9Equilibrina et al.Chromosome Science 18: 9-14, 2015

Regular Article

The role of phosphorylation of histone H3 at serine 10 in chromatin condensation in vitro

Ilma Equilibrina, Elena Krayukhina, Kota Inoue, Yuya Ishikawa, Akihiro Kawamoto, Takayuki Kato, 　　　Isao Suetake, Shoji Tajima, Hideaki Takata, Susumu Uchiyama and Kiichi Fukui

Received: September 30, 2014 / Accepted: April 03, 2015© 2015 by the Society of Chromosome Research

Abstract

Post-translational modifications on histone tails play essential roles in modulating chromatin higher order structure. Phosphorylation of histone H3 at serine 10 (H3S10ph) is associated with chromosome condensa-tion. However direct evidence and the molecular mech-anism underlying chromosome condensation induced by H3S10ph in human is unclear. Herein, we employed in vitro reconstituted nucleosomal arrays mimicking H3S10ph by substitution S10 to aspartic acid (H3S10D) using human recombinant histones and highly tandem repeats widom 601 sequence. Using analytical ultracen-trifugation sedimentation velocity (AUC-SV) analysis and transmission electron microscopy (TEM) observation, we found that there is no significant difference in the local folding and condensation between WT H3 and H3S10D containing nucleosomal arrays. Thus, our results suggest that H3 phosphorylation at serine 10 does not directly play a role in chromatin condensation and thus higher order structure.

I. Introduction

The first fundamental step of compaction critical to packing the whole genomic DNA in a higher eukaryotic nucleus is the formation ofa nucleosome, which consists of 147-bp of DNA wrapping 1.75 times around a core histone octamer composed of pairs of histones H2A, H2B, H3, and H4. Nucleosomesare connected by 10-80 bp of linker DNA to form a chromatin fiber (Luger et al., 1997). The

chromatin fiber then forms much condensed structure called chromosome during mitosis, crucial for faithful segregation of the duplicated genomic DNA into two new daughter cells (Belmont, 2006).

Chromosome condensation is highly influenced by post-translational modifications of histone tails (Carruthers & Hansen, 2000; Shogren-Knaak et al., 2006).The amino acids in the positive N-terminal tails of histone molecules extended out of the nucleosome core are subjected to a variety of covalent post-translational modifications. This can lead to chromosome condensation state by each of two possible ways. First, modification at the histone tail can directly alter the local charge of the nucleosome thus directly modifing chromatin tertiary structure and leading to condensation. Second, it can act as a marker or substrate for binding of other histone modifying or chromatin remodeling factors, thereby altering the chromatin structure.It has been shown that histone tails are responsible for in vitro nucleosomal array condensation, whereas H3 and H4 tailless nucleosomal arrays are unable to fold maximally even at high MgCl2 concentration(Carruthers & Hansen, 2000; Lu et al., 2009). H3 N-terminal tail was shown to contribute to oligomerization through interaction with the DNA of neighboring nucleosomes during inter-molecular folding. These facts show that the H3 N-terminal tail is important in chromatin condensation (Pepenella et al., 2013). However, even though nucleosome core structure has been determined at near-atomic resolution using X-ray crystallography, due to its flexible conformation, the details structure of histone N-terminal tail regions and its interaction with neighboring nucleosomes are still undefined (Luger et al., 1997).

Histone H3 phosphorylation on serine 10 (H3S10ph) has emerged as an important modification factor in chromosome condensation in vivo (Wei et al., 1998, Nowak & Corces, 2004). However, evidence whether H3S10ph is directly required for chromosome condensation in human has not been obtained. A correlation between H3S10ph and chromosome condensation is assumed mainly based on temporal and spatial distribution patterns of H3S10ph during mitosis. H3S10phis highly detected during metaphase and is reduced during telophase when chromosomes undergo decondensation. Chromosome condensation event in mitosis is accompanied by H3S10ph in a variety of organisms (Hendzel et al., 1997; Wei et al., 1998; Hsu et al., 2000). Contribution of H3S10ph on chromosome condensation was observed in Tetrahymena

Ilma Equilibrina, Elena Krayukhina ,Kota Inoue, Yuya Ishikawa, Hideaki Takata ,Susumu Uchiyama and Kiichi Fukui (*)Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, JapanTEL: +81-6-6879-7440FAX: +81-6-6879-7741E-mail: kfukui@bio.eng.osaka-u.ac.jp

Akihiro KawamotoGraduate School of Frontier Bioscience, Osaka University, 2-1Yamadaoka, Suita, Osaka 565-0871, Japan

Isao SuetakeInstitute for Protein Research, Graduate School of Science, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565- 0871, Japan

10 Phosphorylation of histone H3 in chromatin condensation

and S. cerevisiae, in which mutant containing non-phosphorylatable histone H3 showed no chromosome condensation (Wei et al., 1998; Wilkins et al., 2014).

In this study, we examined the effects of a negative charge to H3S10on the higher-order chromatin structure by amino acid substitution using reconstituted nucleosomal arrays. Thus, direct effect of a negative charge at serine 10 on chromatin higher order structure can be revealed. Previous work showed that H3S10ph modification by chemical ligation containing nucleosomal arrays using Xenopus histones and 5S rDNA 208-12 did not alter the chromatinphysical structure (Fry et al., 2004). Here, we reconstituted nucleosomal arrays composed of human recombinant histones and the widom 601 sequence. They produce more homogenous population of nucleosomal arrays than those compared with 5S rDNA 208-12. Because it comes from a tandemly repeated single high affinity nucleosome position that leads to more homogenous linker DNA lengths (Lowary & Widom, 1997; Lowary & Widom, 1998). Consequently, higher homogeneity of nucleosomal arrays will result in lower background on sedimentation analysis AUC-SV and other biophysical methods. In addition, herein human recombinant histones (instead of Xenopus) were used. In spite of highly conserved histone sequences between human and Xenopus, crystal structure of human nucleosome core showed remarkably different nucleosomal DNA path from Xenopus nucleosome core structure. This DNA path alteration is accompanied by 1 bp shift which induces space invasion between DNA duplexes within a single nucleosome. Hence, human nucleosomal DNA attains looser packing(Tsunaka et al., 2005). However details of nucleosomal array folding containing human and Xenopus recombinant histones have not been known. Furthermore, H3 N-terminal tail was shown to engage in chromosome condensation through interaction with neighboring nucleosomal DNA (Pepenella et al., 2013). Accordingly, looser conformation of human nucleosomal DNA might give different impacts on condensation of nucleosomal array. These facts prompt us to investigate the condensation of nucleosomal array containing human recombinant histone mimicking H3S10p.

In our current study, we aimed at clarifying the specific role of H3S10p to chromosome condensation in humans by using in vitro reconstituted chromatin mimicking H3S10ph. We incorporated a negative charge on the H3S10 by substituting serine to aspartic acid (H3S10D). To address the folding of H3S10D containing nucleosomal array, we coupled folding measurements using AUC with transmission electron microscopy (TEM). We found that H3S10D nucleosomal arrays have a similar higher-order chromatin structure to those composed of histones without a negative charge. On the basis of present results we showed that a negative charge on H3S10 solely does not play a direct role on higher order chromatin structure.

II. Materials & Methods

2.1 Reconstitution of nucleosomal array using 12 copies of (12-mer) tandem repeats

Nucleosomal array was assembled with purified recombinant histone octamers, containing human WT H3 and substituted mutated H3S10D (serine to aspartic acid), with 12-mer repeats of a 207 bp of Widom 601 nucleosome- positioning DNA sequence (12-207 x 601

DNA) using a salt dialysis method with end buffer (10 mMTris-HCl, 10 mMKCl, 0.1 mM EDTA). The assembly was titrated by mixing histone octamer and template DNA at stoichiometric ratios of 0.8-1.2 to 1 mol DNA repeat. Saturation of the 12-mer nucleosomal array was then examined by ScaI and AluI digestions for 16 hrs at RT. Nucleosomal array was purified using phenol-chloroform, and then the digested DNA pattern was observed by 5% native PAGE.

2.2 Microscopic analysisChromatin samples were fixed with 0.1% glutaraldehyde

and negatively stained using 2% uranylacetate. Transmission electron microscopy (JEO LJEM-1011) was used for structural observations of reconstituted chromatin.

2.3 Precipitation assaySelf-association of nucleosomal arrays was analyzed by

mixing chromatin with equal volumes of increasing MgCl2

concentration (0–5 mM). After centrifugation, the A260

units of nucleosomal arrays in remaining supernatant was measured and plotted.

2.4 Analytical ultracentrifugation-sedimentation velocity (AUC-SV)

Nucleosomal arrays in TEN buffer (10 mM Tris-HCl, 10 mMKCl, 0.1 mM EDTA) were mixed with different MgCl2 concentrations. Sedimentation velocity experiments were performed on an AUC (ProteomeLab XL-I Beckman Coulter) using an An-60Ti rotor. Samples with an initial absorbance of 0.6-0.8 at 260 nm were equilibrated at 20°C under vacuum in the centrifuge prior to sedimentation. The absorbance was then measured during sedimentation at 20,000 rpm in 12-mm double sector cells. Sedimentation velocity data were analyzed using UltraScan v.9.9 software employing the Van Holde-Weischet method.

III. Results

3.1 Reconstitution of WT and H3S10D nucleosomal arrays

To examine the mechanism of how H3S10ph induces chromosome condensation,direct alteration of chromatin higher-order structure was checked using reconstituted nucleosomal arrays. Wild-type H3 (WT) and H3S10D (serine substitution to aspartic acid) containing-nucleosomal arrays were reconstituted by adding the four core histones at the appropriate molar ratios to defined DNA templates consisting of the nucleosome positioning sequence in order to saturate all of the nucleosomes binding-sites (Luger et al., 1997). Following salt dialysis assembly from 2 M to 10 mM KCl, configuration of nucleosomal array was then checked. First, WT and H3S10D 12-mer nucleosomal arrays were tested whether they were properly reconstituted using gel shift assay (Fig.2A). Both WT and H3S10D nucleosomal arrays were shifted compared to 12-207x601 DNA control indicating that both nucleosomal arrays were properly reconstituted (Fig. 1A). Subsequently, we checked if nucleosomal arrays were fully saturated using ScaI and AluI digestions. Scheme of ScaI and AluI restriction sites were shown: ScaI restriction sites are located in the linker DNA and AluI in the core sequence (Fig. 1B). Following ScaI and AluI digestions, nucleosomal DNA was purified by phenol-

11Equilibrina et al.

chloroform extraction and was run in 5% native PAGE. ScaI digestion on saturated nucleosomal arrays showed digested band of 207 bp, whereas AluI digestion showed in undigested DNA band. The resulted DNA digestion patterns indicated that nucleosomal arrays are fully saturated and histone octamers are positioned well on the core sequence (Fig. 1C).

3.2 Analytical ultracentrifugation-sedimentation coefficient (AUC-SV) analysis on H3S10D nucleosomal array

Chromosome condensation requires physical alteration of chromatin higher order structure through intra- and/or inter-nucleosomal array interactions (Pepenella et al., 2013). Incorporation of a phosphate group at H3S10

Figure 1. Saturation analysis of WT and H3S10D containing nucleosomal arrays.(A) Gel shift assay of H3- and H3S10D- containing nucleosomal arrays. Following reconstitution, nucleosomal array was run in 0.7% agarose gel. (B) Scheme of 12-mer nucleosomal arrays with ScaI and AluI digestion sites. Well positioned histone octamers in nucleosomal arrays are shown as undigested nucleosomal arrays by AluI. Fully saturated nucleosomal arrays are shown by ScaI digestion of linker histones, resulted in a 207 bp band.(C) H3- and H3S10D- containing nucleosomal arrays were digested by ScaI and AluI at RT for 16 hrs, DNA was purified using phenol-chloro-form, and ran in a 5% native gel.

12 Phosphorylation of histone H3 in chromatin condensation

Figure 2. Effect of H3S10ph on nucleosomal array structure and folding.(A) Precipitation assay of WT and H3S10D nucleosomal arrays. After centrifugation assay using the given concentration (0–5 mM) of Mg2+, A260 of the supernatant was measured and plotted (♦ : WT, ■ : H3S10D)(B) Sedimentation coeffecient distibution plot of WT and H3S10D containing nucleosomal arrays in 0 and 1.5 mM Mg2+ ( ♢ : WT 0 mM Mg2+, ♦ :H3S10D 0 mM Mg2+,□ :WT 1.5 mM Mg2+, ■ :H3S10D 1.5 mM Mg2+) (C) TEM observation of negatively-stained of WT and H3S10D nucleosomal arrays. Scale bars: 100 nm.

13Equilibrina et al.

may trigger chromosome condensation by either of two possible ways, First, H3S10ph directly alters the structure of H3 N-terminal tail, and may directly modify nucleosomal arrays interactions and cause condensation. Second, H3S10ph acts to recruit other histone modifying or chromatin remodeling factors, which consequently alters nucleosomal array interactions and condensation. To investigate the direct effect of phosphorylation on chromatin higher-order structure, inter- and intra-array interactions were assessed for WT and H3S10D nucleosomal array. In vitro nucleosomal array oligomerization has previously been shown to be a manifestation of long range fiber-fiber interactions (inter-array) that leads to higher-order chromatin structure (Hansen, 2002). A high Mg2+ concentration can induce nucleosomal arrays oligomerization, which causes arrays to precipitate from a solution (Schwarz et al., 1996). To test inter-array folding, a precipitation assay using 0-5 mM Mg2+ ranges was used. After centrifugation, using 0-5 mM Mg2+ the percentage of soluble nucleosomal arrays in supernatant was measured. At around 1.8-2 mM Mg2+ H3S10D nucleosomal arrays have tendency of less precipitation than WT arrays. However, this tendency is marginally observed only at around 1.8-2 mM Mg2+ and disappeared at higher Mg2+, concentration. Both arrays similarly showed 50% precipitation [Mg50] at ~2.3 mM and further complete oligomerization (indicated by 100% precipitation) at 3 mM Mg2+ (Fig. 2A). These observations suggest that WT and H3S10D arrays have the similar chromatin inter-array interaction.

To determine if phosphorylation at H3S10 directly af fects intra-array interactions, chromatin intra-array condensation (interaction between neighboring nucleosomes and/or DNA in single nucleosomal array) was tested using the AUC-SV. AUC-SV provides information on the size distribution and hydrodynamic shape of nucleosomal array based on the sedimentation coefficient. In a solution, 12-mer nucleosomal array exist in an equilibrium statesbetween unfolded (29 s), moderately folded (40 s), maximally folded (55 s), and oligomeric structures (>100 s) (Hansen, 2002). Without Mg2+ fully-saturated and extended 12-mer nucleosomal array has a sedimentation coefficient of ~29 s (Hansen, 2002). We obtained sedimentation coefficient of WT and H3S10D nucleosomal arrays of 28.2 s and 29.3 s, respectively, conforming that both nucleosomal arrays are fully saturated and in extended (beads on a string) conformations (Fig. 2B). Next, we compared the folding and condensation of both arrays under the presence of Mg2+. With 1.5 mM Mg2+, nucleosomal array form a folded structure which can be indicated by sedimentation coefficients of >29 s. Under this condition, the WT and H3S10D arrays condensed similarly, with maximum sedimentation coefficients of 53 s and 59 s, respectively (Fig. 2B). These results strongly suggest that incorporation of a negative charge (mimicking the phosphorylation event) on H3S10 does not directly alter chromatin folding and condensation, and thus does not affect the higher-order chromatin structure.

Subsequently, to confirm the structure, the nucleosomal arrays were visualized using negative-stained TEM. Without Mg2+ (0 mM), the WT and H3S10D arrays demonstrated adopted extended structure (Fig. 2C, upper figures). In high Mg2+concentration, chromatin will fold and form a structure of 30-nm fibers (Hansen, 2002). In

the results of negative-stained TEM observations with 1.5 mM Mg2+both nucleosomal arrays were maximally folded, with the similar condensation levels (Fig. 2C, lower figures) consistent with AUC-SV analysis. These data confirmed our AUC-SV analysis i.e., WT and H3S10D nucleosomal arrays folding and higher order structure are the similar. These indicate that a negative charge at H3S10 does not directly affect higher order chromatin structure.

IV. Discussion

Previous reports have implied the importance of H3S10ph in chromosome condensation mainly due to its concurrent presence with chromosome condensation during mitosis in vivo (Wei et al., 1998; Wilkins et al., 2014). In this study, we employed reconstituted nucleosomal arrays, so that uniformity of modified histones or DNA of the long chromatin fiber can be generated. We substituted H3 serine10 to aspartic acid to incorporate a negative charge which mimics the electrostatic behavior of phosphorylated H3S10. The tendency of less precipitation of H3S10D array compared with WT array indicates that Inter-array chromatin interaction may be slightly influenced (weaken) by the phosphorylation at specific Mg2+ (1.8-2 mM Mg2+). However this difference is disappeared at higher Mg2+ concentration. Even if the interchromatin interactions are slightly influenced by the phosphorylation at around 1.8-2 mM Mg2+ concentration, considering that the physiological Mg2+concentration at interphase and mitotic nuclei (chromatin) is 2-4 mM and 5-17 mM, respectively (Strick et al., 2001), the observed small difference in the precipitation level between WT and H3S10D at 2 mM Mg2+would not reflect the chromatin condensation event in vivo. AUC-SV analyses demonstrate overlapping sedimentation profiles of WT and H3S10D nucleosomal arrays (Fig. 2B). Furthermore, TEM observations confirm that H3S10D arrays have the similar condensation level to the WT arrays (Fig. 2C). The results suggest that a negative charge at H3S10 does not change H3 N-terminal tail stable interactions and conformation during chromosome condensation (Zhou et al., 2012). These results further suggest that even though human nucleosomal DNA path attains looser structure compared to Xenopus, condensation profiles of the arrays are relatively similar, indicating that nucleosomal DNA path might not contribute significantly to the chromatin folding under the given Mg2+ condition (Fry et al., 2004). We show that a negative charge, and not a phospho-group per-se, at serine 10 does not alter chromatin higher order structure directly.

It should be noted that aspartic acid is not chemically similar to phosphoserine, despites its vast utilization as a phosphorylation mimic. The carboxylic acid functionality of aspartic acid has different chemical properties to those of a serine-phosphate group, including charge state, number of oxygen atoms and pKa (Sieracky & Kamarova, 2013). Despite these differences, functional and structural similarities have been shown for the proteins with phosphorylated serine and a serine substituted to aspartic acid (Wittekind et al., 1989; Leger et al., 1997). Our strategy has given an insight into how a negative charge contributed by a phosphate group can affect chromatin higher-order structure.

Our results show that H3S10ph might indirectly regulates chromatin structure and promotes chromosome

14 Phosphorylation of histone H3 in chromatin condensation

condensat ion. H3S10ph might recruit or change chromatin affinity for factors important for modification of nucleosomal arrays structure, such as chromatin remodeling factors or other histone modifiers. Further studies are required to determine the downstream components in the chromosome condensation pathway initiated by H3S10ph. In the current reports using S. cerevisiae, H3S10ph recruits hst2p, a NAD-dependent histone deacetylase, in order to deacetylate H4 lysine 16 (H4K16) (Perrod et al., 2001; Wilkins et al., 2014). Nucleosomal array containing and mimicking H4K16ac have been shown to inhibit array folding whereas deacetylation at this amino acid can promote interaction of the H4 tail with H2A-H2B acidic patch inthe neighboring nucleosomes (Shogren-Knaak et al., 2006; Zhou et al., 2012). Subsequently, H3S10ph dependent-deacetylation of H4K16 would induce chromatin folding, hence, chromosome condensation during mitosis (Wilkins et al., 2014).

V. Acknowledgement

We would like to thank Prof. Timothy. J. Richmond from ETH Zurich for kindly providing us with 12-207x601 DNA. This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) (21248040 and 2525064, and 18681032 to K.F. and S.U. respectively) and Senri Life Science Foundation (S.U.).

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