Volume 25, Issue 1, Pages 51-56.e6 (January 2018) Engineered Multivalent Sensors to Detect Coexisting Histone Modifications in Living Stem Cells  Aurore M.-F. Delachat, Nora Guidotti, Andreas L. Bachmann, Antonio C.A. Meireles-Filho, Horst Pick, Carolin C. Lechner, Cédric Deluz, Bart Deplancke, David M. Suter, Beat Fierz  Cell Chemical Biology  Volume 25, Issue 1, Pages 51-56.e6 (January 2018) DOI: 10.1016/j.chembiol.2017.10.008 Copyright © 2017 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2018 25, 51-56. e6DOI: (10. 1016/j. chembiol Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Design of Multivalent Probes to Detect Coexisting Histone PTMs (A) Scheme of the cMAP concept. (B) Structures of the readers, Pc CD (PDB: 1PDQ) bound to H3K27me3 (red) and TAF3 PHD (PDB: 5C13) bound to H3K4me3 (green). In both structures the highlighted residues show the binding site. (C) Structure of a bivalent nucleosome (PDB: 1KX5). See also Figure S1. Cell Chemical Biology 2018 25, 51-56.e6DOI: (10.1016/j.chembiol.2017.10.008) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Engineering of cMAPs Selective for Bivalent Chromatin (A) Domain arrangement of cMAP1–4. Linker length and amino acid (aa) composition are indicated (for exact linker sequences and cMAP5–8 see Figure S1). (B) SDS-PAGE analysis of purified cMAP1-4. (C) Synthesis of methylated nucleosomes. (i) In situ peptide thioester formation by oxidation of C-terminal hydrazide using sodium nitrite (NaNO2) followed by thioester conversion with trifluoroethanethiol. (ii) Ligation to N-terminally truncated H3 followed by (iii) radical cysteine desulfurization. (iv) Refolding of histone octamers with recombinant core histones H4, H2A, and H2B. (v) Nucleosome reconstitution. (D) Pull-down of cMAP3 using modified nucleosomes (a–e, compare with C). Probe retention was visualized by western blot (WB) using an anti-FLAG antibody. For full blot, see Figure S3D. IP, immunoprecipitation. (E) Selectivity profile of cMAP1–4 to nucleosomes a, b, d, and e. Relative WB band intensity was quantified, normalized, and averaged. Error bars denote SD; n = 3. (F) cMAP3 binding to indicated peptides determined by microscale thermophoresis. Fit curves represent single-site binding isotherms with KD of 7.9 μM (5b), 8.5 μM (5d), and 0.17 μM (5e). Error bars denote SD; n = 2. See also Figures S2 and S3. Cell Chemical Biology 2018 25, 51-56.e6DOI: (10.1016/j.chembiol.2017.10.008) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Visualizing Bivalent Chromatin Domains in ESCs (A) Immunofluorescence imaging of H3K4me3 and H3K27me3 in fixed ESCs. DNA was stained using DRAQ5. Scale bars, 5 μm. (B) Localization of cMAP3 and indicated mutant proteins in mouse ESCs. Refer to Figure S4D for more cell images. Scale bars, 5 μm. (C) Quantification of fluorescent foci in mouse ESCs for cMAP3 and reader domain mutants. Number of cells analyzed: cMAP3, n = 79; CD mutant, n = 81; PHD mutant, n = 41; CD-PHD mutant, n = 54. Lines denote mean; boxes show 25%–75% of the data, whiskers 10%–90%. *p < 0.05, Student’s t test. See also Figure S4. Cell Chemical Biology 2018 25, 51-56.e6DOI: (10.1016/j.chembiol.2017.10.008) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Small-Molecule Disruption of Bivalent Domains (A) Employed epigenetic modulators. (B) Localization of cMAP3 in mouse ESCs treated and untreated with UNC1999. Scale bars, 5 μm. (C) Average number of fluorescent foci per nucleus in UNC1999-treated versus untreated cells (untreated, n = 79; UNC1999-treated, n = 44). Lines denote mean; boxes show 25%–75% of the data, whiskers 10%–90%. *p < 0.05, Student’s t test. For more cell images, see Figure S4D. (D) Upper panel: Anti-H3K27me3 and anti-H3K9me3 western blot (IB) of acid-extracted histones from mouse ESCs, treated and untreated with UNC1999. Lower panel: Coomassie brilliant blue (CBB)-stained gel of the extracted histone samples. For full blots and gels, see Figures S4L–S4N. (E) Immunofluorescence (IF) of H3K27me3 in mouse ESCs treated and untreated with UNC1999. Scale bars, 5 μm. See also Figure S4. Cell Chemical Biology 2018 25, 51-56.e6DOI: (10.1016/j.chembiol.2017.10.008) Copyright © 2017 Elsevier Ltd Terms and Conditions