Supplemental Information Combinatorial Readout of Unmodified H3R2 and Acetylated H3K14 by the Tandem PHD Finger of MOZ Reveals a Regulatory Mechanism for HOXA9 Transcription Yu Qiu 1, Lei Liu 1, Chen Zhao 1, Chuanchun Han 1, Fudong Li 1, Jiahai Zhang 1, Yan Wang 2, Guohong Li 2, Yide Mei 1, Mian Wu 1, Jihui Wu 1 *, Yunyu Shi 1 * Figure S1. 1
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Figure S10. Figure S11. 8
Figure S12. Supplemental Figure Legends Figure S1. PHD12 Does not Bind to Histone H4 with acetylated K16. No obvious interactions between PHD12 and histone H4 peptide with acetylated K16 were observed from the NMR titration experiment. Figure S2. PHD12 Binds to unmodified Histone H3 Peptides Chemical shift perturbations in the 2D 1 H- 15 N HSQC spectra and ITC titration curves introduced by the binding of H3 (1-8) (A) and unh3 (1-18) (B) peptides respectively. Figure S3. PHD12 Binds to Histone H3 Peptides with Modifications at K4 or K9 Chemical shift perturbations in the 2D 1 H- 15 N HSQC spectra and ITC titration curves introduced by binding of H3 (1-18)K4me3 (A), H3 (1-18)K9ac (B), H3 (1-18)K9me3 (C), and H3 (1-18)K9acK14ac (D) peptides respectively. 9
Figure S4. Side Chain of Arg299 of Another PHD12 Molecule from an Adjacent Asymmetric Unit Blocks the Acetylated Lys14 Binding Owing to Crystal Packing Cartoon (A) or surface (B) show of the crystal structure of PHD12 in H3K14ac peptide bound state. The central PHD12 (purple-blue) is surrounded by other PHD12 molecule (grey) in the same unit cell. The dot lines colored in red enclose the side chain of Arg299 of another PHD12 molecule from an adjacent asymmetric unit and the acetate group (green) from reservoir. The bound H3 peptide is colored in yellow. Figure S5. Dynamic Properties of PHD12 Backbone Investigated by NMR Relaxation Experiments 15 N longitudinal (T1), transversal (T2) relaxation time and 1 H- 15 N heteronuclear NOE are represented for residues of PHD12. The red box encloses the flexible loop between Gly272 and Asn277. Figure S6. PHD12 Does not Bind to Histone H3 (9-18) Chemical shift perturbations in the 2D 1 H- 15 N HSQC spectra and ITC titration curves introduced by the binding of unh3 (9-18) (A) and H3 (9-18)K14ac (B) peptides respectively. Figure S7. PHD12 Binding to Histone Does not Affect the HAT Activity of MOZ in Vitro (A-B) The abilities of wild-type and mutant PHD12-MYST to acetylate histone octamer were determined by a 3 H-based radioactive HAT assay. The results are shown by 15% SDS-PAGE (A), and by liquid scintillation counter (B). The concentration of protein in (B) is indicated on the x-axis, and the HAT activity is indicated on the y-axis. All the reactions performed in triplicate and averaged. The corresponding autoradiography is shown above. (C-D) The same HAT activity assay as in (A) and (B) for substrate of wild-type or H3R2A reconstituted histone octamer which is indicated below. (E) Addition of K14ac peptide to the unmodified recombinant octamers clarifying the catalysis is independent of the PHD12-H3 binding. (F)The HAT assay with nucleosomes or octamers extracted from HeLa cells. Figure S8. The Tandem PHD Finger Does not Interact with the MYST Domain in MOZ ITC measurement of interaction between the tandem PHD finger and the MYST domain of MOZ. Figure S9. Mutations of PHD12 Do not affect the nuclear localization of MOZ The expression plasmids for wild-type and mutant FLAG-MOZ were transfected into H1299 cells. MOZ expression was detected by immunostaining with anti-flag antibody and a rhodamine-labeled secondary antibody. Figure S10. MOZ association with BRPF1 in vivo is independent of PHD12 binding to histone Immunofluorescent staining of H1299 cells after co-transfection with the indicated versions of GFP-BRPF1 (the 2nd panels; green) and FLAG-MOZ (the 3rd panels; red). The nucleus were stained with Hoechst (the 1st panels; blue); merged images are shown in right-hand panels. Figure S11. Sequence Alignment of Tandem PHD Fingers Multiple sequence alignment of tandem PHD fingers involved in H3R2me0 (highlighted in orange), H3K4me0 (highlighted in green), and H3K14ac (highlighted in blue for hydrophobic pocket and yellow for hydrogen bond formation). Four groups of Zn-chelating residues are connected by solid lines. The 10
secondary structural elements of PHD12 are indicated below the sequence. Cys residues coordinating the Zn atoms are highlighted in grey. Figure S12. Comparison of H3K4me0 recognition by the different PHD fingers (A) H3K4me0 recognition sites in PHD fingers are illustrated by showing the side chains of the PHD finger residues (stick and surface representation) that interact with the unmethylated H3K4 ligand residue. (B) Sequence alignment of the PHD fingers of MOZ, DPF3b, AIRE, BHC80 and BRPF2. The same color scheme from Figure S6 is used, except the red background highlighting conserved residues. 11
Supplemental Tables Table S1. Structural statistics for the 20 NMR structures of PHD12 in free state NMR distance and dihedral constraints Distance constraints Total 1916 Intraresidue 561 Interresidue 1335 Sequential( i-j =1) 516 Medium-range(1< i-j <5) 337 Long-range( i-j > 5) 482 Hydrogen bonds 20 Total dihedral angle restraints a φ 55 ψ 56 Structure Statistics Mean energies (kcal mol -1 ) E total -301.70 ± 8.10 E vdw -411.93 ± 7.51 E noe 9.36 ± 0.95 E angle 78.94 ± 2.72 E bond 14.94 ± 1.20 E improper 6.91 ± 0.49 E dihedral 0.09 ± 0.03 Violations(mean ± SD) Distance constraints(å) 0.0081 ± 0.0004 Dihedral angle constraints(º) 0.1162 ± 0.0189 Deviations from idealized geometry Bond lengths(å) 0.0030 ± 0.00012 Bond angles(º) 0.5700 ± 0.0266 Impropers(º) 0.2274 ± 0.0081 PROCHECK Ramachandran Plot analysis (%) b Residues in most favored regions 79.7% Residues in additionally allowed regions 19.9% Residues in generously allowed regions 0.3% Residues in disallowed regions 0.1% Structural r.m.s.d..for secondary structures regions c (Å) Backbone heavy atom (N, Cα, and C ) 0.77 Heavy atom 1.15 a The φ and ψ angle restraints are generated from secondary structures by Talos+. b All non-gly residues, φ/ψ of most favored, and additional allowed regions are given by PROCHECK (Laskowski et al., 1996). c Atoms of well-defined secondary structure regions: residues 208-312. 12
Table S2. Crystallographic Data Collection and Refinement Statistics of PHD12 in complex with H3K14ac Crystal MOZ PHD-H3 (1-18)K14ac Beamline SSRF BL17U Wavelength 0.9793 Space group P2 1 2 1 2 1 Unit cell a, b, c (Å) 35.08, 57.63, 76.51 α, β, γ ( o ) 90, 90, 90 Resolution (Å) 38.26-1.47 (1.55-1.47) a R sym 0.063 (0.46) I/ σ (I) 24.0 (1.60) Completeness (%) 99.6 (97.8) Redundancy 13.6 (12.9) Number of unique reflections 27138 R work /R free (%) 15.29/17.62 Number of nonhydrogen atoms Protein 874 Peptide 53 Water 157 Zn 8 Average B factors (Å 2 ) Protein 17.5 Peptide 18.3 Water 34.9 Zn 15.0 Rmsd Bond lengths (Å 2 ) 0.010 Bond angles ( o ) 1.386 a Values in parentheses are for highest-resolution shell 13
Table S3. Peptide sequences used in this study Peptide name Sequence unh3(1-18) ARTKQTARKSTGGKAPRK H3(1-18)K4me3 ART-Kme3-QTARKSTGGKAPRK H3(1-18)K9ac ARTKQTAR-Kac-STGGKAPRK H3(1-18)K9me3 ARTKQTAR-Kme3-STGGKAPRK H3(1-18)K14ac ARTKQTARKSTGG-Kac-APRK H3(1-18)K9acK14ac ARTKQTAR-Kac-STGG-Kac-APRK H3(1-18)K14ac_T11A ARTKQTARKSAGG-Kac-APRK H3(1-18)K14ac_R2A AATKQTARKSTGG-Kac-APRK H3(1-18)R2me2s AR-Kme2s-TQTARKSTGGKAPRK H3(1-18)R2me2a AR-Kme2a-TQTARKSTGGKAPRK unh3(1-8) ARTKQTAR unh3(9-18) KSTGGKAPRK H3(9-18)K14ac KSTGG-Kac-APRK H4(11-21)K16ac GKGGA-Kac-RHRKV Table S4. DNA primers used in this study Gene Forward primer (5-3 ) Backward primer (5-3 ) Size (bp) MOZ CTTCAGTGAGAGCAGCGAGGAG GTGGTGTTTGCGCTTTCGGACT 139 HOXA9_0 HOXA9_1 HOXA9_2 HOXA9_3 HOXA9_4 HOXA9_5 HOXA9_6 CGACCCACGGAAATTATGAA GTCAGACTATTCTGGCTCC CTCAGAGGCCTGGCGGACTG GCACAGTATCCACACGTGAA CTCCCAGGTTCCGGAGCTGC CCTCGCTCCAGGCGGGTAGC CCTTCTTGATGGCGTGATTA CGTTGGCCACAATTAAAACA TATGCCTGAGAAGACACTG GATACCGACTGGGTGCCCCT GAACGGGGAGGGGTAAAAGG TGGCAATCAGGATTCCCAGG GACTTGGAAAGGTTAGACTG ACGCGTAAATCACTCCGCAC 273 212 203 207 207 231 246 HOXB1 CTCTGGTCCCTTCTTTCC GGCCAGAGTTTGGCAGCC 154 HOXC5 GGCAGGGATTGAGCGATTC GGGTATCTGCAGCCATTCGG 474 HOXD12 GAACCTGCAGGCAAAGTTTC AGAGACTGCGCTCACACATC 120 Supplemental References Laskowski, R.A., Rullmannn, J.A., MacArthur, M.W., Kaptein, R., and Thornton, J.M. (1996). AQUA and PROCHECK NMR: programs for checking the quality of protein structures solved by NMR. Journal of biomolecular NMR 8, 477 486. 14