and NIH-MIRA (R35GM124806) to X.Z.; the NIH (GM059785-15/P250VA) to J.M.D.; and the NIH-NCI (R01CA193481) to L.M.S.

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Z.Y., R.L., X.D., and X.L. performed the structural analysis. S.Q., R.N.S., and X.C. conducted all of the functional experiments. L.L. performed the bioinformatic analysis. M.S. and L.M.S. contributed to mass spectrometry analysis. M.D.B. and J.M.D. provided the peptide array. J.D. and X.Z. designed this study and wrote the manuscript.

Corresponding authors

Correspondence to Jiamu Du or Xuehua Zhong.

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Integrated supplementary information

Supplementary Figure 1 EBS specifically binds methylated H3K4 and H3K27 marks.

a,c, Systematic profiling of histone binding preferences of full-length GST-EBS on a histone peptide microarray. A representative array image shows EBS binding to H3K4me3 (a) and H3K27me3 (c). b,d, Relative intensity of selective H3K4me3-containing (b) and H3K27me3-containing (d) peptide species. Relative signal intensity is calculated by normalizing each mean signal intensity at 635 nm for triplicate spots to the highest signal on the individual subarray, after subtracting background signals (derived from empty spots) for all spots. e, Relative intensity of EBS binding with different peptides. Peptide species containing the same PTMs are grouped together.

Supplementary Figure 2 Structure of the EBS–H3K27me3 complex.

a, Interactions between the BAH and PHD domains. The interacting residues are highlighted in the stick representation. b, An omit map for the H3K27me3 peptide is shown in magenta mesh. c, ITC binding curves between H3K27me3 and various EBS point mutants showing that the aromatic residues and His95 are essential for recognition of H3K27me3. The ITC experiments were repeated twice independently with similar results.

Supplementary Figure 3 Structure of EBSΔC in complex with H3K4me2 peptide.

a, Superposition of the EBSΔC–H3K4me2 complex (in magenta) with the EBS–H3K27me3 complex (in silver) showing that they have similar overall structures. The two peptides are shown in a space-filling representation. b, An omit map for the H3K4me2 peptide is shown in a cyan mesh. c, Superposition of the two structures shows that Pro211 of the C-terminal loop overlaps with H3K4me2, resulting in an autoinhibition mode that blocks binding of H3K4me3 and H3K4me2. d, Enlarged view of the aromatic cage that accommodates H3K4me2. e, ITC binding curves between H3K4me3 and various EBS mutants showing that the aromatic cage is essential for H3K4me3 binding. f, ITC binding curves between EBS and a doubly methylated H3(1–35)K4me3K27me3 peptide. g, ITC binding curves between EBSΔC and a doubly methylated H3(1–35)K4me3K27me3 peptide. N values in f and g represent binding stoichiometry. The ITC experiments were repeated twice independently with similar results. h, Superposition of the EBSΔC–H3K4me2 complex (in color) with the EBS–H3K27me3 complex (in silver). The distance between H3A7 and H3K23 is measured to be 33 Å, which makes it hard to accommodate the spanning 15 residues considering the orientations of the two peptides. Thus, EBS prefers to bind H3K4me3 and H3k27me3 independently and not simultaneously.

Supplementary Figure 4

Chromosomal views showing that EBS colocalizes with H3K4me3 (red) and H3K27me3 (blue) along five Arabidopsis chromosomes.

Supplementary Figure 5 EBS and PRC proteins are co-enriched at similar target genes.

a, ChIP–qPCR analysis showing relative enrichment of EBS and CLF at SOC1, EMF1, and FLC. The TA3 locus serves as a negative control. Bars denote the mean of two independent experiments. b, Venn diagram showing the number of genes overlapping for EBS with LHP129 and EMF131. c,d, Metplots showing average H3K27me3 (c) and H3K4me3 (d) levels along the transcription units of 301 EBS-bound genes co-marked with H3K4me3 and H3K27me3.

Supplementary Figure 6 Phenotypic analysis of EBS mutants with disrupted H3K4me3 or H3K27me3 binding.

ad, Flowering time analysis of long-day-grown plants from six independent T2 ebs transgenic plants expressing wild-type EBS-FLAG (a), H3K27me3-binding-defective triple-mutant EBS Y49A W70A Y72A (b), H3K4me3-binding-defective mutant EBS Y155A (c), and C-terminus deletion mutant EBSΔC (d). Black horizontal lines are the mean, and error bars represent s.d. from the number of plants (indicated by n) for each line (white dots). e, Western blot analysis of protein expression levels from the plants indicated in ad using an anti-FLAG antibody. Rubisco serves as a loading control. Two independent lines for each transgene are shown (uncropped images in Supplementary Fig. 8).

Supplementary Figure 7 Genome-wide H3K4me3 levels in EBS and EBSΔC.

a,b, Metaplot showing the average levels of EBS and EBSΔC occupancy (a) and H3K4me3 levels (b) over EBS and EBSΔC common target genes in transgenic plants expressing EBS-FLAG and EBS∆C-FLAG. Two independent biological replicates are shown as Rep1 and Rep2. TSS, transcription start site; TTS, transcription termination site. –2 K and + 2 K represent 2 kb upstream of the TSS and 2 kb downstream of the TTS, respectively. The y axis represents read density after normalization with total reads. c, Schematic representation of the workflow for sequential ChIP. d,e, ChIP–qPCR of H3K4me3 (d) or H3K27me3 (e) enrichment relative to input materials in plants expressing EBS-FLAG and EBS∆C-FLAG. Bars denote the mean of two independent experiments.

Supplementary Figure 8

Original uncropped scans of representative immunoblots displayed in this article.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8

Reporting Summary

Supplementary Table 1

Summary of histone peptide arrays

Supplementary Table 2

Data collection and refinement statistics

Supplementary Table 3

Summary of EBS and CLF ChIP-seq

Supplementary Table 4

Summary of EBS∆C ChIP-seq

Supplementary Table 5

Summary of EBS mass spectrometry

Supplementary Table 6

Summary of EBS mass spectrometry

Supplementary Table 7

List of primers used in this study

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Yang, Z., Qian, S., Scheid, R.N. et al. EBS is a bivalent histone reader that regulates floral phase transition in Arabidopsis. Nat Genet 50, 1247–1253 (2018). https://doi.org/10.1038/s41588-018-0187-8

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