dual modiﬁed antiphospho (ser10)-acetyl (lys14)-histone h3

c© Indian Academy of Sciences

RESEARCH ARTICLE

Dual modified antiphospho (Ser10)-acetyl (Lys14)-histone H3predominantly mark the pericentromeric chromatin during mitosis

in monokinetic plants

SANTOSH KUMAR SHARMA1∗, MAKI YAMAMOTO2 and YASUHIKO MUKAI1

1Laboratory of Plant Molecular Genetics, Division of Natural Sciences, Osaka Kyoiku University, 4-698-1 Asahigaoka,Kashiwara, Osaka 582-8582, Japan

2Department of Rehabilitation Sciences, Kansai University of Welfare Sciences, Kashiwara, Osaka 582-0026, Japan

AbstractEpigenetic regulatory posttranslational histone modification marks not only function individually but also capable to act incombination as a unique pattern. A total of 16 plant species belonging to 11 genera of eight families (five dicots and threemonocots) including land plants, epiphytes (orchids) and the holokinetic taxa (Drosera spp.) were analysed for chromosomaldistribution of dual modified antiphospho (Ser10)-acetyl (K14)-histone H3 (H3S10phK14ac) to understand the combinato-rial chromatin dynamics during mitotic cell division in plants. The anti-H3S10phK14ac evidently mark the pericentromericchromatin on mitotic chromosomes of the plants excluding the holokinetic Drosera species, which revealed the immuno-labelling of whole chromosomes all along the arms. The dual modified immunosignals were absent during early stages ofmitosis, appeared intensively at metaphase and remained visible until late-anaphase/telophase however, labelled the wholechromosomes during meiotic metaphase I. Colocalization of anti-H3S10phK14ac with an onion’s CENH3 antibody on mitoticchromosomes of Allium revealed the chromosomal location of anti-H3S10phK14ac in the region between signals for CENH3detection. Overall analysis suggests that the unique localization of combinatorial histone modification mark at pericentromericchromatin might have attributed through ‘phospho-acetyl’ cross talk that ultimately facilitate the sister chromatid cohesion atpericentromeres following condensation events in mitotic chromosomes. Here, we propose that dual modified H3S10phK14achistone may serve as an additional cytogenetic landmark to identify pericentromeric chromatin during mitosis in plants. Theplausible role of histone cross talk and future perspectives of combinatorial histone modification marks in plant cytogeneticswith special reference to chromatin dynamics have been discussed.

[Sharma S. K., Yamamoto M. and Mukai Y. 2016 Dual modified antiphospho (Ser10)-acetyl (Lys14)-histone H3 predominantly mark thepericentromeric chromatin during mitosis in monokinetic plants. J. Genet. 95, 965–973]

Introduction

Histones, as the core structure of the nucleosome, are sub-jected to numerous types of posttranslational modifications(PTMs), such as acetylation, phosphorylation, methylation,ubiquitnation, ribosylation and sumolyation to facilitate thepackaging of eukaryotic DNA in nucleoprotein complexknown as chromatin (Xu et al. 2009). Out of such PTMs,histone methylation, acetylation and phosphorylation haveimmense importance in regulation of gene expression, deve-lopment and other biological functions (Fuchs et al. 2006).

∗For correspondence. E-mail: [email protected];[email protected], MY and YM designed the experiments and analysed the results. SKSconducted the experiments and wrote the manuscript.

Histone acetylation and phosphorylation are transient anddynamic events (Jackson et al. 1975; Barth and Imhof2010) that constitute integral components of the chromatin-signalling pathway. Additionally, multimodifications and/orcombinations of histones modifications have also been foundto be associated with the alteration of the chromatin struc-ture by varying protein–DNA or protein–protein interactions(Wang and Patel 2011). Combinations of modifications inter-act particularly when they are located on the same histone tailand on different histones within a nucleosome. According tothe ‘histone code’ hypothesis (Strahl and Allis 2000), spe-cific histone modification(s) acting alone or in combination,can stimulate or inhibit the binding of a ‘reader’ protein tothe nucleosome. The ‘readers’ can promote another histonemodification by establishing a histone crosstalk that gener-ates a different binding platform for the further recruitment

Keywords. histone modification; mitosis; pericentromere; immunostaining; plants.

Journal of Genetics, DOI 10.1007/s12041-016-0723-1, Vol. 95, No. 4, December 2016 965

Santosh Kumar Sharma et al.

of proteins that regulate gene expression as a combinatorialtarget. For example, histone H2B ubiquitination found to beassociated with promotion of methylation of histones H3K79and H3K4 (Briggs et al. 2002; Sun and Allis 2002). Sim-ilarly, ubiquitin ligase is also required for histone methyl-transferase localization in fission yeast (Jia et al. 2005) andacetylation of H3K18 stimulates the association of argininemethyltransferase CARM1 and the methylation of H3R17in humans (Daujat et al. 2002). It is interesting to note thatin humans, the coexistence of several other modificationsalong with H3S10/28ph have also been documented on thesame tail of core histone during mitosis (Garcia et al. 2005).Remarkably, the dual modifications that eventually includephosphorylation and acetylation of K9/K14 on the same tailpromote alteration/modulation in the individual modificationpattern and ultimately may introduce a neo-modification tar-get (Sawicka and Seiser 2014). In this context, site-directedchromatin immunoprecipitation (ChIP) analysis using dualmodification-specific antibodies revealed the presence of theH3S10phK14ac mark at activated promoters in vivo (Winteret al. 2008; Brunmeir et al. 2010; Simboeck et al. 2010) andraised an important question about the mechanism under-lying the simultaneous targeting of the two PTMs to thesame histone H3 tail. Therefore, it is assumed that the theoryof ‘histone code’ now is being expanded to a ‘nucleosomecode’, suggesting that the presence of all posttranslationalhistone modifications in one nucleosome regulate the under-lying DNA sequence (Jenuwein and Allis 2001; Barth andImhof 2010). Particular attention should also be focussed onso-called ‘binary switch modules’ or ‘modification cross-talkswitch’ where two neighbouring modification residues influ-ence/control the activation and/or regulation of each other ata particular location (Fischle et al. 2003).

In the last decade, molecular cytogenetics through immu-nostaining technique has revolutionized the field of plantepigenetics by uncovering the global cytogenetic landscapeof epigenetic signatures of histone PTMs in plants. Severalhistone modifications especially methylation, acetylation andphosphorylation have been cytologically characterized andlinked to various biological, functional and developmentalaspects during cell division in plants (Fuchs et al. 2006;Sharma et al. 2015). However, attempts have not been madeyet to identify the spatio-temporal cytogenetic distributionof dual modified histones aiming at their distinct functionsas a combined unit and contribution to the maintenanceof epigenetic states through cell division. Therefore, in thepresent study, immunostaining was performed to identifychromosomal allocation of a dual modified antibody againsthistone H3 phosphorylated (Ser 10) and acetylated (K14)(H3S10phK14ac) in 16 plant species belonging to 11 generaof eight families (three monocots and five dicots includingholokinetic taxa) to understand the underlying combinatorialtarget of differentially modified histone chromatin-associatedevents on plant chromosomes. The study may serve as a basicmanifesto to introduce cytogenetic characterization of differ-ent histone modifications/combinations or chromatin ‘cross

talk’ in plants to understand underlying chromatin-associatedevents vis-à-vis cell cycle and other cellular functions.

Materials and methods

Plant materials

Plants were selected based on their cytological importance(crop/model/holokinetic plant), chromosome sizes (small tobigger), cytological preparations (easy to difficult) as well astheir habitat (land/epiphytes). Sixteen plant species belong-ing to 11 genera of eight plant families including monocotsand dicots were used for this study. The details of the plantmaterials are as follows: eudicots: Vicia faba (field bean),Nicotiana tabacum (tobacco), N. sylvestris, N. tomentosi-formis, Daucus carota (carrot), Brassica oleracea (cabbage)and a holokinetic taxa i.e. Drosera spp.; monocots: Hordeumvulgare (barley), Triticum aestivum (common wheat), Alliumcepa (onion), A. fistulosum (welsh onion), A. sativum (gar-lic), A. tuberosum (garlic chives), Oryza sativa ssp. japonicacv. Nipponbare (rice) and Cymbidium aloifolium, Dendro-bium nobile (orchids). The plant material used in the presentstudy has also been listed along with their taxonomical status,habitat and chromosome numbers in table 1.

Chromosome preparation and immunostaining

Seeds (field bean, carrot, cabbage, barley, wheat, rice andall onion species) were germinated on moistened filter paperat room temperature to obtain actively growing roots. Thein vitro raised plantlets of tobacco and its allied species wereused for root tips collection (kindly provided by Prof. GoSuzuki, Osaka Kyoiku University, Japan), whereas activelygrowing roots were excised from epiphytic orchids (C.aloifolium and D. nobile) and Drosera species. Flower budswere used for meiotic analysis in D. nobile. All the plantmaterials (root tips/flower buds) were fixed in PHEMESbuffer (50 mM PIPES, 25 mM HEPES, 5 mM MgCl2 and5 mM ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 0.35 M Sorbitol, pH 6.9) containing3% (w/v) paraformaldehyde and 0.2% (v/v) Triton X-100,and washed twice in phosphate-buffered saline (PBS, pH 7.4)as described earlier by Nagaki et al. (2012). The fixed tipswere digested for 30 min–2 h (depending upon the rootmorphology) at 37◦C with 1% (w/v) cellulase Onozuka RS(Yakult Pharmaceutical Industry, Tokyo, Japan) and 0.5%(w/v) pectolyase Y-23 (Seishin Pharmaceuticals, Tokyo,Japan) mixture dissolved in PBS, washed twice in PBS,and then squashed onto slides coated with poly-L-lysine(Matsunami, Osaka, Japan). The slides were incubated forminimum of 12 h at 4◦C with a rabbit polyclonal anti-H3S10phK14ac (diluted 1 : 500 in PBS containing 3% BSA;Merck Millipore, 07-081; synonym: H3K14me3S10ph; vali-dated in ChIP, ICC, WB and ChIP-seq as per supplier’s infor-mation). After washing in PBS, the slides were incubated for1 h at 37◦C with an Alexa Fluor 546-labelled goat antirabbitantibody (1 : 100 diluted in 3% BSA/PBS; Molecular Probes,

966 Journal of Genetics, Vol. 95, No. 4, December 2016

H3S10phK14ac marks pericentromeric chromatin in plants

Table 1. List of plant materials used in this study.

Plant Common name Family Monocot/dicot Habitat land/epiphyte 2n

Vicia faba Field bean Fabaceae Dicot Land 12Nicotiana tabacum Tobacco Solanaceae Dicot Land 48N. sylvestris – Solanaceae Dicot Land 24N. tomentosiformis – Solanaceae Dicot Land 24Daucus carota Carrot Apiaceae Dicot Land 18Brassica oleracea Cabbage Brassicaceae Dicot Land 18Hordeum vulgare Barley Poaceae Monocot Land 14Triticum aestivum Common wheat Poaceae Monocot Land 42Allium cepa Onion Amaryllidaceae Monocot Land 16A. fistulosum Welsh onion Amaryllidaceae Monocot Land 16A. sativum Garlic Amaryllidaceae Monocot Land 16A. tuberosum Garlic chives Amaryllidaceae Monocot Land 32Oryza sativa ssp. japonica Rice Poaceae Monocot Land 24Cymbidium aloifolium Cymbidium orchids Orchidaceae Monocot Land/epiphyte 40Dendrobium nobile Dendrobes (orchids) Orchidaceae Monocot Epiphyte 38Drosera spp. (Holocentric taxa) Sundews Droseraceae Dicot Land 40

11035, MA, USA). Further, to confirm the specific chromosomallocation of the anti-H3S10phK14ac, an antiAllium fistulosumcentromere-specific histone H3 (Afi-CENH3) that can recog-nize CENH3 orthologues of other Allium species too (Nagakiet al. 2012), was also employed on A. cepa chromosomesalong with a mouse monoclonal anti-H3S10phK14ac (MerckMillipore, 05-1315). The immunosignals were detected byusing an Alexa Fluor 488-labelled goat antirabbit antibody(Molecular Probes, 11034) and Alexa Fluor 546-labelledgoat antimouse antibody (Molecular Probes, 11030). Afterfinal wash with PBS, the preparations were counterstainedwith 4′,6-diamidino-2-phenylindole hydrochloride (DAPI)and mounted with ProLong gold antifade mountant (LifeTechnologies, P10144, California, USA). Immunosignals andstained chromosomes were captured using a CCD camera(Hamamatsu Photonics, Shizuoka, Japan) equipped withAxioscope fluorescence microscope (Zeiss, Shizuoka, Japan)and image analysis was performed as described previously(Suzuki et al. 2010).

ResultsCytogenetic detection of combinatorial immunosignals on plantchromosomes

Only nonambiguous mitotic chromosome preparations wereselected and the chromosome count could be precisely deter-mined in all the plants under investigation (figures 1 and 2).Various stages of mitotic divisions ranging from interphase totelophases were investigated microscopically for detection ofimmunosignals in all the plant species. The immunosignalswere localized on chromosome domains that coinciding thecentromere on each and every metaphase with uniform label-ling intensity throughout metaphase, anaphase until telophasein all the plants including monocot and dicots (figures 1and 2). A universal pattern of hyperphosphoacetylationrestricted to the pericentromeric chromatin was observed inmost of the plant chromosomes (figures 1, a–i; 2, a–f; 3). Theimmunosignals were found to be absent in interphase and

prophase stages in all the plants during mitosis (figure 1, a′and i; figure 2a′; arrow marked). The immunosignals couldeasily identify the position of centromere at opposite polestoo during segregation of the chromosomes at anaphasesuntil telophases (figure 1, a′–d′, e, I′–I′′). No consistent dif-ference was observed in the temporal and spatial phospho-acetyl combinatorial signalling pattern between angiospermland plants and epiphytic orchids. Similarly, monocot anddicot plants also confirmed unanimous phosphorylation pat-tern during mitotic divisions. Further, the immunocytoge-netic distribution of H3S10phK14ac found to be universalirrespective of size of the chromosomes and marked peri-centromeric chromatin in small (rice) to larger- (onion/fieldbean) sized chromosomes as well.

Distribution of anti-H3S10phK14ac in holokinetic chromosomesand meiosis I

To investigate whether the type of centromere (mono-centric and holocentric) affects the distribution of dualmodified H3S10phK14ac, mitotic chromosomes of the holo-centric species of Drosera were investigated and foundthat ultimately they showed hyperphosphoacetylation alongthe entire chromosome arm and/or whole chromosome(figure 4, a–a′′). Similarly, metaphase I of meiotic division inthe pollen mother cells (D. nobile) revealed hyperphospho-acetylation all along the chromosomes arms / whole chromo-somes (figure 4, b–c). Interestingly, the early stages of themeiosis I did not show any evidence of immunosignals inany form (figure 4b, arrow marked). We could not observemetaphase II of meiosis II in PMCs due to lack of plantmaterial (flower buds) in orchids.

Colocalization of anti-H3S10phK14ac with a centromeric histoneH3 (CENH3) antibody

The colocalization of anti-Afi-CENH3 and H3S10phK14acwas also performed on mitotic chromosomes of onion thatevidently revealed the significant differences in size, intensity

Journal of Genetics, Vol. 95, No. 4, December 2016 967


Figure 1. Phosphorylation pattern of dual modified anti-H3S10phK14ac targeting pericentromeric region (red) in mitotic chromosomecomplements of monocots. (a) metaphase, (a′) anaphase (A. cepa, 2n = 16); (b) metaphase, (b′) anaphase (A. sativum, 2n = 16);(c) metaphase, (c′) anaphase (A. tuberosum, 2n = 32); (d) metaphase, (d′) late anaphase (A. fistulosum, 2n = 16); (e) anaphase (Hordeumvulgare, 2n = 14); (f) metaphase (Oryza sativa, 2n = 24); (g–h) (epiphytic orchids), (g) metaphase (Cymbidium aloifolium, 2n = 40);(h) metaphase (D. nobile, 2n = 38); (i) metaphase, (i′) anaphase and (I′′) late telophase (Triticum aestivum, 2n = 42, photographs havebeen obtained in different focuses and merged as different layers resulting in different colour, i.e. green). Cells were counter-stained withDAPI (blue); scale bar = 10 μm.

Figure 2. Phosphorylation pattern of dual modified anti-H3S10phK14ac targeting pericentromericregion (red) in mitotic chromosome complements of dicots. (a) metaphase, (a′) anaphase (Nicotianasylvestris, 2n = 24); (b) metaphase (Nicotiana tomentosiformis, 2n = 24); (c) metaphase (Nicotianatabacum, 2n = 48); (d) metaphase, (d′) anaphase (Vicia faba, 2n = 12); (e) metaphase (Brassica oler-acea, 2n = 18); (f) metaphase (Daucus carota, 2n = 18). Cells were counter-stained with DAPI (blue);scale bar = 10 μm.



(a)

(a′)

Figure 3. Phosphorylation pattern of dual modified anti-H3K14me3S10ph during (a) mitoticmetaphase, (a′) enlarged section of chromosomes marked with the box in figure (a) (Allium tubero-sum, 2n = 32); arrow marks show a fine contraction that divides the hyperphosphorylation zoneinto equal halves corresponding to the centromere.

and most importantly the position of the immunosignalson the chromosomes. The different fluorophore labelledimmunosignals corresponding to Afi-CENH3 as well asanti-H3S10phK14ac were precisely localized at the primaryconstrictions of the chromosomes (figure 4, d–d′). The Afi-CENH3 appeared mainly at the periphery of duplicatedcentromeres of each pair of sister chromatids and anti-H3S10phK14ac was colocalized more towards the innerdomain of the centromere and coincide between the signalsfor Afi-CENH3 detection thereby directing the pericen-tromeric position (figure 4d, enlarged section). During chro-mosome segregation at anaphases, the immunosignals ofboth Afi-CENH3 and anti-H3S10phK14ac divide into twoequal halves and localized towards the opposite poles(figure 4, d′′ and e).

Discussion

Anti-H3S10phK14ac as a cytogenetic landmark for centromericchromatin in plants

Centromeres are dynamic assemblies of chromatin and arerequired for accurate segregation of chromosomes duringcell division. Therefore, the position of the centromere pro-vides a useful landmark for dividing chromosomes to ensurefaithful segregation and transmission of chromosomes dur-ing cell division (O’Connor 2008). Unlike telomeres, cen-tromeres lack highly conserved and specific DNA sequences,and therefore, microscopic detection of centromere without

specified centromeric marker is quite a difficult task inconventional plant cytogenetic techniques. In general, cen-tromeric and pericentromeric regions are known to be epi-genetically modified by histone phosphorylation, acetylationand methylation at different amino acid positions. Forinstance, the dynamic events of phosphorylation of S10/S28of histone H3 and Thr120/Thr133 of histone H2A have beencorrelated with the pericentromeric/centromeric position dur-ing both mitosis and meiosis II in plants (Houben et al. 2007;Granot et al. 2008; Dong and Han 2012; Demidov et al.2014). Histone phosphorylation is an evanescent chromatinmodification and its spatio-temporal cytogenetic distributionhas been documented for a broad spectrum of eukaryotesincluding plants (Hendzel et al. 1997; Van Hooser et al.1998; Wei and Allis 1998; Wei et al. 1998; Goto et al. 1999;Houben et al. 1999; Manzanero et al. 2000; Marcon-Tavareset al. 2014). The abundance of histone phosphorylationevents can be marked from entry to exit of the cell intothe cell divisions that eventually follow the chromosomecondensation events (Houben et al. 2007; Marcon-Tavareset al. 2014; Sharma et al. 2015). In general, the level of histonephosphorylation of serine residue increases at the end of theG2 phase, reaching a high level at metaphase, and declinesduring anaphase–telophase (Houben et al. 2007). How-ever, unlikely, the dual modified anti-H3S10phK14ac didnot show any immunosignal during early stages of mitosisand meiosis. The hyperphosphoacetylation on centromericchromatin of the mitotic chromosomes was observed until



Figure 4. Distribution of anti-H3S10phK14ac in mitotic chromosome complements of holocentric taxa Drosera spp. (2n = 40) (a–a′′);(a) mitotic metaphase, (a′) anaphase and (a′′) telophase showed hyperphosphoacetylation along the whole chromosomes; (b–c) meioticmetaphase I (Dendrobium nobile, 2n = 38) show hyperphosphorylation of whole chromosome (red), while arrow mark (b) indicating earlymeiotic stages (interphase–prophase I) where immunosignal were absent. (d–d′′) Colocalization of H3S10phK14ac with an A. fistulosum’scentromere-specific histone H3 (Afi-CENH3) antibody confirms the centromeric position of H3S10phK14ac (red) which formed a bridgebetween CENH3 signals (green), (d–d′) metaphase (d-enlarged section showing the location of H3S10phK14ac on metaphase chromosomeswhich occupied bridge-like centromeric positions (red) in between CENH3 (green), (d′′) early anaphase (A. cepa, 2n = 16); (e) anaphase(A. fistulosum, 2n = 16); cells were counter-stained with DAPI (blue); scale bar = 10 μm.

telophases in all the plants investigated excluding holocen-tric species of Drosera. The holokinetic chromosomes ofDrosera revealed uniform distribution along the entire lengthof chromosomes. Such kind of ‘diffuse centromere’ orga-nization is the key feature associated with other holoki-netic plants too, for example Luzula (Nagaki et al. 2005)and Rhynchospora tenuis (Guerra et al. 2006). Such holoki-netic plants eventually displayed a cell cycle-dependent uni-form histone H3S10/S28 phosphorylation mark, illustrating achromosome-wide ‘pericentromeric-like’ structure (Houbenet al. 2007). More importantly, one of the noteworthy fea-tures associated with the present study is that the centromeresfrom sister chromatids facing in opposite directions (biorien-tation) could be marked clearly and even countable duringsegregation of the chromosomes during mitotic anaphases(for e.g. wheat, field bean, tobacco, etc.). Such immunola-belled centromeres might be useful for analysing abnormalityin segregating chromosomes at anaphases–telophases in hybridsand polyploid plants. Really, the mitotic chromosomal dis-tribution of H3S10ph primarily targets pericentromeric chro-matin in land plants (Marcon-Tavares et al. 2014); however,

its chromosomal distribution may differ to some extentamong different species/taxa and/or cell types (Houben et al.2007; Sharma et al. 2015). Further, such histone modifica-tion not only marks function individually, but also capableto act in combination as a unique pattern. In this connec-tion, our study indicated that the combination of H3S10phand H3K14ac precisely targeted the centromeric chromatinthat bridged between duplicated centromeres of each pair ofsister chromatids. Similar kind of distribution patterns wasreported by Dong and Han (2012), who analysed chromo-somal distribution of Thr133-phosphorylated histone H2Ain maize and suggested that phosphorylation of histone canalso serve as an epigenetic marker for plant centromere acti-vity. On the other hand, recently Demidov et al. (2014) alsoproposed H2AT120ph as a universal marker for the cyto-logical detection of centromeres of monokinetic and holoki-netic plant species. In this connection, it is important to notethat H3S10ph is also known for centromere activity (Prigentand Dimitrov 2003; Han et al. 2006, 2009; Gao et al. 2011)and its firm association with acetylation of H3K14 (Claytonet al. 2000). It was suggested that the double modification



of H3S10phK14ac is necessary for transcriptional activationof specific genes (Cheung et al. 2000; Clayton et al. 2000).Further, Fischle et al. (2005) also advocated that H3S10ph incombination with other modifications might modulate tran-scriptional activity by generating a combinatorial platformfor effector proteins. Therefore, on the basis of chromosomaldistribution of anti-H3S10phK14ac, which eventually targetsthe pericentromeric chromatin, it is proposed that H3S10phby establishing a ‘phospho-acetyl switch’ with the adja-cent H3K14ac may facilitate the transcriptional activationof centromeric genes, sister chromatid cohesion at the peri-centromere followed by chromosome condensation eventsduring mitosis. Therefore, H3S10phK14ac histone eventu-ally offers an additional and important cytogenetic landmarkto identify centromeric chromatin of mitotic chromosomesin plants.

Crosstalk between H3S10ph and H3K14ac, and its rolein chromatin dynamics

Of note, acetylation of histone H3K14 is associated withactive transcription sites and particularly enriched in modi-fications that mark active chromatin, while there are no dataon the chromosomal distribution of methylated H3K14 yet(Fuchs et al. 2006; Sharma et al. 2015). While, hyperphos-phorylation of H3S10 is more or less actively engaged inthe initiation of chromosome condensation events through-out cell cycle progression (Houben et al. 2007). It is quiteapparent that the H3 phosphorylation and acetylation are spa-tially linked, but independent events, where one of the PTMsis not required for the establishment of the other (Thomsonet al. 2001). However, Fischle et al. (2005) suggested thatH3S10ph in combination with other modifications mightmodulate transcriptional activity by generating a combina-torial platform for effector proteins. In particular, Lo et al.(2000) advocated that at least in yeast and mammalian sys-tems, the dependence of H3K14ac for H3S10ph is not uni-versal but such combination usually occurs in a promoterspecific fashion. Simultaneously, Salvador et al. (2001) sug-gested that both H3S10ph and H3K14ac together medi-ate transcriptional activation of mammalian genes in ovarydifferentiation. Further, in case of chromatin dynamics, itis quite apparent that combinatorial histone modificationsalong with their interaction with effector modules/proteinsmight stimulate a cross talk (so called modification switches)among different chromatin modifications that ultimatelyleads the spatiotemporal switch of chromatin-associatedevents. In this connection, Fischle et al. (2005) also advisedthat the H3S10ph trigger the gene activation by establish-ing a ‘methyl/phos switch’ with the adjacent H3K9me3 andregulate the binding of heterochromatin protein 1 (HP1)to form heterochromatin with the help of kinase AuroraB in mammals. Interestingly, 14-3-3 proteins also seem tobe required for transcriptional/gene activation but not nec-essary for H3S10 phosphorylation and/or H3K14 acetyla-tion in mammals (Winter et al. 2008). Such observation

indicated that 14-3-3 proteins might be the readers of theeffector complexes that ultimately regulate transcriptionalactivation of the genes marked with H3S10phK14ac (Winteret al. 2008). Therefore, our study also propose that the com-bination of H3K14ac and H3S10ph establish a cross talkthrough differential kinases/phosphatases-mediated stimula-tion of signalling cascades to actively mark pericentromericchromatin during mitosis in plants. However, the slight tem-poral shift observed with anti-H3S10phK14ac (metaphase tolate telophase) in comparison to H3S10ph (prophase to earlytelophase) during initiation of phospho-acetylation eventsmight be influenced by phospho-acetyl modification switchthat ultimately allow the minor alteration of chromatin-associated events.

Future perspectives of combinatorial histone modification marksin plant cytogenetics

The abundance of specific histone modifications have beencytogenetically characterized, however, their manifestationmay vary dramatically between plant/cell types throughoutinterphase to telophase in both mitosis and meiosis in plants(Xu et al. 2009). The transient nature of histone modifi-cation allows the identification of temporal changes in theelucidation of associated histone marks and its cytologicaldistribution (Sharma et al. 2015, 2016). The dual modifiedanti-H3S10phK14ac can be significantly employed to theplant systems to understand the combinatorial target at chro-mosome level during cell division. The antibody preciselytarget the pericentromeric position in land and epiphyticplants belonging to monocot/dicots, however, its specificityshould also be reconfirmed in other plant systems includ-ing bryophytes, ferns, gymnosperms, diploid/polyploids,different cell types, e.g. vegetative/germinative cells of pol-lens. Further research aiming at immunecytogenetic char-acterization of several other multicombinations of histonesmay uncover the role and functional aspects of ‘modificationswitches’ in cell division by comparing combinatorial targetwith that of associated typical individual modification pat-tern. Several other combined modifications should also beinvestigated in plant/animal systems too to ensure the pre-cise distribution and biological function during cell cycle.Further, employment of dual/multimodified histone antibo-dies, e.g. phospho-acetylated and/or phospho-methylated aswell as phosphor-methyl-acetylated histones in genomewideChIP-seq analysis may uncover the significance and biolog-ical function of the combinatorial histone marks for generegulation both in animal and plant systems. The erraticsegregation of the chromosomes during meiosis may alsobe determined by employing highly specific pericentromericantibodies. Finally, identification of additional combinato-rial modifications at molecular level might lead to the dis-covery of novel functions of histone modification marks inchromatin-associated events and functions.

H3S10phK14ac histone offers a cytogenetic landmarkto identify centromeric chromatin and facilitate the sister



chromatid cohesion at the pericentromeres besides conden-sation of mitotic chromosomes in plants.

Acknowledgements

We thank Japan Society for the Promotion of Science (JSPS), Japan,for providing postdoctoral fellowship and research grant (SKS,no. P13399/2013). Sincere thanks are also to Dr Go Suzuki andall members of Plant Molecular Genetics Laboratory and OsakaKyoiku University, Osaka, Japan, for their constant encouragementand help.

References

Barth T. K. and Imhof A. 2010 Fast signals and slow marks: thedynamics of histone modifications. Trends Biochem. Sci. 35,618–626.

Briggs S. D., Xiao T, Sun Z. W., Caldwell J. A., Shabanowitz J.,Hunt D. F. et al. 2002 Gene silencing: trans-histone regulatorypathway in chromatin. Nature 418, 498.

Brunmeir R., Lagger S., Simboeck E., Sawicka A., Egger G.,Hagelkruys A. et al. 2010 Epigenetic regulation of a murine retro-transposon by a dual histone modification mark. PLoS Genet. 6,e1000927.

Cheung P., Allis C. D. and Sassone-Corsi P. 2000 Signal-ing to chromatin through histone modifications. Cell 103,263–271.

Clayton A. L., Rose S., Barratt M. J. and Mahadevan L. C. 2000Phosphoacetylation of histone H3 on c-fos- and c-jun-associatednucleosomes upon gene activation. EMBO J. 19, 3714–3726.

Demidov D., Schubert V., Kumke K., Weiss O., Karimi-Ashtiyani R., Buttlar J. et al. 2014 Anti-phosphorylatedhistone H2AThr120: a universal microscopic marker for cen-tromeric chromatin of mono- and holocentric plant species.Cytogenet. Genome Res. 143, 150–156.

Daujat S., Bauer U. M., Shah V., Turner B., Berger S. andKouzarides T. 2002 Crosstalk between CARM1 methylation andCBP acetylation on histone H3. Curr. Biol. 12, 2090–2097.

Dong Q. and Han F. 2012 Phosphorylation of histone H2A is associ-ated with centromere function and maintenance in meiosis. PlantJ. 71, 800–809.

Fischle W., Wang Y. and Allis C. D. 2003 Binary switches and mod-ification cassettes in histone biology and beyond. Nature 425,475–479.

Fischle W., Tseng B. S., Dormann H. L., Ueberheide B. M., GarciaB. A., Shabanowitz J. et al. 2005 Regulation of HP1-chromatinbinding by histone H3 methylation and phosphorylation. Nature438, 1116–1122.

Fuchs J., Demidov D., Houben A. and Schubert I. 2006 Chro-mosomal histone modification patterns—from conservation todiversity. Trends Plant Sci. 11, 199–208.

Gao Z., Fu S., Dong Q., Han F. and Birchler J. A. 2011 Inactivationof a centromere during the formation of a translocation in maize.Chromosome Res. 19, 755–761.

Garcia B. A., Barber C. M., Hake S. B., Ptak C., Turner F. B., BusbyS. A. et al. 2005 Modifications of human histone H3 variantsduring mitosis. Biochemistry 44, 13202–13213.

Goto H., Tomono Y., Ajiro K., Kosako H., Fujita M., Sakurai M.et al. 1999 Identification of a novel phosphorylation site on his-tone H3 coupled with mitotic chromosome condensation. J. Biol.Chem. 274, 25543–25549.

Granot G., Sikron-Persi N., Li Y. and Grafi G. 2008 PhosphorylatedH3S10 occurs in distinct regions of the nucleolus in differentiatedleaf cells. Biochim. Biophys. Acta 1789, 220–224.

Guerra M., Brasileiro-Vidal A. C., Arana P. and Puertas M. J. 2006Mitotic microtubule development and histone H3 phosphorylationin the holocentric chromosomes of Rhynchospora tenuis (Cyper-aceae). Genetica 126, 33–41.

Han F., Lamb J. C. and Birchler J. A. 2006 High frequency of cen-tromere inactivation resulting in stable dicentric chromosomes inmaize. Proc. Natl. Acad. Sci. USA 103, 3238–3243.

Han F., Gao Z. and Birchler J. A. 2009 Reactivation of an inac-tive centromere reveals epigenetic and structural componentsfor centromere specification in maize. Plant Cell 21, 1929–1939.

Hendzel M. J., Wei Y., Mancini M. A., Van Hooser A., RanalliT., Brinkley B. R. et al. 1997 Mitosis-specific phosphorylationof histone H3 initiates primarily within pericentromeric hete-rochromatin during G2 and spreads in an ordered fashion coinci-dent with mitotic chromosome condensation. Chromosoma 106,348–360.

Houben A., Wako T., Furushima-Shimogawara R., Presting G.,Kunzel G., Schubert I. and Fukui K. 1999 The cell cycledependent phosphorylation of histone H3 is correlated with thecondensation of plant mitotic chromosomes. Plant J. 18, 675–679.

Houben A., Demidov D., Caperta A. D., Karimi R., Agueci F. andVlasenko L. 2007 Phosphorylation of histone H3 in plants—adynamic affair. Biochim. Biophys. Acta 1769, 308–315.

Jackson A., Shires R., Chalkley D. K. and Granner D. K. 1975 Stud-ies on highly metabolically active acetylation and phosphoryla-tion of histones. J. Biol. Chem. 250, 4856–4863.

Jenuwein T. and Allis C. D. 2001 Translating the histone code.Science 293, 1074–1080.

Jia S., Kobayashi R. and Grewal S. I. 2005 Ubiquitin ligase com-ponent Cul4 associates with Clr4 histone methyltransferase toassemble heterochromatin. Nat. Cell Biol. 7, 1007–1013.

Lo W. S., Trievel R., Rojas J., Duggan L., Allis D., MarmorsteinR. and Berger S. 2000 Phosphorylation of serine 10 in histoneH3 is functionally linked in vitro and in vivo to Gcn5-mediatedacetylation at lysine 14. Mol. Cell 5, 917–926.

Manzanero S., Arana P., Puertas M. J. and Houben A. 2000 Thechromosomal distribution of phosphorylated histone H3 dif-fers between plants and animals at meiosis. Chromosoma 109,308–317.

Marcon-Tavares A. B., Felinto F., Feitoza L., Barros e Silva A. E.and Guerra M. 2014 Different patterns of chromosomal histoneH3 phosphorylation in land plants. Cytogenet. Genome Res. 143,136–143.

Nagaki K., Kashihara K. and Murata M. 2005 Visualizationof diffuse centromeres with centromere-specific histone H3in the holocentric plant Luzula nivea. Plant Cell 17, 1886–1893.

Nagaki K., Yamamoto M., Yamaji N., Mukai Y. and Murata M.2012 Chromosome dynamics visualized with an anti-centromerichistone H3 antibody in Allium. PLoS One 7, e51315.

O’Connor C. 2008 Chromosome segregation in mitosis: the role ofcentromeres. Nat. Edu. 1, 28.

Prigent C. and Dimitrov S. 2003 Phosphorylation of serine 10 inhistone H3, what for? J. Cell Sci. 116, 3677–3685.

Salvador L. M., Park Y., Cottom J., Maizels E. T., Jones J. C.,Schillace R. V. et al. 2001 Follicle-stimulating hormone stimu-lates protein kinase A-mediated histone H3 phosphorylation andacetylation leading to select gene activation in ovarian granulosacells. J. Biol. Chem. 276, 40146–40155.

Sawicka A. and Seiser C. 2014 Sensing core histonephosphorylation—a matter of perfect timing. Biochim. Biophys.Acta 1839, 711–718.

Sharma S. K., Yamamoto M. and Mukai Y. 2015 Immuno-cytogenetic manifestation of epigenetic chromatin modificationmarks in plants. Planta 241, 291–301.



Sharma S. K., Yamamoto M. and Mukai Y. 2016 Distinctchromatin environment associated with phosphorylated H3S10histone during pollen mitosis I in orchids. Protoplasma,(doi: 10.1007/s00709-015-0925-z).

Simboeck E., Sawicka A., Zupkovitz G., Senese S., Winter S.,Dequiedt F. et al. 2010 A phosphorylation switch regulates thetranscriptional activation of cell cycle regulator p21 by histonedeacetylase inhibitors. J. Biol. Chem. 285, 41062–41073.

Strahl B. D. and Allis C. D. 2000 The language of covalent histonemodifications. Nature 403, 41–45.

Sun Z. W. and Allis C. D. 2002 Ubiquitination of histone H2Bregulates H3 methylation and gene silencing in yeast. Nature 418,104–108.

Suzuki G., Shiomi M., Morihana S., Yamamoto M. and MukaiY. 2010 DNA methylation and histone modification in onionchromosomes. Genes Genet. Syst. 85, 377–382.

Thomson S., Clayton A. L. and Mahadevan L. C. 2001 Inde-pendent dynamic regulation of histone phosphorylation andacetylation during immediate-early gene induction. Mol. Cell 8,1231–1241.

Van Hooser A., Goodrich D. W., Allis C. D., Brinkley B. R. andMancini M. A. 1998 Histone H3 phosphorylation is required forthe initiation, but not maintenance, of mammalian chromosomecondensation. J. Cell Sci. 111, 3497–3506.

Wang Z. and Patel D. J. 2011 Combinatorial readout of dual histonemodifications by paired chromatin-associated modules. J. Biol.Chem. 286, 8363–8368.

Wei Y. and Allis C. D. 1998 A new marker for mitosis. Trends CellBiol. 8, 266.

Wei Y., Mizzen C. A., Cook R. G., Gorovsky M. A. and AllisC. D. 1998 Phosphorylation of histone H3 at serine10 is corre-lated with chromosome condensation during mitosis and meiosisin Tetrahymena. Proc. Natl. Acad. Sci. USA 95, 7480–7484.

Winter S., Simboeck E., Fischle W., Zupkovitz G., Dohnal I.,Mechtler K. et al. 2008 14–3–3 proteins recognize a histonecode at histone H3 and are required for transcriptional activation.EMBO J. 27, 88–99.

Xu D., Bai J., Duan Q., Costa M. and Dai W. 2009 Covalent modi-fications of histones during mitosis and meiosis. Cell Cycle 8,3688–3694.

Received 11 March 2016, in revised form 23 April 2016; accepted 27 April 2016Unedited version published online: 29 April 2016Final version published online: 1 December 2016

Corresponding editor: UMESH C. LAVANIA


dual modiﬁed antiphospho (ser10)-acetyl (lys14)-histone h3

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