genes & developmentgenesdev.cshlp.org/.../supplemental_material.docx · web view2014/02/24 ·...
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Supplemental Material:
Molecular basis underlying histone H3 lysine-arginine methylation pattern readout by Spin/Ssty repeats of Spindlin1
Xiaonan Su,1,2,6 Guixin Zhu,1,3,6 Xiaozhe Ding,1,2 Shirley Y. Lee,4
Yali Dou,4 Bing Zhu,5 Wei Wu,1 and Haitao Li1,2
1MOE Key Laboratory of Protein Sciences, School of Life Sciences, and
2Center for Structural Biology, Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing 100084, China
3Tsinghua-Peking Center for Life Sciences, Beijing, 100084, China
4Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA
5National Institute of Biological Sciences, Beijing 102206, China
6These authors contributed equally to this work:
Xiaonan Su & Guixin Zhu
Correspondence should be addressed to:
Haitao Li ([email protected]) and Wei Wu ([email protected])
Supplemental Material and Methods
Recombinant protein preparation
Spindlin1 50-262 (SPIN150-262) was cloned on pRSFDuet vector with N-terminal 6xHis tag. Recombinant SPIN150-262 was overexpressed in E.coli BL21 (DE3). After overnight induction by 0.2 mM isopropyl β-D-thiogalactoside (IPTG) at 16 °C in LB medium, cells were harvested and suspended in buffer: 0.2 M NaCl, 20 mM Tris, pH 8.0. After cell lysis and centrifugation, the 6xHis SPIN150-262 was purified to homogeneity over successive HisTrap, anion exchange Q, and Superdex G75 columns (GE Healthcare). The protein was concentrated to 20 mg ml-1 in 0.2 M NaCl, 20 mM Tris, pH 8.0 for future use.
Human MLL1 SET domains, WDR5, RbBP5, and Ash2L were cloned on a pET28a-based vector, containing 6xHis-Sumo tag. Individual plasmids were transformed into E.coli BL21 (DE3) codon plus for expression. Cells were cultured in 1-L LB media at 37 ºC and induced by final IPTG concentration of 0.3 mM at 20 °C. Cells were harvested 16 hours post-induction and resuspended in BC500 buffer (25 mM Tris, pH 8.0, 500 mM NaCl, and 20% glycerol) with 0.01% NP-40, 1 mM PMSF, and 0.1 mM Benzamidine for sonication. Crude lysate was collected after centrifugation and incubated with Ni-NTA resin for 1 hour at 4 ºC. Unbound protein was washed away with BC500 buffer and 20 mM imidazole, and free protein was collected by overnight ULP1 cleavage at 4 ºC.
Human PRMT2 was cloned in the pGEX-6p2 vector. Plasmid was transformed into BL21 (DE3) Codon plus for expression as described above. Protein induction was achieved with 50 µM IPTG at 20 ºC. Cells were harvested after 16 hours of incubation and resuspended in BC500 buffer containing 0.01% NP-40, 1 mM PMSF, and 0.1 mM Benzamidine. After sonication, crude lysate was collected, and ~20 μg of PRMT2 was incubated with 40 µL GST resin for 2 hours at 4 ºC. Unbound protein was washed away with additional BC500 buffer, followed by three washes with 25 mM Tris, pH 8.0 and 150 mM NaCl to equilibrate the resin for subsequent enzymatic reactions.
Crystallization conditions
All crystals were grown by the sitting-drop vapor-diffusion method at 18 °C. Prior to crystallization, protein was mixed with peptide in 1:1.5 ratio and kept on ice for 30 min. Drops were generated by mixing 1 μl of complex solution with 1 ul of reservoir solution. The SPIN150-262-H31-20K4me3 crystal was obtained from a reservoir solution containing: 37.5% MPD_P1K_P3350, 0.1 M 0.1 M bicine/Trizma, pH 8.5, and 0.06 M MgCl2/CaCl2 (Morpheus® Reagents, Molecular Dimensions). Crystals of H31-10K4me3R8me2a and H31-10K4me3R8me2s complexes were obtained from a same reservoir solution containing: 20% PEG8000, 20% PEG400, 0.1 M MgCl2 and 0.1M Tris-HCl, pH 8.5.
Antibodies and reagents
Anti c-Myc (sc-40) was purchased from Santa Cruz Biotechnology. Anti-FLAG M2 and anti-FLAG M2-Agarose were purchased from Sigma-Aldrich. Anti-Spindlin1 (ab118784) and anti-PRMT2 (ab3763) were purchased from Abcam. Anti-H3K4me3 (07-473) was purchased from Millipore. Anti-H3R8me2a (NB21-1062) was purchased from Novus Biologicals. Axin2 (2151S), Histone 3 (9715S) and Lamin A/C (4777S) were purchased from Cell Signaling Technology. Anti-Cyclin D1 (K0062-3) was purchased from MBL.
Transfection and RNA interference
Plasmid DNA and siRNA transfections were performed using VigoFect transfection reagent (Vigorous Biotech) or Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions, respectively. Spindlin1 siRNAs have been validated previously (Wang et al. 2011). siRNA sequences for Spindlin1 and PRMT2 were as follows:
Spindlin1-1: 5’-GCAAAGCAGUGGAACAUAU-3’;
Spindlin1-2: 5’- GCAUUAUGCCUGAUUCCAA-3’;
PRMT2: 5’- GCAGACCAGCCACGAACAA -3’;
Supplemental Table S1 Summary of thermodynamic parameters from isothermal titration calorimetry binding assays
Protein
Peptides
ΔH (kcal/mol)
ΔS
(cal/mol/deg)
Kd (μM)
N
SPIN150-262
H31-6K4me3
-15.5
-20.6
0.147
1.06
H31-10K4me3
-15.5
-20.9
0.147
1.06
H31-20K4me3
-12.6
-14.2
0.750
1.08
H31-10K4me3R8me1
-14.6
-17.7
0.139
1.04
H31-10K4me3R8me2s
-16.8
-23.4
0.066
1.00
H31-10K4me3R8me2a
-18.9
-29.8
0.045
1.00
H31-10K4me2R8me2a
-11.8
-10.9
0.510
1.00
H31-10K4me1R8me2a
-10.9
-13.3
8.40
1.07
H31-10R8me2a
-9.4
-10.3
21.7
1.05
H31-10R8me2s
-8.7
-10.1
71.4
1.00
H31-10un
-7.3
-7.1
156
0.97
TCF4458-469
-4.0
11.4
3.88
1.01
E142A
H31-10K4me3R8me2a
-16.1
-22.3
0.115
1.08
D184A
H31-10K4me3R8me2a
-11.7
-14.4
3.97
1.03
Y170A
H31-10K4me3R8me2a
N.D.
N.D.
N.D.
N.D.
Y177A
H31-10K4me3R8me2a
-2.98
10.6
31.8
1.13
F141A
H31-10K4me3R8me2a
-12.1
-9.04
3.32
0.99
W62A
H31-10K4me3R8me2a
-12.1
-9.15
0.128
0.99
W72A
H31-10K4me3R8me2a
-13.7
-14.6
0.141
0.94
F251A
H31-10K4me3R8me2a
-16.4
-22.4
0.072
0.93
W72R
H31-10K4me3R8me2a
-11.5
-9.44
0.415
1.05
Y98R
H31-10K4me3R8me2a
-8.65
-2.04
1.27
0.98
F251R
H31-10K4me3R8me2a
-9.41
-5.14
1.70
0.99
H31-6K4me3: NH2-ARTK(me3)QT-NH2
H31-20K4me3: NH2-ARTK(me3)QTARKSTGGKAPRKQL-COOH
H31-10un: NH2-ARTKQTAR(me1)KS-COOH
H31-10R8me2s: NH2-ARTKQTAR(me2s)KS-COOH
H31-10R8me2a: NH2-ARTKQTAR(me2a)KS-COOH
H31-10K4me3R8me1: NH2-ARTK(me3)QTAR(me1)KS-COOH
H31-10K4me3R8me2s: NH2-ARTK(me3)QTAR(me2s)KS-COOH
H31-10K4me3R8me2a: NH2-ARTK(me3)QTAR(me2a)KS-COOH
H31-10K4me1R8me2a: NH2-ARTK(me1)QTAR(me2a)KS-COOH
H31-10K4me2R8me2a: NH2-ARTK(me2)QTAR(me2a)KS-COOH
TCF4458-469: NH2-RRKKKCVRYIQG-COOH
N.D. not detectable
PRIMER ID
SEQUENCES
APLICATION
Axin2_F
AGTGTGAGGTCCACGGAAAC
cDNA
Axin2_R
CTTCACACTGCGATGCATTT
cDNA
Cyclin D1_F
CCGTCCATGCGGAAGATC
cDNA
Cyclin D1_R
ATGGCCAGCGGGAAGAC
cDNA
ID2_F
TCAGCCTGCATCACCAGAGA
cDNA
ID2_R
CTGCAAGGACAGGATGCTGAT
cDNA
Tiam1_F
AAGACGTACTCAGGCCATGTCC
cDNA
Tiam1_R
GACCCAAATGTCGCAGTCAG
cDNA
RPLP0_F
ACCCAGCTCTGGAGAAACTGC
cDNA
RPLP0_R
TGAGGTCCTCCTTGGTGAACA
cDNA
Spindlin1_F
ACCCCATTCGGAAAGACACC
cDNA
Spindlin1_R
CCATTCCCCTCTTTCCACCC
cDNA
PRMT2_F
GAGTCCATCCTGTATGCCCG
cDNA
PRMT2_R
GTGCACGGTTCAGAGAGACA
cDNA
Axin2_C_F
CTGGAGCCGGCTGCGCTTTGATAA
ChIP
Axin2_C_R
CGGCCCCGAAATCCATCGCTCTGA
ChIP
Cyclin D1_C_F
GGGCTTTGATCTTTGCTTAAC
ChIP
Cyclin D1_C_R
ACTCTGCTGCTCGCTGCTAC
ChIP
Negative control_C_F
CTCCTCCTCCCCTCTGGTCTTTCC
ChIP
Negativecontrol_C_R
CCAATCTGTTCTGCCCACTCCATC
ChIP
Sat2_C_F
CATCGAATGGAAATGAAAGGAGTC
ChIP
Sat2_C_R
ACCATTGGATGATTGCAGTCAA
ChIP
β-globin_C_F
AGGACAGGTACGGCTGTCATC
ChIP
β-globin_C_R
TTTATGCCCAGCCCTGGCTC
ChIP
c-Myc_C_F
ACAGGCAGACACATCTCA
ChIP
c-Myc_C_R
GCCACGTATACTTGGAGA
ChIP
Supplemental Table S2 Primer sequences used in this paper
Supplemental Figure S1 Alignment of H3 peptides in different H3K4me3-Spindlin1 complexes. (A) Simulated annealing Fo-Fc omit map (1.7 Å, countered at 2.5 σ level) around the H3K4me3 peptide. H3K4me3 peptide was omitted for simulated annealing (starting temperature 2500 K and 500 cooling steps) omit maps generation by PHENIX (B) The C-terminal segment of histone H3K4me3 peptide adopts different conformations in the SPIN50-262-H31-20K4me3 complex reported here compared to the previous one24 (PDB: 4H75). Notably, H3R8 adopts an orientation pointing to pocket 1 in the current structure (colored in yellow), while in the reported one, H3R8 points oppositely to pocket 2 and forms hydrogen bonding interactions with D173 and Y177 (colored in magenta).
Supplemental Figure S2 Structure and sequence alignment of Spin/Ssty repeat with other ‘Royal’ family members. (A) Superimposition of Spindlin1 Spin/Ssty1 with PHF1 Tudor (PDB: 4HCZ; RMSDcα = 2.95 Å, 48 residues), HP1 Chromo (PDB: 1KNA; RMSDcα = 3.13 Å, 40 residues), HDGF PWWP (PDB: 3QBY; RMSDcα = 3.51 Å, 48 residues), and L3MBTL1 MBT (PDB: 2RHZ; RMSDcα = 5.57 Å, 56 residues) domains. Spin/Ssty1 is colored in cyan. Spin/Ssty displays the highest similarity with Tudor domain. Signature differences in secondary structural elements are highlighted by black arrows. (B) Structure-based sequence alignment among Spin/Ssty, Tudor, PWWP, Chromo, and MBT domains.
Supplemental Figure S3 Structural comparison of triangular-shaped triple-repeats proteins: Spindlin1 and L3MBTL1. (A) Global structure of Spindlin1 triple Spin/Ssty repeats. (B) Global structure of L3MBTL1 triple MTB repeats. The opening of the β-barrel is facing outwards in Spindlin1, and inwards in L3MBTL1. Also note the ‘clockwise’ and ‘anti-clockwise’ arrangement of the triple modules in the case of Spindlin1 and L3MBTL1, respectively.
Supplementary Figure 3
Supplemental Figure S4 Simulated annealing Fo-Fc omit map around (A) the H3K4me3R8me2a peptide (2.1 Å, countered at 2.5 σ level) and (B) the H3K4me3R8me2s peptide (2.2 Å, countered at 2.5 σ level). In both cases, H3 peptide was omitted for simulated annealing (starting temperature 2500 K and 500 cooling steps) omit maps calculation by PHENIX. Note the poor electron densities after H3 Q5 in (B). T6 and A7 was modeled based on electron density at lower signal level.
Supplementary Figure 3
Supplemental Figure S5 Dot-blot assay of anti-H3K4me3 and anti-H3R8me2a antibodies against dually modified H3K4me3R8me2a peptides. Though sensitivities were slightly compromised, both antibodies were tolerable to additional methylations adjacent to their primary epitope.
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