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Planta (2013) 238:23–33 DOI 10.1007/s00425-013-1885-1

ORIGINAL PAPER

The dynamics of histone H3 modifications is species-specific in plant meiosis Cecilia Oliver • Mo´nica Pradillo • Eduardo Corredor Nieves Cun˜ado



Received: 17 January 2013 / Accepted: 11 April 2013 / Published online: 27 April 2013 Ó Springer-Verlag Berlin Heidelberg 2013

Abstract Different histone modifications often modify DNA-histone interactions affecting both local and global structure of chromatin, thereby providing a vast potential for functional responses. Most studies have focused on the role of several modifications in gene transcription regulation, being scarce on other aspects of eukaryotic chromosome structure during cell division, mainly in meiosis. To solve this issue we have performed a cytological analysis to determine the chromosomal distribution of several histone H3 modifications throughout all phases of both mitosis and meiosis in different plant species. We have chosen Aegilops sp. and Secale cereale (monocots) and Arabidopsis thaliana (dicots) because they differ in their phylogenetic affiliation as well as in content and distribution of constitutive heterochromatin. In the species analyzed, the patterns of H3 acetylation and methylation were held constant through mitosis, including modifications associated with ‘‘open chromatin’’. Likewise, the immunolabeling patterns of H3 methylation remained invariable throughout meiosis in all cases. On the contrary, there was a total loss of acetylated H3 immunosignals on condensed chromosomes in both meiotic divisions, but only in monocot species. Regarding the phosphorylation of histone H3 at Ser10, present on condensed chromosomes, although we did not observe any difference in the dynamics, we found slight differences between the chromosomal distribution of this modification between Arabidopsis and cereals (Aegilops sp. and rye). Thus far, in Electronic supplementary material The online version of this article (doi:10.1007/s00425-013-1885-1) contains supplementary material, which is available to authorized users. C. Oliver  M. Pradillo  E. Corredor  N. Cun˜ado (&) Departamento de Gene´tica, Facultad de Biologı´a, Universidad Complutense, C/Jose´ Antonio Nova´is 12, 28040 Madrid, Spain e-mail: [email protected]

plants chromosome condensation throughout cell division appears to be associated with a particular combination of H3 modifications. Moreover, the distribution and dynamics of these modifications seem to be species-specific and even differ between mitosis and meiosis in the same species. Keywords Arabidopsis  Aegilops  Chromosome condensation  Histone H3 modification  Meiosis  Mitosis  Secale Abbreviations BSA Bovine serum albumin DAPI 40 ,6-Diamidino-2-phenylindole H3K9K14ac Histone H3 acetylation at lysine 9 and/or lysine 14 H3K4me2 Histone H3 di-methylation at lysine 4 H3K4me3 Histone H3 tri-methylation at lysine 4 H3K9me2 Histone H3 di-methylation at lysine 9 H3K27me3 Histone H3 tri-methylation at lysine 27 H3S10ph Histone H3 phosphorylation at serine 10 PBS Phosphate-Buffered Saline Meiotic stages L Leptotene Z Zygotene P Pachytene Dp Diplotene Dk Diakinesis MI Metaphase I AI Anaphase I TI Telophase I Dd Dyad PII Prophase II MII Metaphase II AII Anaphase II

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TIIt Td

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Telophase II Tetrad

Somatic cell stages I Interphase I* Interphase of tapetum binucleated cells P Prophase M Metaphase A Anaphase T Telophase

Introduction Histones can undergo a great variety and number of covalent post-translational modifications of their amino-termini tails, including acetylation, methylation, phosphorylation, sumoylation, ubiquitination and ADP ribosylation (see, for review, Kouzarides 2007). These modifications influence DNA-histone interactions and are believed to regulate the level of chromatin compaction, either by disrupting chromatin contacts or by recruiting non-histone proteins to chromatin (Fischle et al. 2003; Cerutti and Casas-Mollano 2009; Lauria and Rossi 2011). Different combinations of histone modifications are very important for the fine-tuning of transcriptional regulation and DNA metabolism-related processes. In general, acetylation correlates with transcriptional activated genes and also facilitates DNA repair and recombination. By contrast, high methylation is present in transcriptionally silent chromatin although it is highly dependent on the type of modified residue (Pfluger and Wagner 2007; Roudier et al. 2009, 2011). Even though histones and their modifications are highly conserved, recent data have shown that chromosomal distribution of individual modifications can be different among groups of eukaryotes. This fact implies the possibility of evolutionary divergence in the histone language, at least at whole chromosomal level. For example, in plants the patterns of acetylated H3 and H4 isoforms often do not coincide with those found in mammals (Wako et al. 2002). Similarly, the distribution of different methylated histone isoforms can vary between yeasts, Neurospora, Drosophila and mammals, as well as among plant species (Lachner et al. 2004; Loidl 2004). Numerous studies have demonstrated that these modifications play a key role in maintaining normal transcription patterns during interphase, by directly or indirectly affecting the structural properties of the chromatin (Prigent and Dimitrov 2003). However, there is much more to be learned regarding whether or not these histone covalent modifications are involved in achieving global chromosome condensation in nuclear divisions, mainly in meiosis (see, for review, Xu et al. 2009).

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Phosphorylation is one of the best studied post-translational histone modifications during mitosis and meiosis, particularly the phosphorylation of histone H3 at Ser10 (H3S10ph). This dynamic post-translational modification is believed to be involved in both permissive and restrictive chromatin in interphase (Prigent and Dimitrov 2003; Granot et al. 2009). It could also play a role in chromosome condensation since there is a significant increase in the levels of H3 phosphorylation at the beginning of cell division and a decrease during both mitotic and meiotic telophases (Van Hooser et al. 1998; Wei et al. 1999; Houben et al. 1999; Manzanero et al. 2000). However, in some cereals, the phosphorylation levels are heterogeneously distributed along chromosomes during mitosis and second meiotic division (high in pericentromeric regions but very low in chromosome arms), while in the first meiotic division chromosomes are highly phosphorylated throughout (Houben et al. 1999; Manzanero et al. 2000). Another peculiarity of the H3 phosphorylation distribution in these plants is that single chromatids, which result from equational divisions of univalents during anaphase I, do not show immunolabeling all over the second meiotic division (Manzanero et al. 2000). In order to clarify the relationship between chromosome condensation and histone modifications in different plant species, the present study analyzes the chromosomal distribution of selected histone H3 modifications associated with transcriptional regulation and differentiation of euchromatin and heterochromatin (e.g. acetylation, phosphorylation, and methylation of diverse residues) throughout all phases of both meiosis and mitosis. The plant material chosen for this study was the dicot Arabidopsis thaliana and different types of Gramineae (the monocot species Aegilops sp. and Secale cereale) because of their different phylogenetic affiliation, genome size and heterochromatin content. The results show that the patterns of the H3 modifications studied diverge in the chromosomal distribution/ location (e.g. methylation of different residues), and during the different cell-cycle stages (e.g. phosphorylation or acetylation). Moreover, they vary between meiosis and mitosis (e.g. acetylation) and/or between the plant species, revealing the existence of species-specific chromatin-based regulatory mechanisms.

Materials and methods Plant material The plant materials used were the dicot species Arabidopsis thaliana L. (ecotype Columbia) (2n = 10), and several monocot species as the diploid Aegilops uniaristata

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Vis. (2n = 14, NN), the tetraploid Ae. ventricosa Tausch (2n = 49 = 28, DDNN), the synthetic amphiploid Ae. ventricosa Tausch–Secale cereale L. (2n = 69 = 42, DDNNRR), and the hybrid Ae. cylindrica Host 9 Ae. caudata L. (2n = 39 = 21, DCC). The source of the natural species was the Aula Dei Experimental Station, CSIC, Zaragoza, Spain, whereas seeds of the synthetic amphiploid were kindly supplied in 1980 by Dr. F. Dosba (INRA, France). The Aegilops species were selected because all they shared one or two genomes (D and/or N) and so it was possible to compare their behavior in different situations. The plants were grown in a conditioned greenhouse under identical conditions, with a 16-h day/8-h night photoperiod. Immunostaining Immunolocalization of modified H3 histones was performed according to Manzanero et al. (2000), with some modifications. Young buds and anthers in the florets of the emerging spikes were fixed for 20 min in freshly prepared 4 % (w/v) paraformaldehyde, 0.1 % (v/v) Triton X-100 in phosphate-buffered saline (PBS, pH 7.3). Buds and anthers were then washed at room temperature for 30 min in PBS that was changed twice. Buffer was removed before incubation at 37 °C during 20–40 min with an enzyme mixture of 1 % pectinase, 1 % cellulase and 1 % cytohelicase (w/v) (Sigma), dissolved in PBS. Buds or anthers, immersed in a small volume of PBS, were transferred to slides with a Pasteur pipette, macerated with a needle and squashed between a glass slide and cover slip. After freezing in liquid nitrogen, the cover slips were removed and the slides were transferred immediately into PBS. Prior to immunostaining experiments the slides were washed twice in PBS, 0.1 % (v/v) Triton X-100 for 5 min each. To avoid non-specific antibody binding, slides were incubated for 30 min in PBS with 1 % BSA (w/v) and 0.1 % Triton X-100 at room temperature. The incubation with the primary antibody was carried out in a humidified chamber. The primary antibodies used were mouse anti-phosphorylated-Histone H3 (Ser 10) (H3S10ph) (Upstate, cat. no. 05-598, diluted at 1:300); rabbit anti-dimethyl-Histone H3 (Lys 4) (H3K4me2) (Upstate, cat. no. 07-030, diluted at 1:200); rabbit anti-trimethyl-Histone H3 (Lys 4) (H3K4me3) (Abcam, cat. no. ab8580, diluted at 1:200); rabbit anti-dimethyl-Histone H3 (Lys 9) (H3K9me2) (Upstate, cat. no. 07-212, diluted at 1:200); rabbit antitrimethyl-Histone H3 (Lys 27) (H3K27me3) (Upstate, cat. no. 07-449, diluted at 1:200); rabbit anti-acetyl-Histone H3 (Lys 9 and/or 14) (H3K9K14ac) (Upstate, cat. no. 06-599, diluted at 1:200). All the primary antibodies were diluted in PBS, 1 % BSA, 0.1 % Triton X-100.

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After overnight incubation at 4 °C and washing for 15 min in PBS with 0.1 % Triton X-100, the slides were incubated for 1 h at room temperature with anti-mouse IgG Cy3 conjugate (Sigma, C2181) or anti-rabbit IgG FITC conjugate (Sigma, F7512) antibody, diluted 1:150 in 1 % BSA, 0.1 % Triton X-100 in PBS. Then the slides were washed in PBS, 0.1 % Triton X-100, before they were stained with the fluorochrome 40 ,6-diamidino-2-phenylindole (DAPI, 10 lg/ml) in Vectashield antifade mounting medium (Vector Laboratories). Fluorescent signals were observed using an epifluorescence microscope Olympus BX-61. Images were captured with an Olympus DP71 digital camera controlled by analySIS software (Soft Imaging System), analyzed, and processed with Adobe Photoshop CS4 software. Single DAPI images were presented in gray as the best way to discriminate the subtle contrast between the different chromosome regions (heterochromatin/euchromatin). In the merged images, all immunosignals were shown in green color, and the DAPI staining in red.

Results We have analyzed the chromosomal distribution of some H3 histone modifications during mitosis and both meiotic divisions in different plant species (the dicot species as Arabidopsis thaliana and the monocot species Aegilops sp. and Secale cereale). We have selected different H3 modifications associated not only with regulation of transcriptional activity but also with the differentiation of euchromatin and heterochromatin (H3K9K14ac, H3K4me2/3, H3K9me2, and H3K27me3) and with chromosome condensation (H3S10ph). On the other hand, Arabidopsis thaliana and Gramineae species (Aegilops and Secale) were selected because of their significant differences in genome size and heterochromatin content (small vs large size genome, and low vs high percentage of repetitive sequences, respectively). In addition, the monocot plants analyzed, named Aegilops uniaristata (NN), Ae. ventricosa (DDNN), synthetic amphiploid Ae. ventricosa–Secale cereale (DDNNRR), and the hybrid Ae. cylindrica 9 Ae. caudata (DCC), are quite different regarding heterochromatin content and distribution as detected by C-banding. Thus, rye chromosomes have prominent C-bands at one or both distal regions, whereas in Aegilops species, C-heterochromatin usually appears in centromeric, pericentromeric and interstitial regions. In addition, there are also qualitative and quantitative differences among Aegilops genomes: the N chromosomes of Ae. uniaristata (NN) and Ae. ventricosa (DDNN) show prominent C-bands located at the centromeric and pericentromeric regions, whereas the D genome

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shows thin centromeric and interstitial bands (Teoh and Hutchinson 1983; Cermen˜o et al. 1985; Cun˜ado 1992). Besides, it was possible to analyze the labeling patterns of some specific genomes (N and D) in a diploid or polyploid background (natural or artificial). Finally, we have also included the Ae. cylindrica 9 Ae. caudata (DCC) hybrid and the Ae. ventricosa 9 S. cereale amphiploid to analyze whether the immunostaining differs among chromosomes forming bivalents or univalents at first meiotic division. In all species, the mitotic division was analyzed in cells obtained from anthers. The immunosignals pattern was analyzed in a minimum of either 20 pollen mother cells or somatic cells in each stage; all the cells displayed the same pattern (summarized in Table 1). Histone H3 phosphorylation at serine 10 The spatial and temporal course of serine 10 H3 phosphorylation (H3S10ph) during mitosis and meiosis has been previously characterized in the monocot species S. cereale and T. aestivum (Manzanero et al. 2000). In the present work the immunolabeling patterns along the chromosomes obtained in Arabidopsis thaliana were slightly different (Table 1). In a similar way, in Arabidopsis, the H3S10ph pattern was hardly observed during interphase of somatic cells (Fig. 1a). The immunosignals appeared not at the initiation of chromosome condensation, but at mid-late prophase (Fig. 1a). At metaphase and anaphase, the whole chromosomes showed strong

immunolabeling which disappeared at the end of telophase (Fig. 1b). Regarding early stages of first meiotic division, in Arabidopsis, H3 phosphorylation was detected for the first time at diplotene, the pericentromeric regions appearing slightly more labeled than other parts of the chromosomes (Fig. 1d). From diakinesis to anaphase I chromosomes were homogeneous and strongly immunolabeled (Fig. 1e). At telophase I, the phosphorylation of H3 gradually disappeared. Between the first and second meiotic divisions there is not DNA replication and the chromatin displayed no immunostaining (Fig. 1f). Unexpectedly, the pattern of H3S10ph at second meiotic division was similar to that observed in the first one and during the mitotic division; the entire chromosomes were immunolabeled from late prophase II to metaphase II (Fig. 1g). The phosphorylation status remained unchanged after the separation of the sister chromatids at anaphase II. At telophase II, at the same time the chromosomes decondensed, the phosphorylation of histone H3 disappeared and was not observed in tetrads (Fig. 1h). Histone H3 acetylation at lysines 9 and/or 14 In contrast to the dynamic H3 phosphorylation, the present study revealed that in Arabidopsis and monocot plants (Aegilops and Secale) the acetylation state of H3 at lysines 9 and/or 14 (H3K9K14ac) was maintained constant throughout mitosis, independent of chromatin

Table 1 The immunolabeling patterns of H3 histone modifications throughout all phases of mitosis and meiosis in Arabidopsis thaliana and Gramineae species analyzed (Aegilops sp. and Secale cereale) Modified histone

H3S10ph

Plant species

A. thalianab Gramineae

a

Mitotic cells

Pollen mother cells

I

P

M-A

T

L-P

Dp

Dk

MI-AI

TI

Dd

PII

MII-AII

TII-Td

-

-?

?(b)

?-



-?

?

?(b)

?-



-?

?(b)

?-



-?

?

(d)

(b)

?



?

?(d)

? -?

? ?

? ?-

? –

? ?

?

?

?

?

?



?

?

?

H3K9K14ac

A. thalianac Gramineaec

? ?

? ?

? ?

? ?

? ?

? ?

? ?-

? –

H3K4me2/me3

A. thalianac

?

?

?

?

?

?

?

?

b

?

?

?

?

?

?

?

?

?

?

?

?

?

A. thalianad

?

?

?

?

?

?

?

?

?

?

?

?

?

Gramineaeb

?

?

?

?

?

?

?

?

?

?

?

?

?

A. thalianab

?

?

?

?

?

?

?

?

?

?

Gramineaee

?

?

?

?

?

?

?

?

?

?

Gramineae H3K9me2 H3K27me3

?

?

?

?

?

?, Immunosignals visible; -, immunosignals not detectable; -?, gradual transition from a slight signal to a strong one; ?-, gradual transition from a strong signal to a slight one a

Data taken from Manzanero et al. (2000)

b

Labeling distributed throughout the entire chromosomes

c

Labeling distributed along the chromosome arms, whereas the pericentromeric regions showed reduced or no signals

d

Labeling clustered at pericentromeric domains

e

Labeling detected at distal regions of chromosome arms

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locations) showed reduced histone acetylation signal (Supplementary Fig. S1). Once more, in Aegilops and Secale chromosomes the H3K9K14ac immunostaining pattern during meiosis clearly differed from that observed in Arabidopsis meiosis, but it was also different from that of mitosis. Thus, in Arabidopsis the overall H3 acetylation levels remained stable over time at meiosis (Fig. 2a–e), although the pericentromeric heterochromatin regions were largely devoid of signals (Fig. 2a). On the contrary, in monocot plants, the immunosignals were distributed all over the decondensed chromosomes at early phases of both meiotic divisions, but a general reduction of acetylation was observed at diplotene/diakinesis transition, when chromosomes are in a highly compact state (Fig. 2f–j). At metaphase I and anaphase I, both bivalents and univalents, last ones observed in the synthetic amphiploid, did not show any labeling (Fig. 2h and Supplementary Fig. S1). The immunosignals reappeared at telophase I and were distributed over the entire decondensed chromosomes (Fig. 2i). The spatial and temporal course of H3 acetylation during the second meiotic division resembled that of the first one (Fig. 2j and Supplementary Fig. S1). Histone H3 methylation at lysines 4, 9 and 27

Fig. 1 Immunolocalization of histone H3 phosphorylated at serine 10 at representative stages of mitotic (a–b) and meiotic (c–h) cells of Arabidopsis thaliana. Mitosis: metaphase (a) and anaphase (b). Meiosis: zygotene-pachytene (c), diplotene (d, a detail of a centromeric region is shown), metaphase I (e), dyad (f), metaphase II (g), and tetrad (h). Left Immunostaining with antibodies against H3S10ph shown in green. Middle Nuclei counterstained with DAPI shown in gray. Right Merged, DAPI shown in red. Bars represent 10 lm

condensation (Supplementary Fig. S1). In Arabidopsis, during mitosis, the H3K9K14ac immunosignals appeared more or less uniformly distributed all over the interphase chromatin and condensed chromosomes. In Aegilops and rye, the H3 acetylation was also scattered along chromosomes at metaphase and anaphase, although heterochromatic regions (at pericentromeric and distal

The immunolabeling patterns obtained with antibodies that discriminate between di- and trimethylation at lysine 4 of histone H3 (H3K4me2/me3) were similar in all the species analyzed, and also between mitotic and meiotic divisions (Fig. 3 shows H3K4me3; H3K4me2, data not shown). In all cases, methylation levels of H3K4me2/me3 remained high and constant throughout the cell cycle and the immunosignals were distributed all over the chromosomes. Only in Arabidopsis, the labeling of methylated H3K4 was reduced in pericentromeric heterochromatin regions in stages as zygotene or pachytene (Fig. 3b). In contrast, the labeling remained uniform along the chromosomes in Aegilops and Secale (Fig. 3e–h). Moreover, the univalents showed the same immunolabeling that of the bivalents in the triploid Ae. cylindrica 9 Ae. caudata (DCC) hybrid (Fig. 3g). On the other hand, the labeling obtained using an antibody against dimethylated H3 at lysine 9 (H3K9me2) showed that there are remarkable differences in the chromosomal distribution between Arabidopsis, with the signals restricted to pericentromeric regions (Fig. 4a–d), and Aegilops, whose chromosomes were labeled throughout their length (Fig. 4e–h). In both cases, the spatial and temporal course of H3K9me2 remained constant throughout mitosis and meiosis (Fig. 4 and Supplementary Fig. S2).

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Fig. 2 Immunolocalization of histone H3 acetylated at lysine 9 and/ or lysine 14 at some meiotic stages in Arabidopsis thaliana (a–e), Aegilops ventricosa–Secale cereale amphiploid (f, g), and Ae. uniaristata (g–j). Left Immunostaining with antibodies against H3K9K14ac (green). Middle Nuclei counterstained with DAPI shown in gray. Right Merged, DAPI shown in red. Bars represent 10 lm.

A. thaliana: pachytene (a, a detail of a centromeric region is shown), diplotene (b), diakinesis (c), dyad (d), anaphase II (e). Ae. ventricosa– S. cereale amphiploid (f, h) and Ae. uniaristata (g, i–j): zygotenepachytene (f), diplotene and an arrow indicates one pro-metaphase I (g), metaphase I (h, arrowheads indicate some univalents), dyad (i), anaphase-telophase II (j)

Once more, the immunostaining pattern corresponding to histone H3 trimethylation at lysine 27 (H3K27me3) clearly differed between Arabidopsis and monocot plants. In Arabidopsis, the signals were uniformly dispersed over interphase nuclei, excluding nucleolus, and along the mitotic chromosomes (Fig. 5a and Supplementary Fig. S3). In contrast, in Aegilops and rye, H3K27 trimethylation labeling was clearly polarized toward one side of the interphase nuclei (Fig. 5e). Also, H3K27me3 staining was restricted to distal regions on metaphase and anaphase chromosomes (Fig. 5f and Supplementary Fig. S3). As well as other methylated histones, the chromosomal distribution of H3K27me3 was stable throughout mitosis and meiosis in both types of plants (Fig. 5 and Supplementary Figs. S3–S4). In addition, the univalents presented in the amphiploid Ae. ventricosa–S. cereale and in the triploid hybrid Ae. cylindrica 9 Ae. caudata showed the same pattern distribution than that displayed by bivalents (Figs. 3g, 5h and Supplementary Figs. S3p, S4a).

Discussion

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Histone H3 has been in the chromatin spotlight for many years and is currently considered to bear the greatest number of known modifications out of the four core histones (see, for review, Xu et al. 2009). Both data obtained from this study (Table 1) and by other authors show that the chromosomal distribution of individual H3 modifications (acetylation, methylation, and phosphorylation) can differ along the cell cycle as well as among plants (see, for review, Fuchs et al. 2006). This diversity can be understood taking into account the variability in genome size, composition and organization between the plants under study (Lachner et al. 2004; Bennett and Leitch 2005; Fuchs et al. 2006). Arabidopsis thaliana has an extremely small genome and only about 15 % of the nuclear genome consists of repetitive sequences and transposons (Leutwiler et al. 1984), mainly located at nucleolus-organizing regions and within the pericentromeric heterochromatin regions

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Fig. 3 Immunolocalization of histone H3 trimethylated at lysine 4 at selected stages of mitosis and meiosis of Arabidopsis thaliana (a–d), Aegilops cylindrica 9 Ae. caudata hybrid (e–g), and Ae. uniaristata (h). Left Immunostaining with antibodies against H3K4me3 (green). Middle Nuclei counterstained with DAPI shown in gray. Right Merged, DAPI shown in red. Bars represent 10 lm. A. thaliana,

Mitosis: metaphase (a); Meiosis: zygotene-pachytene (b, a detail of a centromeric region is shown), metaphase I (c), metaphase II (d). Ae. cylindrica 9 Ae. caudata hybrid (e–g) and Ae. uniaristata (h), Mitosis: metaphase and anaphase cells (e); Meiosis: zygotene (f), metaphase I (g, arrowhead indicates one univalent), metaphase II and telophase II (h)

Fig. 4 Immunolocalization of histone H3 dimethylated at lysine 9 at representative stages of mitotic and meiotic cells of Arabidopsis thaliana (a–d), Aegilops ventricosa (e), and Ae. uniaristata (f–h). Left Immunostaining with antibodies against H3K9me2 (green). Middle Nuclei counterstained with DAPI shown in gray. Right Merged, DAPI

shown in red. Bars represent 10 lm. A. thaliana, Meiosis: leptotene (a), pachytene (b), metaphase I (c), metaphase II (d). Ae. ventricosa (e) and Ae. uniaristata (f-h). Mitosis: metaphase (e); Meiosis: zygotene (f), metaphase I (g), metaphase II (h)

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Fig. 5 Immunolocalization of histone H3 trimethylated at lysine 27 at representative stages of mitotic and meiotic cells of Arabidopsis thaliana (a–d) and Aegilops cylindrica 9 Ae. caudata hybrid (e–h). Left Immunostaining with antibodies against H3K27me3 (green). Middle Nuclei counterstained with DAPI shown in gray. Right Merged, DAPI shown in red. Bars represent 10 lm. A. thaliana.

Mitosis: late prophase (a); Meiosis: zygotene-pachytene (b), metaphase I (c), metaphase II (d). Ae. cylindrica x Ae. caudata hybrid. Mitosis: interfase of tapetum binucleated cells (e), metaphase and anaphase (f); Meiosis: zygotene (g), diakinesis and metaphase I (h, arrowheads indicate some univalents)

corresponding to brightly DAPI-stained and Giemsabanding positive (Ambros and Schweizer 1976; Bauwens et al. 1991; Maluszynska and Heslop-Harrison 1991). In accordance with this fact, heterochromatin-specific H3 modifications (H3K9me2) were restricted mainly to the pericentromeric regions (Soppe et al. 2002) (Fig. 4 and Supplementary Fig. S2), coinciding with the absence of euchromatin-specific signals (H3K4 methylated and H3 acetylated) (Probst et al. 2004; Zhang et al. 2009; Lauria and Rossi 2011), which appear at high levels along the chromosome arms (Figs. 2, 3 and Supplementary Fig. S1). In contrast, rye and Aegilops sp. present large genomes, full of different families of repetitive sequences and mobile elements (together at least 85 % of the nuclear genome) that are scattered throughout the chromosome length, and/ or clustered in regions that appear as constitutive heterochromatin identified by C-banding (Flavell et al. 1974; Cermen˜o et al. 1985; Cun˜ado 1992; Heslop-Harrison and Schwarzacher 2011). These dispersed repetitive sequences (mainly retroelements) interspersed with potentially active genes are also silenced by ‘heterochromatinization’, and thereby the degree of condensation of the chromatin in these regions would be intermediate between the one of the constitutive heterochromatin and that of the euchromatin of small genomes (Soppe et al. 2002; Houben et al. 2003; Jasencakova et al. 2003). In Aegilops and rye, chromosome

regions showing this organization appeared associated with a more or less uniform labeling, because of the overlapping distribution of H3K9me2 (heterochromatic mark) with H3K9K14ac and H3K4me2/me3 (related to gene-rich regions), excluding the centromeric and pericentromeric regions in which H3K9me2 prevails (Figs. 2, 3, 4 and Supplementary Figs. S1–S2) (Jasencakova et al. 2001). Finally, H3K27me3 has been reported as a euchromatinspecific mark though to be mainly associated with genes under repression in Arabidopsis (Roudier et al. 2011) and, in agreement with this, the immunostaining was evenly distributed along the chromosomes. Conversely, in both Aegilops and Secale, the H3K27me3 labeling was stronger at distal chromosome regions, visible in metaphase and interphase nuclei (Fig. 5 and Supplementary Figs. S3–S4), even though the location of regions defined cytologically as Giemsa-banded heterochromatin was divergent in both species, being mainly pericentromeric and distal, respectively (Teoh and Hutchinson 1983; Cermen˜o et al. 1985; Cun˜ado 1992). Thus, H3K27me3 displayed two different distribution patterns: in Aegilops the immunostaining marked the gene-rich termini of metaphase chromosomes, whereas in Secale cereale, it overlapped with distal C-heterochromatin blocks. The functional meaning of this fact is until now an open question, although recent publications have provided new insights. In this context, the

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small intraspecific variation in H3K27me3 found between two Arabidopsis accessions could be explained by the insertion of TEs at the flanks of H3K27me3 targets (Dong et al. 2012). This epigenetic mark has been also suggested to be a factor in the evolution of expression patterns after gene duplication (Berke et al. 2012). H3 modifications in cell division Chromatin condensation is a common and essential process during cell division to ensure faithful segregation of the duplicated genome into daughter cells although chromosome dynamics in meiosis displays some peculiarities with respect to that in mitosis. For this reason, it is not surprising that we have found differences in the epigenetic profiles during meiotic chromosome morphogenesis. The chromatin has to undergo structural changes in meiotic prophase I, during which homologous chromosomes pair recombine and synapse (Zickler and Kleckner 1999; Kouzarides 2007) and diverse genetic, biochemical and cytological assays have established that several histone modifications play fundamental roles in these processes (Borde et al. 2009; Buard et al. 2009; Herna´ndez-Herna´ndez et al. 2010; Perrella et al. 2010). Until now, detailed dynamic changes of these H3 modifications have not been thoroughly examined at latter meiotic stages in plants. Regarding histone methylation, primarily associated with regulation of gene transcription (Kouzarides 2007; Xu et al. 2009; Zhang et al. 2009), it does not seem to be associated with mitotic or meiotic chromosome condensation and segregation, considering that the levels of H3 methylated at different lysine residues remained constant throughout the cell cycle in all plant species analyzed (Figs. 3, 4, 5 and Supplementary Figs. S2–S4). By contrast, the relationship between H3 phosphorylation and chromatin condensation during nuclear division is highly conserved among eukaryotes. Surprisingly, the distribution of this modification over the condensed chromosomes in Arabidopsis is different from that observed for S. cereale and T. aestivum (Table 1; Fig. 1). It cannot be ruled out that this difference might be due to the small size and low heterochromatin content of Arabidopsis chromosomes. Anyhow, all the collected data call into question whether H3 phosphorylation is an absolute requirement for the initiation and/or maintenance of chromosome condensation (Wei et al. 1998, 1999; Kasza´s and Cande 2000). First, H3 phosphorylation begins late in prophase when the chromosomes are already quite condensed. Second, it has been reported that H3 phosphorylation can be uncoupled from mitotic and meiotic chromosome condensation, after experiments in Tetrahymena mutants and mice spermatocytes treated with okadaic acid (Wei et al. 1999).

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Moreover, the distribution of H3S10ph along the chromosomes varies between both mitotic and meiotic divisions in monocot plants (Houben et al. 1999; Manzanero et al. 2000) and respect to the pattern observed in Arabidopsis (Fig. 1). Unlike the dynamic phosphorylation of H3, in Arabidopsis and monocot plant (Aegilops and Secale) the pattern of H3K9K14ac is held apparently constant regardless of the chromosome condensation through mitosis (Supplementary Fig. S1). These data diverge from those observed not only in animals but also in plants as tobacco, in which chromosomal packaging has been associated with H3 deacetylation (Li et al. 2005; Xu et al. 2009; Kheir and Lund 2010), although different chromosome regions show H3 acetylation in other dicot plants as Vicia (Belyaev et al. 1998). Accordingly, it might be possible that despite the general H3 acetylation reduction coinciding with chromosome compaction during mitosis, specific residues or chromosome regions remain acetylated in different animal and plant species. On the other hand, the meiotic pattern of H3 acetylated obtained in Secale and Aegilops species was different from that observed not only in Arabidopsis but also during mitotic division (Fig. 2 and Supplementary Fig. S1). In monocot species, chromosomes in a highly compact state during both meiotic divisions do not show any labeling, whereas in Arabidopsis, the overall levels of histone H3 acetylation remain stable throughout meiosis, over both decondensed and condensed chromosomes. As indicated before, this diversity in the labeling pattern may reflect differences in genome size, repetitive sequences content and organization, and may be related to previous data suggesting that the compaction of chromatin in Arabidopsis, at least in pachytene, was significantly lower than in other plant species (Lo´pez et al. 2008). Here we have found, for the first time, the existence of reversible changes in the labeling of one H3 modification throughout meiosis, coinciding with the progression of chromosome compaction, although the same does not seem to occur during mitosis. This difference raises the question whether chromosome condensation depends on changes in H3K9K14ac levels, since they appear to be meiotic and species-specific. Remarkably, our data, together with previous reports, point out that several H3 post-translational modifications, such as H3S10ph and H3K9K14ac, are not only reversible and highly dynamic, allowing quick changes in chromosome organization during both types of cellular division, but also independent of the DNA replication process, at least in plants (Pe´rez-Cadahı´a et al. 2009; Kheir and Lund 2010). All these data reveal that the patterns of histone modifications are not identical between organisms and even differ between cell types. Moreover, these modifications

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are not always well correlated with a specific chromatin conformation. Some combinations of histone H3 modifications described above appear to be associated with two opposed chromatin states in plants: the highly condensed mitotic and/or meiotic chromosomes and the more accessible chromatin structure observed during interphase (e.g. combination of H3S10ph and H3K9K14ac, associated generally with open chromatin and active transcription and highly condensed chromosomes in Arabidopsis) (Pe´rezCadahı´a et al. 2009; Kheir and Lund 2010). Likewise, we have found that in most cases, various methylations of H3 are present in either the same or different chromosomal domains throughout mitosis and meiosis. In conclusion, the mitotic and meiotic chromosome condensation appears to be associated with different combination of H3 modifications in plants, in agreement with differences in chromosome structure in both types of nuclear divisions. The meiotic temporal patterns of some H3 modifications might be species-specific and, also differ from that of mitosis found in the same species (Table 1). Further research will be required to elucidate cross-talks among different types of histone modifications to understand their implication in higher order condensation during cell division, as well as the evolutionary divergence in the language of the epigenetic marks in different plant species. Acknowledgments This work was supported by the Ministerio de Ciencia e Innovacio´n of Spain [Grant number BFU2008-00459/ BMC], the Universidad Complutense-Banco Santander of Spain [Grant number 910452] and the European Union Framework Program 7 [Meiosys-KBBE-2009-222883].

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