Systematic Profiling of Histone Readers in Arabidopsis thaliana Shuai Zhao, Baichao Zhang, Mo Yang, Jinsong Zhu, Haitao Li  Cell Reports  Volume 22, Issue 4, Pages 1090-1102 (January 2018) DOI: 10.1016/j.celrep.2017.12.099 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2018 22, 1090-1102DOI: (10.1016/j.celrep.2017.12.099) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 Proteins Containing Histone Reader Domain in Arabidopsis thaliana (A–F) Domain architecture of proteins containing PHD finger domains (A), “Royal Family” (Tudor, Chromo, PWWP) domains (B), BAH domains (C), 14-3-3 and BRCT domains (D), Bromo domains (E), and CW-Zf domains (F). Bottom-right: the color code of each domain type. In total, 92 PHD finger domains, 31 Tudor domains, 21 Chromo domains, 19 PWWP domains, 11 CW-Zf domains, 14 14-3-3 domains, 21 BRCT domains, 28 Bromo domains, and 20 BAH domains were used in this study. See also Figure S1 and Table S1. Cell Reports 2018 22, 1090-1102DOI: (10.1016/j.celrep.2017.12.099) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 SPRi Signals of Potential Histone Readers toward H3K4me3, H4K5acK8ac, and H3S10ph Peptides The SPRi signals of each protein (204 expressed proteins in total) toward H3K4me3, H4K5acK8ac, and H3S10ph peptides are shown in black squares. The ITC-confirmed positive hits are shown in red stars; the ITC-confirmed negative hits are shown in green hexagons; the sticky proteins (defined as more than four different peptides having significant signals toward one protein, which indicates potential non-specific absorptions) are shown in white circles; and the less pure proteins (defined as purity <50%, as shown in Figure S1; protein bands labeled with yellow asterisks) are shown in gray triangles. In the panel of H3K4me3, the average signal value of ITC-confirmed positive hits is labeled with a red line; the average signal value of ITC-confirmed negative hits is labeled with a green line. The numbering of each protein and the original SPRi data are listed in Table S2. A summary of all ITC parameters is presented in Table S3. See also Figures S1, S2, S3, and S4 and Tables S2 and S3. Cell Reports 2018 22, 1090-1102DOI: (10.1016/j.celrep.2017.12.099) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 Confirmation of Histone Mark-Reader Interaction Pairs by ITC and TSA (A–E) ITC curves of ING1/2 (A), AL1/2/3/4 (A), EBS (B), At3g54460 (C), SHH2 (C), EML1 (D), and MSH6 (E) binding to H3K4me3 peptide. (F) ITC curves of Chromo domain protein LHP1 binding to H3K27me3 peptide. (G) ITC curves of Tudor domain protein MSH6 binding to H3K36me3 peptide. (H) ITC curves of Bromo domain proteins binding to H4K5ac peptide. (I) ITC curves of 14-3-3 domain proteins binding to H3Sph peptide. (J) TSA curves of EBS, ING1, and SHH2 binding to H3Kme peptides. (K) TSA curves of LHP1 binding to H3Kme peptides. (L) TSA curves of GRF2 binding to H3Sph peptides. See also Table S3. Cell Reports 2018 22, 1090-1102DOI: (10.1016/j.celrep.2017.12.099) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 The Single Tudor Domain of EML1 Is a Plant-Specific H3K4me2/3 Reader (A) ITC curves of EML1 single Tudor domain binding to H3 peptides with methylation on different sites. (B) Sequence alignment of EML1 single Tudor domain among different plant species. The conserved aromatic cage residues are labeled with a blue asterisk. (C) ITC curves of EML1 single Tudor domain from different plant species binding to H3K4me3 peptide. (D) Structure model of AtEML1 single Tudor domain. The potential aromatic cage is shown in this model. (E) ITC curves of mutant AtEML1 single Tudor domain binding to H3K4me3 peptide. See also Figure S5. Cell Reports 2018 22, 1090-1102DOI: (10.1016/j.celrep.2017.12.099) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 Site Specificity of Histone H3K4me2/3 Reader AL1 (A) ITC curves of AL1-PHD finger domain binding to H3 peptides with methylation on different sites. (B) The crystal structure of AL1-H3K4me3 complex. The PHD finger domain of AL1 is shown in gray; the histone H3K4me3 peptide is shown in yellow. AL1 residues involved in H3K4me3 recognition are labeled. (C) The binding pocket of H3R2 and H3K4me3 on the AL1 PHD finger domain. See also Figure S6 and Table S4. Cell Reports 2018 22, 1090-1102DOI: (10.1016/j.celrep.2017.12.099) Copyright © 2017 The Author(s) Terms and Conditions

Figure 6 State Specificity of H3K4me2/3 Readers in Arabidopsis thaliana (A) ITC curves of AL1/2/3/4 PHD finger domain binding to H3K4me0/1/2/3 peptides. (B) Sequence alignment of AL1/2/3/4 PHD finger domain. Y/D mutation determines H3K4me2/3 preference and is indicated with a red asterisk. (C) The structural alignment of AL3-H3K4me2 and AL3-H3K4me3 complexes. The H3K4me2 peptide is shown in orange, and the H3K4me3 peptide is shown in white. (D) ITC curves of mutant AL1 and AL3 PHD finger domain proteins binding to H3K4me2/3 peptides. See also Figure S7, Table S3, and Table S4. Cell Reports 2018 22, 1090-1102DOI: (10.1016/j.celrep.2017.12.099) Copyright © 2017 The Author(s) Terms and Conditions

Figure 7 Systematic Profiling of Histone Combinatorial Readout Pattern in Arabidopsis thaliana (A) ITC fitting curves of histone H3K4me2/3 readers binding to H3T3phK4me3, H3K4me3T6ph, and H3K4me3K9ac peptides. (B) ITC fitting curves of histone H3K27me3 reader LHP1 binding to H3K27me3S28ph peptide. (C) ITC fitting curves of histone H3K36me3 reader MSH6 binding to H3S28phK36me3 peptide. (D) ITC fitting curves of histone Sph reader GRF2 binding to H3K9acS10ph, H3K27me3S28ph, and H3S28phK36me3 peptides. (E) ITC fitting curves of histone Kac reader AT5G55040 binding to H4K5acK8ac peptide. (F) ITC fitting curves of histone Kac reader AT1G58025 binding to H4K5acK8ac peptide. For each fitting curve, the unit of x axis is “molar ratio” and the unit of y axis is “kcal mol−1 of injectant.” Bottom right: cartoon diagram highlighting three molecular recognition consequences of modification combinatorial pattern readout. See also Table S3. Cell Reports 2018 22, 1090-1102DOI: (10.1016/j.celrep.2017.12.099) Copyright © 2017 The Author(s) Terms and Conditions