A Molecular Threading Mechanism Underlies Jumonji Lysine Demethylase KDM2A Regulation of Methylated H3K36. Supplementary Materials

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1 A Molecular Threading Mechanism Underlies Jumonji Lysine Demethylase KDM2A Regulation of Methylated H3K36 Zhongjun Cheng 1, Peggie Cheung 2, Alex J. Kuo 2, Erik T. Yukl 3, Carrie M. Wilmot 3, Or Gozani 2,# and Dinshaw J. Patel 1,# Supplementary Materials 1

2 Extended materials and methods Protein cloning, expression and purification Two fragments (residues and ) of mouse KDM2A were cloned into a modified vector PRSFduet with a SUMO tag at the N terminus. The truncated KDM2A was expressed in Escherichia coli BL21(DE3). Cell lysis was loaded onto a Ni-NTA affinity column and the SUMO tag was removed by adding Ulp protease. The protein was dialyzed to remove imidazole and loaded onto a Ni-NTA column again to absorb the SUMO tag and further purified by Q-column and gel filtration. Site-directed mutations were introduced using the Stratagene Quikchange Mutagenesis kit, and all mutations were confirmed by DNA sequencing. Synthetic peptides were prepared at Memorial Sloan-Kettering Cancer Center Protein Core Facility and Anaspec Inc. Fluorescence-based lysine demethylase assays We tested the enzyme kinetic parameters and lysine demethylation activities using the formaldehyde dehydrogenase (FDH)-coupled continuous fluorescent assay as described in the literature (Horton et al. 2010). For enzymatic assays, Ni 2+ was removed by EDTA and prior to assays, Fe 2+ was added to the assay buffer. Briefly, assays were done in triplicate at 37 C in 50 mm HEPES, ph 7.0 buffer with 50 µm (NH4) 2 FeSO 4, 1 mm αkg and 2 mm ascorbic acid, 1 mm 3-acetylpyridine adenine dinucleotide (APAD+) (Sigma) and 0.05 units of FDH (Sigma), 10 µm KDM2A and 100-1,500 μm histone H3 peptide substrate. For the KDM2A and peptide mutants, 200 μm peptide and 10 µm enzyme was used in the assay. Reactions were initiated by adding peptides and monitored in an infinite M1000 (Tecan) fluorescence reader at an excitation wavelength of 363 nm and an emission wavelength of 482 nm. The fluorescence increase was then converted to product concentration by a formaldehyde calibration standard curve. Kinetic parameters were produced by plotting initial velocities against substrate concentration and fitting to the Michaelis-Menten equation. Reagents, plasmids, and antibodies Full-length mouse KDM2A cdna was cloned into pcag-flag for overexpression in 293T cells. For retroviral transduction, full-length KDM2A was cloned into pwzl-3xflag vector, and viral particles were prepared with packaging plasmids pvsvg and pgagpol. KDM2A mutants were generated by site-directed mutagenesis (Stratagene). Antibodies used in the study were: H3, H3K36me1, H3K4me2, H3K9me2 (Abcam); H3K36me2 (Active Motif); Flag (M5) (Sigma). 2

3 Cell culture and transfection 293T, HT1080, and p19arf-/- MEFs were cultured in DMEM (Gibco) with 10% fetal bovine serum (ATCC), glutamine and penicillin/streptavidin (Gibco). Transfection of plasmid DNA was performed using TransIT-LT1 or TransIT-293 (Mirus) following the manufacturer s protocol. RNAi knock down strategy Oligos targeting KDM2A UTR were synthesized and cloned into plentilox3.7 to generate lentivirus particles for stable transduction. The two shrnas target KDM2A transcripts at untranslated region (UTR) at 5 -GCG TGT CTC TCC TCC GTT AAA -3 (shrna#1) or 5 -GCT GTC CAA CAC TCA TAA TAC -3 (shrna#2). Transduced HT1080 cells were selected with 10ug/mL puromycin. Immunofluorescence HT1080 cells were fixed with 4% Paraformaldehyde (Sigma) for 15 mins, wash with PBS (Gibco), permeabilized with 0.2% TritonX100 (Sigma) for 10 mins, blocked with 10% Normal goat serum (Sigma) and mounted with DAPI-Prolonged Gold antifade reagent (Invitrogen). Soft agar colony formation assay For soft-agar assays, cells were plated in triplicate in 0.8% (wt/vol) agarose on a base agar medium of 3% (wt/vol) agarose. Colony formation was quantified after 21 days as the numbers of colonies per field. 3

4 Table S1. Data collection and refinement statistics of methylated H3K36 peptide-kdm2a complexes with Ni 2+ and NOG. H3K36me1-KDM2A H3K36me2-KDM2A H3K36me3-KDM2A Data collection Ni 2+ + NOG Ni 2+ + NOG Ni 2+ + NOG Space group P P P Cell dimensions a, b, c (Å) 54.6, 86.5, , 87.5, , 86.7, Resolution (Å) 2.20 ( ) 1.75 ( ) 1.60 ( ) R merge 6.4 (43.5) 6.1 (32.6) 6.6 (52.7) I/σI 12.1 (2.54) 18.9 (2.0) 29.6 (2.4) Completeness (%) 96.2 (98.6) 99.0 (92.8) 99.5 (99.5) Redundancy 3.8 (3.9) 3.9 (3.5) 6.6 (6.5) Refinement No. reflections R work /R free 18.5/ / /21.6 No. atoms Overall B-factors (Å 2 ) r.m.s. deviations Bond length (Å) Bond angles ( ) Values in parentheses are for the highest resolution shell. 4

5 Table S2. Data collection and refinement statistics of methylated H3K36 peptide-kdm2a complexes with Ni 2+ and αkg. H3K36me1-KDM2A Ni 2+ + αkg H3K36me2-KDM2A Ni 2+ + αkg H3K36me3-KDM2A Ni 2+ + αkg Data collection Space group P P P Cell dimensions a, b, c (Å) 54.3, 86.5, , 86.5, , 84.6, Resolution (Å) 2.35 ( ) 2.35 ( ) 1.70 ( ) R merge 10.9 (41.6) 10.9 (41.6) 4.9 (49.2) I/σI 19.2 (3.2) 19.2 (3.2) 27.8 (2.3) Completeness (%) 98.8 (97.3) 98.8 (97.3) 96.6 (95.5) Redundancy 6.5 (4.6) 6.5 (4.6) 4.6 (4.6) Refinement No. reflections R work /R free 18.6/ / /22.3 No. atoms Overall B-factors (Å 2 ) r.m.s. deviations Bond length (Å) Bond angles ( ) Values in parentheses are for the highest resolution shell. 5

6 Table S3. Data collection and refinement statistics H3K36me1-KDM2A Data collection Space group Cell dimensions Fe 2+ + αkg + NO P21 a, b, c (Å) 55.1, 158.0, 48.7, β=89.9 Resolution (Å) 2.20 ( ) R merge 12.2 (48.2) I/σI 9.1(1.8) Completeness (%) 97.9 (97.5) Redundancy 2.9 (2.8) Refinement No. reflections R work /R free 24.3/27.6 No. atoms 7164 Overall B-factors (Å 2 ) 29.5 r.m.s. deviations Bond length (Å) Bond angles ( ) Values in parentheses are for the highest resolution shell. 6

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8 Figure S1. Structural details of H3K36me peptides bound to KDM2A. (A) Plot of B-factors at individual amino acid positions of the H3(A29-P43)K36 peptide in the H3K36me2-KDM2A complex. (B-D) Comparison of cutaway views of KDM2A catalytic pockets containing αkg and H3K36me1 (panel B), H3K36me2 (panel C) and H3K36me3 (panel D). (E) Stereo view of superposition of the H3K36me3-KDM2A complex containing αkg (in green) and NOG (in magneta). 8

9 Figure S2. Sequence alignments of H3 peptides and KDMs. (A) Sequence alignments centered about K36, K4, K9, K27 and K79 in H3 and K20 in H4. (B) Comparison of sequence alignment at positions 212, 214 and 222 in KDM2A with corresponding positions in PHF8, KIAA1718 (specificity for lower lysine methylation states) and KDM4A, KDM5A and KDM6B (specificity for higher lysine methylation states). 9

10 Figure S3. Sequence insertions in the KDM family of lysine demethylases and their structural implications. (A) Structure-based sequence alignment of core JmjC domains of histone lysine demethylases KDM2A/B, PHF8, KIAA1718, KDM6A/B, KDM4A and KDM5A. The insertion in the KDM2 subfamily is framed in magenta, while the insertions in the KDM4, KDM5 and KDM6 subfamilies are framed in blue. Stars indicate the metal binding ligands. (B) Stereo view of the 10

11 core JmjC domain structure of KDM2A showing the insertion loop that includes α4, in magenta. For other KDMs in panel A, the position of their insertion loop between α5 and α6 is boxed in blue. Metal-coordinating residues are shown in a stick representation. 11

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13 Figure S4. Comparison of crystal structures of H3K36me3-KDM2A and H3K36me3-KDM4A complexes. (A) Crystal structure of the H3K36me3-KDM2A complex. The bound H3K36me3 peptide is shown in yellow in a stick representation with the trimethyl group of K36 shown as dotted balls. The KDM2A backbone is colored in silver. The insertion loop of KDM2A is shown in magenta. (B) Crystal structure of the H3K36me3-KDM4A complex (PDB code: 2P5B). The bound H3K36me3 peptide is shown in beige in a stick representation with the trimethyl group of K36 shown as dotted balls. The KDM4A backbone is colored in green. The insertion loop of KDM4A is shown in blue. (C) A stereo view superposition of the catalytic pockets and inserted K36me3 side chain of the H3K36me3 (in yellow)-kdm2a (in silver) and H3K36me3 (in beige)- KDM4A (in green) complexes. Spheres denote the position of the metal ion. 13

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15 Figure S5. Comparison of crystal structures of H3K36me2-KDM2A and H3K4me3K9me2-PHF8 complexes. (A) Crystal structure of the H3K36me2-KDM2A complex. The bound H3K36me2 peptide is shown in yellow in a stick representation with the dimethyl group of K36 shown as dotted balls. The KDM2A backbone is colored in silver. The insertion loop of KDM2A is shown in magenta. (B) Crystal structure of the H3K4me3K9me2-PHF8 complex (PDB code: 3KV4). The bound H3K4me3K9me2 peptide is shown in beige in a stick representation with the trimethyl group of K4 and dimethyl group of K9 shown as dotted balls. The PHF8 backbone is colored in green. The PHD finger of PHF8 is shown in blue zinc ions are represented by yellow spheres. (C) A stereo view superposition of the catalytic pockets and inserted K36me2 side chain of the H3K36me2 (in yellow)-kdm2a (in silver) and H3K9me2 side chain of H3K4me3K9me2 (in beige)-phf8 (in green) complexes. Spheres denote the position of the metal ion and water molecule, as labeled. 15

16 Figure S6. Schematic of a proposed model of KDM2A-mediated demethylation as a function of H3K36 methylation state. Panel 1: Native Fe 2+ -containing KDM2A structure (PDB code: 2YU2). Panel 2: αkg bound structure of KDM2A in the 'off-line' binding mode (PDB code: 2YU1). Panels 3 and 4: αkg-bound structures of KDM2A with H3K36me2 and H3K36me3 peptides, respectively. The C1 carboxylate oxygen of αkg coordinates at axial position a, representative of the off-line binding mode. Panels 5 and 6: Proposed transition from off-line to in-line mode for oxygen and αkg-bound structures of KDM2A with H3K36me2 and H3K36me3 peptides, 16

17 respectively. The C1 carboxylate oxygen of αkg switches to in-plane position b, representative of the in-line mode. The off-line to in-line transition can occur for K36me2 (panel 3 to 5), but is likely hindered for K36me3 (panel 4 to 6) because of steric clash between the additional CH 3 group and the C1 carboxylate oxygen of αkg. Panels 6 and 7: Proposed reaction intermediates during the H3K36me2 demethylation process. 17

18 Figure S7. Anomalous maps identifying metal bound in the active center of the H3K36me3- KDM2A in the presence of αkg. The anomalous maps of Fe 2+ (in red) and Ni 2+ (in green) for the H3K36me3-KDM2A complex in the presence of αkg, both shown at 3σ. One data set was collected at the Fe K edge using a wavelength of Å and another was collected at the Ni K edge using a wavelength of Å on the same crystal of the complex. 18