Regulation of Stability on Histone H2A H2B Dimer by H2A Tyr57 Phosphorylation

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1 Supporting information Regulation of Stability on istone 2A 2B Dimer by 2A Tyr57 Phosphorylation Takuma Sueoka, Gosuke ayashi, and Akimitsu kamoto*, Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, ongo, Bunkyo-ku, Tokyo , Japan, Research Center for Advanced Science and Technology, The University of Tokyo, Komaba, Meguro-ku, Tokyo , Japan. Corresponding author: Akimitsu kamoto Department of Chemistry and Biotechnology, The University of Tokyo, ongo, Bunkyo-ku, Tokyo , Japan. Tel: Fax: Table of contents Scheme S1 Figure S1. Figure S2. Figure S3 Figure S4 Figure S5 Figure S6 Figure S7 Figure S8 Figure S9 Figure S10 S1

2 Y57 P 2A(1 46) 1 2 S S 2A(48 85) + 2 2A(87 129) CL P S 2A(48 85) S 2A(87 129) 4 P Thz-Cys conversion S 2 2A(48 85) S 2A(87 129) 5 CL P 2A(1 46) S 2A(48 85) 6 S 2A(87 129) Desulfurization Y57 P 2A(1 46) 2A(48 85) 7 2A(87 129) Scheme S1 Synthetic route of histone 2A phosphorylated at Y57. S2

3 Figure S1. Synthesis and identification of peptide 2. (a) PLC charts of crude and purified peptide 2. All the spectra were monitored at 220 nm absorbance. A linear gradient of water-acetonitrile containing 0.1% TFA was applied. The gradient started from 40% to 60% acetonitrile for 20 min. (b) MALDI-TF mass spectrum of peptide 2. [Peptide 2 + ] + : found (calcd ). S3

4 Figure S2. Synthesis and identification of peptide 4 and 5. (a) PLC charts for the ligation reaction and Thz deprotection. All the spectra were monitored at 220 nm absorbance. A linear gradient of water-acetonitrile containing 0.1% TFA was applied. The gradient started from 30% to 70% acetonitrile for 20 min. (b) MALDI-TF mass spectrum of peptide 4. [Peptide 4 + ] + : found (calcd ). (c) MALDI-TF mass spectrum of peptide 5. [Peptide 5 + ] + : found (calcd ). S4

5 Figure S3. Synthesis of peptide 7 using MPAA as a thiol catalyst. All the PLC spectra were monitored at 220 nm absorbance. A linear gradient of water-acetonitrile containing 0.1% TFA was applied. The gradient started from 20% to 70% acetonitrile for 25 min. (a) Synthetic scheme of peptide 7 by using MPAA. Dialysis was performed under 4 C before desulfurization. (b) PLC charts for the ligation reaction. (c) PLC charts of desulfurization reaction. The reaction mixture before and after desulfurization were shown in blue and red line, respectively. (d) MALDI-TF mass spectrum of peptide 7. [Peptide 7 + ] + : found (calcd ). (e) PLC chart of purified peptide 7. S5

6 Figure S4. In vitro refolding of 2A 2B dimer. (a) Size-exclusion chromatography for 2A 2B dimer using recombinant 2A (top), synthetic 2A (middle), and synthetic 2A bearing Y57ph (bottom). All the spectra were monitored at 280 nm absorbance. (b) SDS-PAGE analysis of the purified 2A 2B dimer. Lane 1: Broad Range Protein Molecular Weight Markers (Promega), lane 2 4: 2A 2B dimer containing recombinant 2A, synthetic 2A, and Y57ph-2A, respectively, lane 5: 2A, lane 6: 2B. (c) PLC charts for the confirmation of histone stoichiometry using the purified 2A 2B dimer. All the spectra were monitored at 220 nm absorbance. A linear gradient of water-acetonitrile containing 0.1% TFA was applied. The gradient started from 30% to 60% acetonitrile for 30 min. (d) Peak areas of PLC charts in Figure S4(c). S6

7 Figure S5. Thermal shift assay with 2A 2B dimer by using SYPR range. The assays with 2A-2B dimer containing recombinant 2A and Y57ph-2A are represented by blue circles and red triangles, respectively. (a) Thermal shift assay in 1 M acl and 10 mm Tris-Cl buffer. (b) Thermal shift assay in 0.25 M acl and 10 mm Tris-Cl buffer. S7

8 Figure S6. Thermal shift assay with 2A 2B dimer containing synthetic 2A by using SYPR range. (a) Thermal shift assay in 2 M acl and 10 mm Tris-Cl buffer. (b) Thermal shift assay in 1 M acl and 10 mm Tris-Cl buffer. (c) Thermal shift assay in 0.25 M acl and 10 mm Tris-Cl buffer. S8

9 Figure S7. In vitro refolding of histone octamer. (a) Size-exclusion chromatography for histone octamer using recombinant 2A (top), synthetic 2A (middle), and synthetic 2A bearing Y57ph (bottom). All the spectra were monitored at 280 nm absorbance. (b) SDS-PAGE analysis of the purified octamer. Lane 1 and 5: Broad Range Protein Molecular Weight Markers (Promega), lane 2 4: octamer containing recombinant 2A, synthetic 2A, and Y57ph-2A, respectively. (c) PLC charts for the confirmation of histone stoichiometry using the purified octamer. All the spectra were monitored at 220 nm absorbance. A linear gradient of water-acetonitrile containing 0.1% TFA was applied. The gradient started from 30% to 60% acetonitrile for 50 min. (d) Peak areas of PLC charts in Figure S7(c). S9

10 Figure S8. ative-page analysis of reconstituted nucleosomes. Lane 1: 100 bp DA ladder, lane 2: 193 bp 601 DA fragment, lane 3: nucleosome with 193 bp DA containing recombinant 2A, lane 4: nucleosome with 193 bp DA containing synthetic Y57ph-2A. S10

11 Figure S9. ative-page analysis of Mase assay. The treatment was conducted at 37 C for 5 min. The nucleosome containing recombinant 2A was treated with 0.5, 1, and 2 units of Mase (lane 4, 5, and 6, respectively). Also, the nucleosome containing 2A-Y57ph was treated with 0.5, 1, and 2 units of Mase (lane 7, 8, and 9, respectively). Lane 1 and 10 indicate 10 bp DA ladder. Lane 2 and 3 indicate 193 bp and 145 bp DA fragments. S11

12 Figure S10. (a) ative-page analysis of reconstituted nucleosomes. Lane 1: 100 bp DA ladder, lane 2: 193 bp 601 DA fragment, lane 3: nucleosome with 193 bp DA containing synthetic 2A. (b) ative-page analysis of Mase assay. The treatment was conducted at 23 C for 5 min. The nucleosome containing synthetic 2A was treated with 0.5, 1, and 2 units of Mase (lane 2, 3, and 4, respectively). Lane 1 indicate 10 bp DA ladder. Lane 5 and 6 indicate 145 bp and 193 bp DA fragments. S12