Regulation of the Stability of the Histone H2AâH2B Dimer by H2A

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Regulation of Stability on Histone H2AH2B Dimer by H2A Tyr57 Phosphorylation Takuma Sueoka, Gosuke Hayashi, and Akimitsu Okamoto Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00504 • Publication Date (Web): 16 Aug 2017 Downloaded from http://pubs.acs.org on August 18, 2017

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Regulation of Stability on Histone H2A–H2B Dimer by H2A Tyr57 Phosphorylation Takuma Sueoka†, Gosuke Hayashi†, and Akimitsu Okamoto*†,‡ †

Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of

Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan, ‡

Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba,

Meguro-ku, Tokyo 153-8904, Japan.

Corresponding author: Akimitsu Okamoto Department of Chemistry and Biotechnology, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. Tel: +81-3-5452-5200 Fax: +81-3-5452-5209 E-mail: [email protected]

ABSTRACT: Histone H2A and H2B form a H2A–H2B heterodimer, which is a fundamental unit of nucleosome assembly and disassembly. Several posttranslational modifications change the interface between the H2A–H2B dimer and the H3–H4 tetramer, and regulate nucleosome stability. However, posttranslational modifications associated with the interface between H2A and H2B have not been discussed. In this paper, it is shown that Tyr57 phosphorylation in H2A strongly influences H2A–H2B dimerization. Tyr57-phosphorylated H2A was chemically synthesized and utilized to reconstitute the H2A–H2B dimer and nucleosome as well as canonical H2A. Thermal shift assays showed that phosphorylation destabilized the dimer and facilitated dissociation of H2A and H2B from the nucleosome structure. The proximity between H2A Tyr57 and the H2B αC-helix is assumed to lead the destabilization. DNA accessibility of the nucleosome was estimated by using micrococcal nuclease. The phosphorylated nucleosome did not change DNA accessibility compared to canonical nucleosome. It is demonstrated that phosphorylation at Tyr57 changes the H2A–H2B dimer interaction, but does not interfere with histone–DNA interactions. This work on the destabilization of the H2A–H2B dimer by Tyr57 phosphorylation is a promising step to elucidate control mechanisms of dynamic behavior of H2A and H2B through posttranslational modifications.

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Introduction Posttranslational modifications (PTMs) in histone proteins have essential roles for DNA replication, transcription, and repair. PTMs generally affect three types of interactions: (1) histone–DNA interactions, (2) histone–histone interactions, and (3) histone–chaperone interactions.1 An example of a histone–DNA interaction is acetylation at H3 Lys56 (H3-K56ac), which is located near the DNA entry-exit site and facilitates nucleosome unwrapping.2 Compared with many reports in which PTMs control histone–DNA and histone–chaperone interactions, there are relatively few reports on PTMs associated with histone–histone interactions. Acetylation and ubiquitination at H4-K91 have a potential to disturb the interface between the H2A–H2B dimer and the H3–H4 tetramer.3,4 Recently, Davis and co-workers investigated the biochemical properties of H2A-T101GlcNAc and showed that it destabilized the nucleosome structure; they also suggested a ‘trapping’ model in which T101GlcNAc keeps the H2A–H2B dimer unbound to tetrasome.5 These several PTMs alter the interaction between the H2A–H2B dimer and the H3–H4 tetramer. However, to our knowledge, PTMs that affect the interaction between H2A and H2B have not been reported. Phosphorylation of Tyr57 in H2A (H2A-Y57ph) is related to the regulation of transcriptional elongation.6 The phosphorylation affects the level of ubiquitination of lysine in H2B (H2B-K120ub), and trimethylation of lysine in H3 (H3-K4me3 and H3-K79me3), which is a marker of active gene expression. One of the suggested mechanisms of transcriptional elongation is that H2A-Y57ph inhibits deubiquitinase activity against H2B-K120ub. Therefore, eviction of the ubiquitinated H2A–H2B dimer by FACT (facilitates chromatin transcription complex) is promoted and then RNA polymerase II (RNA pol II) acts on the nucleosome-unformed region. On the other hand, direct effects of Y57ph against histone complexes such as the H2A–H2B dimer and nucleosome remain unclear. In this study, we demonstrate that H2A-Y57ph strongly regulates the stability of the histone H2A–H2B dimer. Chemically synthesized H2A, with phosphate modification at Y57, was applied to investigate the effect of the phosphorylation on the H2A–H2B dimer and nucleosome stability in vitro. We have also clarified whether the change of histone–histone interaction affects histone–DNA interaction, based on the comparison of the accessibility of nucleosomal DNA to Y57-phosphorylated nucleosome and canonical nucleosome.

MATIRIALS AND METHODS General methods and materials. The MALDI-TOF mass spectra were obtained on microflex (BRUKER), using Protein Calibration Standard I as an external standard. All peptide fragments were synthesized through 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS) using Intavis ResPep SL (Intavis). Reversed-phase (RP) HPLC was performed on a 5C18-AR-300 and Protein-R column (4.6ID × 250 mm for analysis and 10ID × 250 mm for 2


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purification, Nacalai) with a PU-2080 plus Intelligent HPLC Pump (JASCO) and MD-2018 plus Photodiode Array Detector (JASCO) at 195 to 650 nm. Solvents and reagents were purchased from Wako Pure Chemical Industries, Ltd., Watanabe Chemical Industries, Ltd., and MERCK Millipore, and used without further purification. The yields of SPPS or native chemical ligation (NCL)7 reactions were estimated as isolated yields, in which the molecular weights of individual peptides were calculated as trifluoroacetic acid (TFA) salts at Arg and Lys positions. Synthesis of peptide fragments. Peptide fragment 1, 2, and 3 were synthesized as previously described.8 Regarding peptide 2, Fmoc-Tyr(PO(OBzl)OH)-OH (MERCK Millipore) was used for the introduction of Y57ph. After purification, peptide 2 was obtained in 11% yield. 1st NCL. To the powder of peptide 2 (1 eq) and peptide 3 (1.5 eq), ligation buffer (6 M guanidine hydrochloride (Gn·HCl), 0.2 M sodium phosphate, 200 mM 4-mercaptophenylacetic acid (MPAA), and 50 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl), pH 7.2) was added. The final concentration of peptide 2 was fitted to about 3 mM. The reaction was conducted at 37 °C, and was traced by RP-HPLC. The ligation product 4 was identified by MALDI-TOF mass spectrometry. After the completion of the NCL reaction, 400 mM methoxyamine hydrochloride was added to the mixture to convert N-terminal thiazolidine (Thz) group to cysteine (Cys) residue. The reaction was conducted at 37 °C, and was traced by RP-HPLC. The ligation product 5 was identified by MALDI-TOF mass spectrometry. 2nd NCL. To synthesize Y57-phosphorylated H2A (peptide 7), we conducted the second NCL reaction in the presence of MPAA and performed dialysis to remove it prior to desulfurization. Peptide 1 (1.5 eq) and peptide 5 (1 eq) were added to a ligation buffer containing 6 M Gn·HCl, 0.2 M sodium phosphate, 100 mM MPAA, and 50 mM TCEP·HCl. The ligation buffer was fitted to pH 7.2 with 6 M NaOH aq. The concentration of MPAA was reduced compared to the 1st NCL to easily remove MPAA in the subsequent dialysis step. The reaction rate did not change significantly between 100 mM and 200 mM of MPAA.9 The final concentration of peptide 5 was adjusted to 3 mM. The reaction mixture was stirred at 37 °C and monitored by RP-HPLC at 220 nm. After the reaction was completed, the reaction mixture was dialyzed against 100 mL of buffer containing 6 M Gn·HCl and 0.2 M sodium phosphate twice under 4 °C. The desulfurization was subsequently conducted by the addition of the same amount of aqueous solution containing 600 mM TCEP·HCl, 8%

v/v

2-methyl-2-propanethiol,

and

80

mM

2,2’-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (VA-044). pH was fitted to 6.0–7.0 with 6 M NaOH aq. The reaction mixture was stirred at 37 °C and monitored by RP-HPLC at 220 nm. The yield of peptide 7 was 54% from peptide 5. Refolding of H2A–H2B dimer. Equimolar of histone H2A and H2B were dissolved to a concentration of approximately 1.0 mg mL−1 in unfolding buffer (20 mM Tris-HCl (pH 7.5), 7 M Gn·HCl, and 20 mM 2-mercaptoethanol). After the denaturation by incubation for 3 h on ice, the 3


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mixture was dialyzed against 1 L of dialysis buffer (10 mM Tris-HCl (pH 7.5), 2 M NaCl, and 2 mM 2-mercaptoethanol) at 4 °C at least 3 times, by using Oscillatory Cup MWCO 8000 (Cosmo Bio, BTE-212949). The mixture was concentrated by Amicon Ultra (3 K, 0.5 mL) and then, purified by size-exclusion chromatography with a SuperdexTM 200 Increase 5/150 GL column. Histone stoichiometry was confirmed by 18% sodium dodecyl sulfate (SDS) poly-acrylamide gel electrophoresis (PAGE) and HPLC analysis. Nucleosome reconstitution. Equimolar of histone H2A, H2B, H3, and H4 were dissolved to a concentration of approximately 1.5 mg mL−1 in the unfolding buffer described above. As described for refolding of H2A–H2B dimer, denaturation, refolding, and purification were performed to prepare histone octamer. Histone stoichiometry was confirmed by 18% SDS-PAGE and HPLC analysis. After the refolding, nucleosome was reconstituted through salt-dialysis method. Purified octamer solution (1 eq) was added to 145 bp or 193 bp of 601DNA solution (0.7 eq, containing 2 M NaCl, and 10 mM Tris-HCl, pH 7.5). The final concentration of octamer was adjusted to around 0.3 mg/mL. The mixture was dialyzed against 1 L of dialysis buffer (10 mM Tris-HCl (pH 7.5), 2 M NaCl, and 1 mM 2-mercaptoethanol) for 16 h at room temperature. NaCl concentration was gradually decreased by changing dialysis buffer. Prepared nucleosome was analyzed by 6% native PAGE (acrylamide : bis-acrylamide = 29 : 1). Thermal shift assay by using SYPRO Orange (Estimation of histone-histone interaction). To measure the stability of the H2A–H2B dimer, the dimer solution containing the other two different NaCl concentration (1 M and 0.25 M) were prepared through dialysis from the purified dimer solution containing 2 M NaCl. SYPRO Orange (SIGMA-ALDRICH, S5692, 5000× solution) was diluted by the solution of NaCl (2, 1, or 0.25 M) and Tris-HCl (10 mM) to adjust 50×. 18 µL of the dimer solution was added to 2 µL of the diluted SYPRO Orange solution (final SYPRO Orange concentration, 5×). After the incubation of the samples at 15 °C for 5 min, the fluorescence measurements were obtained at each 1 °C step from 15 °C to 95 °C, using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). The temperature of the samples was maintained for 1 min after increasing the temperature. The channel 2 (HEX, excitation wavelength: 515–535 nm, detection wavelength: 560–580 nm) was used for detecting the fluorescence of SYPRO Orange. The Tm values were determined by the maximum of the first derivative of the fluorescence curves. As for the nucleosome, the stability was estimated in the solution containing 0.25 M NaCl, 10 mM Tris-HCl, 1 mM 2-mercaptoethanol, and 5× SYPRO Orange. The total volume was adjusted to 20 µL. The fluorescence measurements were obtained at each 1 °C step from 25 °C to 95 °C. The equipment and the detection conditions were the same as the measurement of H2A–H2B dimer. MNase assay (Estimation of histone-DNA interaction). To the nucleosome solutions composed of 193 bp DNA, MNase buffer (20 mM Tris-HCl (pH 7.5), 20 mM NaCl, and 5 mM 4


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CaCl2) was added, and then 0.5, 1, or 2 units of MNase (TaKaRa) were added to adjust the final volume to 20 µL. After 5 min incubation at 23 °C or 37 °C, the digestion was stopped by addition of 0.5 M EDTA. Subsequently, proteinase K (TaKaRa) was added to the mixture and incubated at 23 °C for 30 min to digest all of the proteins. DNA fragments were purified by ethanol precipitation and then analyzed by 8% native-PAGE (acrylamide : bis-acrylamide = 19 : 1).

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Figure 1. The position of Tyr57 in H2A (PDB ID: 3AFA). Histone H2A, H2B, and Y57 are colored yellow, cyan, and red, respectively. (a) Whole view of the nucleosome. (b) Enlarged view of the H2A–H2B dimer. Y57 is adjacent to the αC-helix of histone H2B.

RESULTS AND DISCUSSION Chemical Synthesis of Y57-Phosphorylated H2A. Crystal structures of the nucleosome show that Tyr57 in H2A is adjacent to the αC-helix of H2B (ca. 3.2 Å) and is located in the center of the handshake motif of the H2A–H2B dimer (Figure 1). Therefore, we assumed that this phosphorylation affects the stability of the H2A–H2B dimer. Moreover, this proximity suggests that Y57ph itself promotes transcriptional elongation by destabilizing the nucleosome structure, in addition to the inhibition of deubiquitination. Given that many research groups have demonstrated that the H2A–H2B dimer is a fundamental unit of nucleosome turnover,10 the biochemical properties of Y57ph and the change of the stability of the H2A–H2B dimer is worth investigating. To estimate the effect of H2A-Y57ph, it was necessary to prepare phosphorylated histone H2A in high purity. Chemical protein synthesis through SPPS and NCL enables the desired PTMs to be introduced in a specific position.11–13 This promising method is particularly advantageous for the introduction of Y57ph, which is located in the histone fold domain. Although casein kinase 2 (CK2) was identified as a kinase of H2A-Y57, the use of CK2 is not suitable for the preparation of pure Y57-phosphorylated H2A because of unselective phosphorylation of the kinase. Recently, our group reported the total chemical synthesis of H2A and its application for in vitro and in cell assays.8 Chemically synthesized H2A has also been applied in studies on H2B deubiquitination14 and H2A glutamine methylation15 by several research groups. To synthesize H2A containing Y57ph, we divided the whole sequence of H2A into three fragments at Ala47 and Ala86 residues (Scheme S1),8 which were mutated to Cys to utilize NCL and subsequent desulfurization reaction.16 All of the fragments were synthesized through Fmoc-SPPS. Phosphorylated tyrosine protected by a benzyl group (Fmoc-Tyr(PO(OBzl)OH)-OH) was introduced at the 57th position in fragment 2 (Figure S1). Fragments 1 and 2 had N-acyl-benzimidazolinone (Nbz) as a C-terminal leaving group for the NCL reaction.17 The first NCL reaction between peptide 2 and 3 completed in 4 h, and one-pot deprotection of the N-terminal Thz group generated peptide 5 in 54% yield (Figure S2). The second NCL was performed in the presence of MPAA, which is widely used as a thiol catalyst.18 After the NCL reaction, MPAA was removed through dialysis, because it inhibits radical reaction such as desulfurization (Figure S3). These steps from the second NCL to desulfurization provided the full-length phosphorylated H2A in around 54% yield.

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Control of the interaction between H2A and H2B by Y57ph. To apply the phosphorylated H2A for in vitro assay, the H2A–H2B dimer was refolded through denaturation of each of the histones and successive dialysis.19 Analytical charts of size-exclusion chromatography showed that the dimer with the phosphorylated H2A eluted at similar retention time compared to the dimer with recombinant H2A (Figure S4). The result indicates that Y57ph does not inhibit the formation of the H2A–H2B dimer under high-salt reconstitution conditions. We examined the thermal stability of the H2A–H2B dimer with Y57ph. Differential scanning fluorimetry (DSF) is one method that can be used to estimate the stability of protein–protein interactions.20 In the assay, SYPRO Orange is typically used as a fluorescent dye that binds to the hydrophobic regions of proteins. The method can also be applied to estimate nucleosome stability. Previous reports studied the effects of histone variants or PTMs on nucleosome structure.8,21 We expected that DSF would provide a powerful way to estimate the effect of H2A-Y57ph on the stability of the H2A–H2B dimer and nucleosome. First, we prepared the recombinant H2A–H2B dimer and the phosphorylated H2A–H2B dimer in 2, 1, 0.25 M NaCl solution, respectively. The results of the assay showed that the melting temperature of the dimer composed of H2A with Y57ph was lower than that of the canonical dimer. In 2 M NaCl solution, whereas the Tm of the recombinant dimer was 70 °C, the Tm of the phosphorylated dimer was 63 °C (Figure 2). In 1 and 0.25 M NaCl conditions, the phosphorylated dimer was also unstable compared with the canonical dimer, with a difference in Tm of about 10 °C (Table 1 and Figure S5). The phosphorylated dimer may be disrupted to some extent under 0.25 M NaCl conditions, because the background fluorescence was high at 15 °C (Figure S5(b)). Both recombinant and phosphorylated dimers became unstable upon decreasing the NaCl concentration from 2 M to 1 and 0.25 M. This trend, together with the Tm values, is consistent with previous reports that cite hydration effects and screening of electric interaction by salts.22,23 The consistency of Tm values indicates that the SYPRO Orange assay is suitable for evaluating H2A–H2B dimer interactions as well as other thermal shift assays such as differential scanning calorimetry (DSC) measurements. It is worth noting that artifact in protein chemical synthesis was negligible. Tm values of the dimer containing synthetic non-modified H2A were identical to those of the recombinant dimer (Table 1 and Figure S6). Taken together, the phosphorylation in Tyr57 clearly destabilizes H2A–H2B interactions.

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Figure 2. Thermal shift assays with the H2A–H2B dimer by using SYPRO Orange. The fluorescence intensity at each temperature is plotted. The measurements were performed in 2 M NaCl buffer. Assays with the H2A–H2B dimer containing recombinant H2A and H2A-Y57ph are represented by blue circles and red triangles, respectively.

Table 1. Tm values of H2A–H2B dimers. NaCl concentration

Entry

a

2M

1M

0.25 M

H2A(recombinant)–H2B

70 °C

62 °C

50 °C

H2A(synthetic)–H2B

71 °C

62 °C

51 °C

H2A(Y57ph)–H2B

63 °C

54 °C

(37 °C)a

The value was calculated from the data containing a high background fluorescence at 15 °C

(Figure S5(b)).

Destabilization of the nucleosome structure by Y57ph. We investigated the effect of the destabilized H2A–H2B dimer against whole nucleosome. To prepare the nucleosome bearing Y57ph, the histone octamer containing the phosphorylated H2A was refolded and purified as described for the preparation of the H2A–H2B dimer (Figure S7). The nucleosome was reconstituted by using 145-bp 601DNA through the salt-dialysis method as described before. After decreasing the NaCl concentration to 20 mM, the formation of nucleosomes was confirmed by native-PAGE (Figure 3(a)). The band from the phosphorylated nucleosome did not show remarkable difference

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compared to that from canonical nucleosome, which demonstrates that the phosphorylated H2A can be utilized for nucleosome reconstitution as well as recombinant H2A. We then performed DSF experiments against nucleosome containing H2A-Y57ph (Figure 3(b)). Canonical nucleosome containing recombinant H2A started to decompose around 65 °C and showed an increase in fluorescence. The first rapid increase in the fluorescence from 65 to 75 °C indicates the removal of H2A and H2B, and the later increase from 75 to 87 °C indicates the removal of H3 and H4 from DNA. In contrast, we found that nucleosome containing H2A-Y57ph started to decompose at approximately 60 °C, which is a lower temperature than that of canonical nucleosome. The Tm values of the first increase were likewise different between recombinant nucleosome (71 °C) and phosphorylated nucleosome (68 °C). This difference in Tm indicates that Y57ph destabilizes the nucleosome structure. The DSF curve also revealed that only the first increase shifted to lower temperature, and that the second increase did not move compared with the spectrum of canonical nucleosome. This typical change of the DSF curves mean that Y57ph strongly promotes the dissociation of H2A and H2B from nucleosome, and is consistent with the crystal structure of nucleosome, in which Y57ph is close to the H2B α-helix. Although the detailed mechanism of the dissociation is yet to be uncovered, a fluctuation of H2B αC-helix by Y57ph may weaken H2B–H4 interactions (i.e. the formation of a four-helix bundle), which are the main interactions between H2A–H2B dimer and H3–H4 tetramer.24

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Figure 3. (a) Native-PAGE analysis of reconstituted nucleosomes. Lane 1: 100 bp DNA ladder, lane 2: 145 bp 601 DNA fragment, lane 3: nucleosome with 145 bp DNA containing recombinant H2A, lane 4: nucleosome with 145 bp DNA containing synthetic Y57ph-H2A. (b) Thermal shift assays with the nucleosome by using SYPRO Orange. The fluorescence intensity at each temperature is plotted. The assays with nucleosome containing recombinant H2A and Y57ph-H2A are represented by blue circles and red triangles, respectively. The average values of three independent experiments are shown with error bars derived from the standard deviation.

Evaluation of histone–DNA interaction using MNase. We next evaluated the effect of Y57ph on another important interaction in nucleosome: histone–DNA interaction. Although the crystal structure of nucleosome shows that Y57ph is distant from nucleosomal DNA and does not have any direct interaction, the conformational change between the α2-helix in H2A and the 10


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αC-helix in H2B may accelerate the extent of DNA unwrapping from the nucleosome, leading to higher DNA accessibility. To estimate the DNA accessibility, we performed a micrococcal nuclease (MNase) assay against nucleosome.25 MNase is a DNase that digests free DNA in preference to nucleosomal DNA. Histone–DNA interactions between proteins with and without Y57ph can thus be differentiated by measuring the length of digested DNA fragments. We prepared canonical and Y57-phosphorylated nucleosomes consisting of a 193-bp DNA fragment to digest the linker DNA region by MNase (Figure S8). Given that the histone octamer is wrapped by about 145–147 bp DNA, it is expected that MNase digestion would result in similar lengths of DNA when canonical nucleosome is used.25 To the canonical and phosphorylated nucleosome solutions, CaCl2-containing buffer, and, subsequently, MNase were added. After incubation of the solutions at either 23 or 37 °C for 5 min, the digestion was stopped by the addition of EDTA. Proteins were degraded through co-incubation with proteinase K, and remaining DNA fragments were analyzed by native-PAGE (Figures 4 and S9). As a result, the length of the DNA became shorter according to the increasing amount of MNase. High temperature evoked frequent digestion because of activation of MNase ability and DNA breathing in the nucleosome. However, there were subtle differences in the lengths of DNA fragments when proteins with and without Y57ph were used. As shown in Figure 4, the bands around 145 bp were the strongest in both nucleosomes upon 2 U MNase digestion. In addition to this band, several bands under 100 bp appeared after incubation at 37 °C with both nucleosomes (Figure S9). Although DNA accessibility near the entry-exit site was changed by temperature and MNase concentration, DNA accessibility around the center of the nucleosome was not altered, even in the presence of Y57ph. The same result was obtained by using the nucleosome containing synthetic non-modified H2A (Figure S10). From the assays using SYPRO Orange and MNase, it is thus clear that Y57ph has a strong effect on H2A–H2B interactions.

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Figure 4. Native-PAGE analysis of the MNase assay. Treatment was conducted at 23 °C for 5 min. The nucleosome containing recombinant H2A was treated with 0.5, 1, and 2 units of MNase (lane 4, 5, and 6, respectively). The nucleosome containing H2A-Y57ph was also treated with 0.5, 1, and 2 units of MNase (lane 7, 8, and 9, respectively). Lanes 1 and 10 indicate 10 bp DNA ladder. Lanes 2 and 3 indicate 193 bp and 145 bp DNA fragments.

Artificial H2A containing Y57ph can be utilized to approach biological issues in living cells. H2A-Y57 is located in the acidic patch, and its phosphorylation increases negative charge. The acidic patch is a region on the surface of the nucleosome that bears several acidic amino acids and acts as a platform for reader proteins.24 The effect of changes in the acidic patch can be examined by using Y57-phosphorylated nucleosome. A biochemical assay can also elucidate whether Y57ph accelerates eviction of the H2A–H2B dimer from the nucleosome as a FACT complex. Current epigenetics studies suggest that H2A and H2B act as a H2A–H2B dimer throughout cell cycles.26 However, details of the assembly and disassembly of H2A and H2B have not been elucidated so far. This work on Y57ph suggests a way to prove whether PTMs regulate the formation of H2A–H2B dimers in live cells. Conclusion. We have shown for the first time a PTM that destabilizes the H2A–H2B interaction. By using chemically synthesized H2A, we demonstrated that H2A-Y57ph influences the stability of the H2A–H2B dimer and nucleosome. The results of thermal shift assays indicate that the both complexes decompose at lower temperature upon the inclusion of Y57ph. MNase assays revealed that the DNA accessibilities of two types of nucleosomes were nearly identical. We 12


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conclude that Y57ph is only involved in histone–histone interaction and does not interfere with histone–DNA interactions. Little is currently known about the assembly and disassembly of H2A and H2B throughout the cell cycle. This work on Y57ph is an important step to elucidating control mechanisms of H2A–H2B dimerization in living cells.

ASSOCIATED CONTENT Supporting information The Supporting Information is available free of charge on the ACS Publications website at DOI: . Scheme S1 and Figures S1−S10 associated with data analyses (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. ORCID Akimitsu Okamoto: 0000-0002-7418-6237 Gosuke Hayashi: 0000-0001-6853-2706 Funding This work was supported by the Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research (A) (15H02190), and JSPS Grant-in-Aid for JSPS Research Fellow (15J08667). Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We thank Prof. H. Kurumizaka and Dr. A. Osakabe (Waseda University) for kindly providing a plasmid containing 193-bp 601DNA. T.S. was supported by Research Fellow of the Japan Society for the Promotion of Science.

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Table of Contents (TOC) Regulation of stability on histone H2A–H2B dimer by H2A Tyr57 phosphorylation

Takuma Sueoka†, Gosuke Hayashi†, and Akimitsu Okamoto*†,‡ †

Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of

Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan ‡

Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba,

Meguro-ku, Tokyo 153-8904, Japan

E-mail: [email protected]

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Regulation of the Stability of the Histone H2AâH2B Dimer by H2A

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