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1 Supplemental Material Molecular basis for oncohistone H3 recognition by SETD2 methyltransferase Shuang Yang, 1, 2 Xiangdong Zheng, 1, 2, 3 Chao Lu, 4 Guo-Min Li, 2 C. David Allis, 4 1, 2, 3, 5* and Haitao Li 1 MOE Key Laboratory of Protein Sciences, Beijing Innovation Center for Structural Biology, 2 Department of Basic Medical Sciences, School of Medicine, 3 Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing , China; 4 Laboratory of Chromatin Biology and Epigenetics, The Rockefeller University, New York, NY 10065, USA; 5 Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu , China To whom correspondence should be addressed: Haitao Li Medical Science Building C228 Tsinghua University Beijing , China Tel: (+) lht@tsinghua.edu.cn Keywords: oncohistone; SETD2 methyltransferase; crystal structure, epigenetic regulation Running title: Oncohistone recognition by SETD2 This file includes: Supplemental Methods Supplemental Figures S1-S5 Supplemental Table S1 1
2 Supplemental Methods Protein purification, crystallization, and data collection The catalytic domains of wide type and mutant human SETD2 (residues ) (SETD2 CD ) were cloned into a modified pet28b vector with an N-terminal 10xHis-SUMO tag. Recombinant proteins were overexpressed in E. coli BL21 (DE3) induced by 0.2 mm isopropyl-1-thio-d-galactopyranoside (IPTG) at 16 C overnight. Cells were harvested and resuspended in buffer containing 50 mm Tris, ph 8.0, 500 mm NaCl, 10 mm imidazole and 5% glycerol. After cell lysis and centrifugation, the resultant His-SUMO-SETD2 CD in supernatant was affinity-purified by a HisTrap column (GE Healthcare). After ULP1 digestion to cleave off the 10xHis-SUMO tag, the SETD2 CD protein was further purified by cation exchange HiTrap SP HP column (GE Healthcare). The protein sample was finally polished over a Superdex /300 GL (GE Healthcare) column in buffer containing 20 mm Tris, ph 8.0 and 200 mm NaCl. For binary complex crystal growth, the purified SETD2 CD protein at ~7 mg/ml was incubated with SAM at the molar ratio of 1:5. For ternary complex crystal growth, the SETD2 protein was incubated with SAM and the histone H K36M/I peptide at the molar ratio of 1:10:5 (protein:sam:peptide). Crystallization was performed via sitting drop method by mixing equal volumes (1μl) of protein and reservoir solution. Binary crystal was grown in solution containing 0.1 M KSCN and 30% PEGMME-2K at 291 K. Ternary crystal was grown in solution containing 0.2 M KSCN, 0.1 M Bis-Tris propane, ph 8.5, and 20% PEG 3350 at 277 K. For data collection, crystals were briefly soaked in a cryo-protectant condition composed of reservoir solution supplemented with 20% glycerol, and were flash frozen in liquid nitrogen for future use. Mononucleosome isolation from HeLa cells HeLa cells were cultured in DME medium supplemented with 10% FBS. When the cells reach 90% confluency, cells were collected and washed three times by PBS. The cells were resuspended by TM2 2
3 buffer (20 mm Tris-HCl, ph 7.4, 2 mm MgCl 2 ), and further lysed with 1.5% NP-40 on ice for 5 min. The nuclei pellets were resuspended in TM2 buffer plus 1 mm CaCl 2, and then digested with micrococcal nuclease (New England Biolabs) at the concentration of 10U/10 7 cell nuclei for 80 min at 37. The reaction was stopped with 2 mm EGTA (ph 8.0) on ice for 10 min. After centrifuging at 2000 rpm for 10 min, the pellet was retained and washed twice by TM2 buffer and then resuspended by STM600 buffer (20mM Tris-HCl, ph 7.4, 2mM MgCl 2, 600mM NaCl, 0.1% Triton X-100) and rotated at 4 C overnight. After centrifuging at rpm for 10 min, the mononucleosome was collected as the supernatant. Nucleosomes were analyzed by 1.2% agarose TBE gel electrophoresis and 18% SDS-PAGE separately to check the size and purity of DNA and histone. In vitro methyltransferase assay The methyltransferase assay was carried out in a 20 μl reaction system containing 0.3 μm SETD2 CD (wide type or mutants), 5 μg mononucleosome isolated from HeLa cells and 1μl S-adenosyl- [methyl- 3 H]-l-methionine ( 3 H SAM, 5-15 Ci/mmol, 0.55 mci/ml; PerkinElmer), in buffer containing 20 mm Tris, ph 9.0, 0.01% Triton-X100, 1.5 mm MgCl 2, and 6 mm TCEP. The reaction was performed at 30 C for 1 hour. For scintillation counting, reactions were bound to filter paper (Millipore P81), and quantification by a scintillation counter (MicroBeta Jet Microplate Scintillation and Luminescence Counter; PerkinElmer). The inhibition assay was performed in the same reaction system, except that SETD2 was pre-incubated with H peptides with K36me3 modification or K36 mutations. 3
4 α8 Figure S1. Comparison of the L IN Post-SET region between the two open states of SETD2 CD structures. The peptide-bound structure reported here is colored magenta; the Pr-SNF bound structure (PDB code: 4FMU) is colored light cyan. The H3 peptide is shown as yellow ribbon with side chains shown in stick mode. Note the conformation differences of key L IN loop residues: Q1667, F1668, Q1669, R1670, Y1671, K1673 (G1672 not labelled). Side chains of Q1667, R1670, and K1673 in the structure of 4FMU are only partially modelled due to their flexibility. Note the ordering of the C-terminal loop-α8 fragment of Post-SET in the peptide-bound complex. 4
5 Figure S2. LIGPLOT diagram listing critical contacts between the H3K36M peptide and SETD2 CD. H3 segment (orange) and key residues of SETD2 CD (green) are depicted in ball-and-stick mode. Grey ball, carbon; blue ball, nitrogen; red ball, oxygen; big cyan ball, water molecules. Bonding distances are shown in the unit of angstrom. 5
6 Figure S3. Modeling of H3G34 mutants in the G33-G34 tunnels of SETD2 (A, B, C) and modeled NSD1/2/3 (D, E). Key G33-G34 tunnel residues of SETD2 and modeled NSD1/2/3 are represented as blue (A, B, C) and cyan sticks (D, E), respectively. Steric clashes are calculated by the software PyMol and shown as red plates. 6
7 Figure S4. (A) Positioning of H3K36M in the SETD2 lysine access pocket. Figure is prepared in stereoview. The H3K36M peptide and SAH are shown as space-filling spheres. The lysine access pocket is shown in cavity mode with half-transparency. Key residues that form the pocket are shown as cyan sticks. (B, C) Cut-away surface (blue) view of the lysine access pocket with H3K36M (B) or H3K36I (C) and SAH depicted as yellow and white sticks, respectively. Key SETD2 residues that embrace K36M or K36I are represented as cyan sticks. Red arrow highlights the positioning of the Cγ2 atom of I36 in a spare space near the pocket entrance. 7
8 Figure S5. Structure-based sequence alignment of the catalytic domains among human histone H3K36 methyltransferase family members including SETD2, NSD1/2/3 and ASH1L. Key residues that participate in H3 peptide recognition are denoted by color-coded triangles. 8
9 Table S1 Data collection and refinement statistics Data collection SETD2 CD -SAH- H3.3K36M SETD2 CD -SAH- H3.3K36I SETD2 CD -SAH Space group P P P Cell dimensions a, b, c (Å) 60.2, 76.7, , 76.8, , 77.2, 78.7 α, β, γ ( ) 90, 90, 90 90, 90, 90 90, 90, 90 Resolution (Å) ( )* ( ) ( ) R merge 12.6 (96.7) 7.8 (75.4) 8.1 (84.1) IIσI 24.0 (2.8) 55.4 (3.9) 41.0 (3.6) Completeness (%) 100 (100) 100 (100) 99.4 (100) Redundancy 9.0 (9.1) 9.3 (7.2) 7.0 (7.3) Refinement Resolution (Å) No. reflections 22,948 58,391 12,819 R work /R free 17.8/ / /25.0 No. atoms Protein Peptide/SAH/Zn 108/26/3 109/26/3 -/26/3 Water Others B-factors (Å 2 ) Protein Peptide/SAH/Zn 39.5/23.9/ /17.1/20.6 -/62.9/67.7 Water Other R.m.s. deviations Bond lengths (Å) Bond angles ( ) * Values in parentheses are for highest-resolution shell. 9
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