Tree Genetics & Genomes

, Volume 9, Issue 5, pp 1247–1256 | Cite as

Epigenetic characterization of chromatin in cycling cells of pedunculate oak, Quercus robur L.

  • V. Vičić
  • D. Barišić
  • T. Horvat
  • I. Biruš
  • Vlatka Zoldos
Original Paper


Cycling cells of Quercus robur have a simple nuclear organization where most of the heterochromatin is visible as DAPI-positive chromocenters, which correspond to DAPI bands at the (peri)centromeric region of each of the 24 chromosomes of the oak complement. Immunofluorescence using 5-mC revealed dispersed distribution of the signal throughout the interphase nucleus/chromosomes without enrichment within DAPI-positive chromocenters/bands, suggesting that DNA methylation was not restricted to constitutive heterochromatin, but was associated with both euchromatic and heterochromatic domains. While H3K9ac exhibited typical euchromatin-specific distribution, the distributional pattern of histone methylation marks H3K9me1, H3K27me2, and H3K4me3 showed some specificity. The H3K9me1 and H3K27me2, both heterochromatin-associated marks, were not restricted to chromocenters, but showed additional dispersed distribution within euchromatin, while H3K27me2 mark also clustered in foci that did not co-localize with chromocenters. Surprisingly, even though H3K4me3 was distributed in the entire chromatin, many chromocenters were enriched with this euchromatin-specific modification. We discuss the distribution of the epigenetic marks in the context of the genome composition and lifestyle of Q. robur.


Interphase chromatin Chromocenters DNA methylation Epigenetics Histone acetylation Histone methylation Quercus robur 


DNA methylation, histone modifications and histone variants, nucleosome positioning, action of small noncoding RNAs, and nuclear architecture are components of epigenetic information superimposed on the information encoded by nucleotide sequence. Modification of DNA, by the addition of a methyl group on carbon 5 position of cytosine (5-mC), has an important role in the regulation of gene transcription and in ensuring genome stability by repressing transcription of mobile elements. This is especially prominent in plant genomes, especially large ones (such as Hordeum vulgare, Triticum aestivum, Zea mays, etc.), which contain large proportions of transposable elements (TEs) (Wicker et al. 2001, 2005; Schnable et al. 2009), and DNA methylation is the main genome defense mechanism (Yoder et al. 1997). Posttranslational modifications of histone tails (methylation, acetylation, phosphorylation, ubiquitination, etc.) and their combinatorial association in the genome affect chromatin condensation and give rise to different chromatin states with different indexing functions, which participate in the selective readout of the genome sequence (Filion et al. 2010; Roudier et al. 2011). Mechanisms establishing DNA methylation and histone modification marks work interdependently, resulting in a formation of heterochromatin, as shown in the case of histone deacetylation and methylation at lysine 9 of H3 implicated in the establishment of DNA methylation patterns (Soppe et al. 2002; Tariq et al. 2003; Johnson et al. 2007; Li et al. 2008).

Even though epigenetic marks and mechanisms that establish them are fairly conserved, the distribution and the association of these marks with chromatin states and gene activity diverged in the plant kingdom, probably due to evolutionary forces related to special genomic needs for a particular taxa (Feng and Jacobsen 2011). The cytogenetic distribution of the epigenetic marks is influenced by several factors such as genome size (Houben et al. 2003; Fuchs et al. 2006), sequence composition, and chromatin organization within a particular genome (Shi and Dawe 2006; Braszewska-Zalewska et al. 2010). The most comprehensive characterization of distribution of epigenetic marks in the interphase nucleus is available for Arabidopsis thaliana (Fransz et al. 2003; Jasencakova et al. 2003; Jackson et al. 2004; Fuchs et al. 2006). More recently, immunostaining experiments to detect distribution of various acetylated and methylated histones were performed on interphase nuclei and metaphase chromosomes of many angiosperm species, mostly on crop plants or other cultivated plants such as Z. mays, H. vulgare, Vicia faba, Brassica species, Raphanus sativus, Glycine max, Allium sativum, or Citrus species (Fuchs et al. 2006; Shi and Dawe 2006; Carchilan et al. 2007; Marques et al. 2010; Suzuki et al. 2010; Fuchs and Schubert 2012). Studies performed on a few gymnosperm species suggest quite different chromosomal distribution and association of histone marks with gene activity from those of angiosperms (Fuchs et al. 2008). From these data, it became clear that the same histone modification marks could assume different roles within different species of the plant kingdom confirming the existence of plant dialects of the histone language (Loidl 2004). In short, methylation of H3K4 is a highly conserved euchromatin-specific mark, while distribution and interpretation of mono-, di-, and three-methylation of Lys 9 and 27 of histone H3 may vary between different plant species. Therefore, although H3K9me1, H3K9me2, and H3K27me1 in most plant species mark heterochromatin, in some species, these marks are also found spread within euchromatin. Furthermore, H3K27me2 and H3K27me3 are variably found in euchromatin or heterochromatin of different plant species (Fuchs et al. 2006, 2008; Feng and Jacobsen 2011).

The level of knowledge concerning tree genomes and epigenomes lags significantly behind the level of knowledge concerning annual and perennial plants or cultivated crop plants. The epigenetic information serves as a mediator between genes and the environment and thus has an extremely important role in plant adaptation. Indeed, roles for epigenetic pathways in plant stress tolerance, acclimation, and possibly adaptation are becoming increasingly evident (Pfluger and Wagner 2007; Boyko et al. 2010; Hauser et al. 2011). Oaks are hardwood trees with an average lifespan of 200 years. Some individuals can be over 500 years old and extreme cases in Europe are estimated to be between 1,200 and 1,500 years old. A very long life span and the ability to adapt to a wide range of soil and climatic conditions in a long time scale (Jones 1986) make oak trees specific, and it is not hard to imagine that epigenetic features of this fascinating trees are also peculiar. Therefore, we aimed to reveal the distribution of cytosine methylation and histone modification marks in pedunculate oak (Quercus robur L.), the predominant component of the northern hemisphere deciduous forests. Almost no epigenetic characterization of euchromatin and heterochromatin is available for any of the angiosperm wild trees. In the genus Quercus, there is only one report of the distribution of some epigenetic marks in the mature pollen cells of Quercus suber (Ribeiro et al. 2009). Here we present the first epigenetic chromatin landscape in Q. robur root tip meristematic cells, which represent plant cycling cells.

Materials and methods

Plant material

Acorns of Q. robur L. (Fagaceae) were obtained from Croatian Forest Research Institute, Jastrebarsko and germinated on moist cotton in Petri dishes in insolated place at room temperature. For fluorescence in situ hybridization (FISH) and immunofluorescence experiments (IF) using 5-methylcytosine (5-mC), root tips were fixed in freshly prepared ethanol–acetic acid (3:1) overnight at 4 °C, then transferred to 70 % ethanol, and stored at −20 °C until use. Prior to fixation, root tips were treated with 2 μM 8-hydroxiquinoline for 4 h at 18 °C. Root tips for IF experiments were fixed in 2 % (w/v) paraformaldehyde, 1 % (w/v) PVP, and 0.5 % (v/v) Triton X-100 in 1× PBS for 20 min at room temperature and then washed extensively in 1× PBS (phosphate buffered saline).

Slide preparation

Chromosome suspensions for FISH and IF using 5-mC were prepared by incubating root tips in the enzymatic mixture [8 % (w/v) cellulase RS, 4 % (w/v) hemicellulase, 4 % (w/v) pectolyase Y-23 in 0.1 M citrate buffer pH = 4.8] for 90 min at 37 °C. Then, root tips were macerated in a droplet of 60 % acetic acid, the obtained macerate was transferred into a 1.5-ml Eppendorf tube, and nuclei were collected by centrifugation at 800×g for 3 min. The supernatant was discarded and the pellet washed twice with freshly prepared fixative. Finally, the nuclei were resuspended in an appropriate volume of fixative, and 2–3 μl of the suspension was dropped on clean slides and air-dried. For immunodetection of histones, meristems were digested with the enzymatic mixture [8 % (w/v) cellulase RS, 4 % (w/v) hemicellulase, 4 % (w/v) pectolyase Y-23 in 0.1 M citrate buffer pH = 4.8] for 90 min at 37 °C, washed in 1× PBS for at least 15 min, transferred into a droplet of dH2O, and squashed. After freezing, the coverslips were removed and slides transferred to 1× PBS.

Immunodetection of 5-methylcytosine

For immunodetection of 5-mC, the slides were denatured in 70 % formamide in 2× SSC at 70 °C for 2 min and dehydrated through an ice-cold ethanol series. The slides were blocked in blocking buffer [1 % (w/v) BSA, 0.1 % (v/v) Triton X-100 in PBS] for 30 min at room temperature and, after washing in 1× PBS for 5 min, incubated for 1 h at 37 °C in primary antibody against 5-mC (anti-5-mC, Abcam, ab 10805) diluted 1:100 in the blocking buffer. After washing in 1× PBS, the slides were incubated with FITC-conjugated secondary antibody (Abcam, ab 6785) diluted 1: 200 in blocking buffer for 1 h at 37 °C. Finally, the slides were washed in 1× PBS, counterstained with 0.5 μg/ml DAPI for 8 min, and mounted in antifade (DAKO Fluorescent Mounting Medium).

Immunodetection of histone modifications

The slides were blocked at room temperature for 30 min in 4 % (w/v) BSA and 0.1 % (v/v) Triton X-100 in PBS and then washed for 5 min in 1× PBS. Then the slides were incubated for 1 h at 37 °C with primary antibodies prepared in the following dilutions: 1:50 anti-H3K9ac (Abcam, ab 10812), 1:100 anti-H3K27me2 (Abcam, ab 24684), 1:200 anti-H3K9me1 (Abcam, ab 89906), 1:200 anti-H3K4me3 (Abcam, ab 71998), and 1:200 anti-H3K9me2 (Abcam, ab1220) in 1 % (w/v) BSA in PBS supplemented with 0.1 % (v/v) Triton X-100. Alternatively, the slides were incubated at 4 °C overnight in 1:300 dilution of anti-H3K9me2 (Millipore, 07–441) in 1 % (w/v) BSA in PBS supplemented with 0.1 % (v/v) Triton X-100. The slides were then washed in 1× PBS and incubated for 1 h at 37 °C with 1:200 dilution of either Cy3- or FITC-conjugated secondary antibody (Abcam, ab 6939 and ab 6785). Finally, the slides were washed in 1× PBS, counterstained with 0.5 μg/ml DAPI for 8 min, and mounted in antifade (DAKO Fluorescent Mounting Medium). All antibodies were applied in the same IF experiments on Q. robur and on A. thaliana cycling cells in order to exclude the possibility of technical problems with oak (Supplementary Fig. S1).

For the combination of histone immunodetection and FISH, 5-mC IF was carried out first. After incubation in the secondary antibody, the slides were dehydrated through an ethanol series and treated with RNaseA solution (50 μg/ml in 2× SSC) for 1 h at 37 °C. After washing in 1× PBS, the slides were postfixed in 4 % (w/v) paraformaldehyde for 30 min at room temperature, washed in 1× PBS, dehydrated through an ethanol series, and air-dried. The hybridization mixture consisted of 50 % (v/v) formamide, 10 % (w/v) dextran sulfate, 50 mM NaH2PO4, and 1.5–2.0 ng/μl Cy3-labeled 18S-5.8S-26S (45S) rDNA probe in 2× SSC. The plasmid containing a 2.4-kb fragment of 18S rRNA gene from Cucurbita pepo (Toress-Ruiz and Hemleben 1994) was labeled with Nick Translation Mix (Roche Applied Science) according to the manufacturer’s protocol. The chromosomes and the probe were denatured simultaneously at 80 °C for 5 min and were left to hybridize overnight in a humid chamber at 37 °C. After posthybridization stringent washes, the slides were counterstained with 0.5 μg/ml DAPI for 8 min and mounted in antifade (DAKO Fluorescent Mounting Medium).

Microscopy, image processing, and analysis

Images were captured on the epi-fluorescence microscopes Axiovert 200M (Zeiss, Germany) and Olympus BX51 (Olympus, Japan) coupled with a CCD camera using appropriate filters. Optical sections were acquired using the Z-stack module of AxioVision software 4.5. Images were processed, pseudo-colored, and merged in ImageJ. Integrated fluorescence intensity analysis was performed using an ImageJ-based plug-in that added together all voxel intensity values for a selected nucleus. The obtained values were normalized to the volume of the nuclei as visualized by DAPI staining. 5-mC signal intensity along chromosomes was measured with the RGB Profiler plug-in for ImageJ in 10 metaphase spreads. Confocal microscopy was performed with a Leica TCS SP2 AOBS microscope. DAPI and FITC signals were recorded separately with excitation wavelengths of 488 and 570 nm.

Co-localization and statistical analyses

Prior to co-localization analyses, images were deconvolved using Parallel Iterative Deconvolution plug-in for ImageJ. Co-localization analyses were performed on 100 chromocenters by measuring the Pearson’s coefficient (PC) using an ImageJ-based plug-in JACoP (Bolte and Cordelieres 2006). For statistical analysis of the obtained values, we used GraphPad Prism 5.0 software. We chose the Mann–Whitney nonparametric test because it does not assume a Gaussian distribution of data. Error bars represent standard error of the mean (SEM). To analyze the simultaneous presence of H3K9me1 and H3K4me3 in the chromocenters, we used the Spearman rank correlation test in GraphPad to compare the obtained PC values at the level of each isolated chromocenter. When under 0.5, the PC value is considered a loose indication of signal co-localization (Bolte and Cordelieres 2006). The confidence interval (CI) for the percentage of H3K4me3-positive chromocenters was calculated according to the binomial proportion CI equation using normal approximation. Relevant data sets were confirmed to come from a normal distribution using the Kolmogorov–Smirnov test.


Heterochromatin in Q. robur cycling cells

Analysis of DAPI-stained nuclei of Q. robur using confocal microscopy revealed three nuclear domains—heterochromatin, euchromatin, and nucleolus. Cytologically detectable heterochromatin was mostly located around centromeres of all 24 metaphase chromosomes, seen as moderately thick pericentromeric DAPI bands (Fig. 1a), whose number matched the number of DAPI-positive chromocenters in 3D interphase nuclei (Fig. 1b). Only one large nucleolus was observed in the majority of the nuclei, formed from one or both sites of the major 45S rDNA (NOR-1) locus (Zoldos et al. 1999).
Fig. 1

DAPI-stained metaphase chromosomes of Q. robur complement (2n = 24) show bright (peri)centromeric AT-rich heterochromatic bands (a), which correspond to chromocenters in interphase nuclei (b). Scale bar is 5 μm

Distribution of 5-methylcytosine in interphase nucleus and metaphase chromosomes of Q. robur cycling cells

5-mC signal was almost uniformly distributed along chromosome arms of all 24 oak chromosomes and without obvious enrichment at pericentromeric regions containing constitutive heterochromatin (Fig. 2a). This observation was further corroborated by analysis of 5-mC signal intensity on four different chromosomes—the largest and the smallest ones, and the two 45S rDNA loci bearing chromosomes—which were chosen for analysis since these are the only chromosomes that can be unambiguously identified in the Q. robur complement (Zoldos et al. 1999). The distribution pattern of 5-mC signal showed no prominent peaks in signal intensity along the chromosomes, unlike prominent peaks corresponding to 45S rDNA loci (Fig. 2c). Similarly, dispersed and evenly distributed 5-mC signals were observed throughout the interphase nucleus, with no significant signal enrichment within the chromocenters (Fig. 2b).
Fig. 2

Immunolocalization of 5-mC in Q. robur cycling cells showing an even distribution of the signal along the entire length of chromosomes (a) and dispersed distribution within the interphase chromatin (b) without enrichment at any specific regions; n = 17. Scale bar is 5 μm. c Distribution of 5-mC signals on the largest and the smallest chromosomes and the two chromosomes bearing 45S rDNA loci of Q. robur complement. Intensity profile for 5-mC is shown in green, for 45S rDNA in red, and for DAPI counterstain in blue. Chromosome length is shown on the x-axis and signal intensity values on the y-axis. Scale bar is 5 μm

Distribution of histone modification marks in interphase nuclei of Q. robur cycling cells

For four out of five histone marks studied (H3K9ac, H3K9me1, H3K9me2, H3K27me2, and H3K4me3), we could detect the immunofluorescent signal in the interphase nuclei of Q. robur. However, after several repeated experiments using two different H3K9me2 antibodies, we could not visualize this histone mark in oak nuclei, while the same protocol labeled chromocenters in A. thaliana (Supplementary Fig. S1).

IF signal corresponding to acetylated Lys 9 in histone H3 (H3K9ac) was evenly distributed throughout the interphase chromatin, with the exception of the nucleolus which showed very weak signals or was devoid of this modification (Fig. 3). Also, we observed more condensed H3K9ac foci randomly distributed throughout the nuclei; however, these were exempt from chromocenters, which was further supported by co-localization analysis (PC value of 0.246, Fig. 4).
Fig. 3

Immunolocalization of histone modification marks in Q. robur interphase nuclei. H3K9ac mark is evenly distributed throughout the interphase chromatin, but is absent from chromocenters and nucleolus; H3K9me1 mark is present throughout the interphase chromatin, with strong enrichment in chromocenters. Arrows indicate rDNA chromatin associated with nucleolus, both enriched with H3K9me1 mark; H3K4me3 mark is distributed in the entire chromatin, including many chromocenters (arrowheads) and nucleolus; H3K27me2 mark is distributed in both euchromatic and heterochromatic domain (excluding nucleolus), with enrichment in foci other than chromocenters. Scale bar is 5 μm

Fig. 4

Co-localization analysis of histone modification marks with chromocenters. PC values demonstrate weak co-localization of H3K9ac and H3K27me2 marks, significant co-localization of H3K4me3 mark, and strong co-localization of H3K9me1 mark with chromocenters. Error bars represent standard error of the mean (SEM); n = 100

The most intense signals of the antibody against H3K9me1 were found within chromocenters (Fig. 3), which was further confirmed by co-localization analysis measuring the Pearson’s coefficient (PC = 0.680, Fig. 4). Nevertheless, a less intense, dispersed signal was also present outside of the chromocenters. In some nuclei, H3K9me1 signal was also visible within the nucleolus and possibly 45S rDNA loci involved in its formation (Fig. 3, arrows). The H3K4 trimethylation mark was distributed in the entire chromatin, including the nucleolus (Fig. 3). Surprisingly, many chromocenters were enriched with H3K4me3 signal (Fig. 3, arrowheads), and PC values (PC = 0.506, n = 100) indicate a mildly positive co-localization trend of the signal with the chromocenters (Fig. 4). The IF signal corresponding to H3K27me2 was found both in euchromatic and heterochromatic domains of the oak nucleus, but was completely devoid from the nucleolus (Fig. 3). Also, the signal was clustered in numerous foci of stronger IF intensity, which however showed very low level of co-localization with the chromocenters (PC = 0.337, Fig. 4).

When we analyzed the distribution of all individual PC values corresponding to individual histone modifications, we observed that H3K9me1 strongly co-localized with chromocenters, with 68 % of them having the PC value above or equal to 0.7. However, this was true only for 35 % of chromocenters in the case of H3K4me3, 16 % in the case of H3K27me2, and 14 % in the case of H3K9ac (Fig. 5a–c). The clear separation of PC values into two populations when comparing H3K9me1 and H3K9ac confirmed the presence of the former and the exclusion of the latter mark from the chromocenters (Fig. 5b). Unexpectedly, Pearson’s coefficient of co-localization of H3K4me3 (considered typically as a euchromatic mark) with the chromocenters was found to be equal to or higher than 0.5 in 58 ± 10 % of chromocenters as calculated according to the binomial proportion CI equation using normal approximation. To test whether individual chromocenters were associated with both H3K9me1 and H3K4me3 at the same time, we performed a paired correlation test and, interestingly, obtained a significant positive correlation (R = 0.379; p = 0.0001).
Fig. 5

ac Distribution frequency of histone marks in chromocenters; n = 100


Genome size and sequence composition both influence nuclear architecture and distribution of epigenetic marks on the cytogenetic level (Houben et al. 2003; Fuchs et al. 2006; Braszewska-Zalewska et al. 2010). Q. robur, containing 0.94 pg (919.3 Mb)/1C (Zoldos et al. 1998) has a very small genome size (according to the classification of Leitch et al. 1998) and shows a simple nuclear chromatin organization similar to that of A. thaliana (157 Mb/1C) or Brassica rapa (283.8 Mb/1C), in which constitutive heterochromatin forms clearly visible DAPI-stained chromocenters (Fransz et al. 2003; Braszewska-Zalewska et al. 2010). In this study, we report for the first time the distribution of 5-mC and histone acetylation and methylation marks in Q. robur genome. According to dispersed 5-mC signal throughout the interphase nucleus/chromosomes without enrichment within chromocenters/DAPI bands of (peri)centromeric position, DNA methylation in Q. robur does not appear to be restricted to constitutive heterochromatin, unlike in A. thaliana and B. rapa where chromocenters contain most of the heavily methylated DNA (Fransz et al. 2003; Jasencakova et al. 2003; Braszewska-Zalewska et al. 2010). Cytosine methylation has a dual localization in plants—it is present predominantly over silent TEs and other repeats and in the body of approximately 30 % of genes with moderate expression levels (Lippman et al. 2004; Zhang et al. 2006; Cokus et al. 2008). 5-mC also forms small domains in euchromatin and large domains in TEs clusters, as recently shown for Arabidopsis (Roudier et al. 2011). The dispersed distribution of 5-mC throughout the oak genome without clustering at chromocenters might therefore suggest that DNA methylation is associated with both euchromatic and heterochromatic domains and that most of the repeated sequences which are silenced by cytosine methylation are rather dispersed within the oak genome and not tandemly repeated.

While distribution of acetylated K9 at histone H3 was consistent with distribution already recorded in other angiosperm species, associating this epigenetic mark with permissive chromatin and gene activity (Zhou et al. 2010), distributional pattern of histone methylation marks showed some peculiarity. H3K9me1 is classified as heterochromatin-specific in many plant species, and this mark strongly co-localized with the oak chromocenters, too. However, H3K9me1, together with H3K27me2 and H3K4me3, was dispersed in the entire interphase chromatin of Q. robur cycling cells. The distribution of these marks is similar to the one found in the large genome of H. vulgare (Fuchs et al. 2006), suggesting that either H3K9me1 might not represent an exclusively repressive mark in Q. robur cycling cells or that it is associated with a considerable fraction of TEs present in the oak genome. H3K9me1 also labels euchromatin of two gymnosperm species. While present in equal amounts in euchromatin and interstitial heterochromatin on chromosomes of Picea abies, it is completely absent from heterochromatin of Pinus sylvestris chromosomes (Fuchs et al. 2008).

H3K27me2 mark was present throughout the interphase chromatin in pedunculate oak; however, signals also clustered in foci that did not co-localize with chromocenters, suggesting the existence of a specific type of heterochromatin not detectable by standard cytogenetic techniques such as Giemsa and fluorochrome banding (see discussion below). Such distribution resembles the H3K27me2 distributional pattern in P. abies and P. sylvestris, where H3K27me2 was enriched at interstitial heterochromatin but absent from pericentromeric heterochromatin and 45S rDNA loci (Fuchs et al. 2008). Even though H3K27me2 was found uniformly distributed over the entire chromatin in the large genome of V. faba, enrichment in heterochromatic regions other than chromocenters was evident (Fuchs et al. 2006). A recent large-scale epigenetic study of the Arabidopsis genome showed that H3K27me2 is, similar to 5-mC, a dual mark associated with clusters of TEs, but also with a fraction of genes (Roudier et al. 2011). Distribution of H3K9me1, H3K27me2, and 5-mC can be discussed in the context of the composition of the Q. robur genome. Cytogenetically detectable heterochromatin in this species is visible in (peri)centromeric regions of all 24 chromosomes as DAPI/CMA-positive C bands (Ohri and Ahuja 1990; Zoldos et al. 1999 and this work, Fig. 1), in CMA-positive NOR regions (Zoldos et al. 1999), and in some tiny intercalary DAPI-positive C bands (Ohri and Ahuja 1990). Therefore, it seems that pedunculate oak contains low amounts of tandemly repeated DNA, concentrated mostly in the (peri)centromeric chromosome region. On the other hand, we identified in its genome several classes of dispersed repetitive sequences possibly of retrotransposable origin (Zoldos et al. 2001). Even though Q. robur, as well as related oak species, is still poorly explored in terms of molecular structure of the genome (Zoldos Pecnik 2008), available data suggest that this species might contain more TEs or other dispersed repeats than satellite DNA (Faivre Rampant et al. 2011). Particular distribution of H3K9me1, H3K27me2, and 5-mC in the oak genome, which all preferentially mark TEs (Roudier et al. 2011), favors this hypothesis. It also suggests the type of genome organization where TEs and other repeats exist in small patches between genes of the euchromatic chromatin domain and have to be silenced by heterochromatinization, as has been seen in large plant genomes characterized by a large fraction of mobile elements (Houben et al. 2003; Shi and Dawe 2006).

H3K4me3 has been associated with gene activity (see Feng and Jacobsen 2011 and references herein) and shows distribution throughout euchromatin of 24 angiosperm plant species (Houben et al. 2003). Even though H3K4me3 was present throughout the interphase chromatin and within the nucleolus of Q. robur cycling cells, our analysis has shown moderate co-localization of this mark with oak chromocenters, probably reflecting random distribution of this modification mark within the oak nucleus. According to the histone code hypothesis, a large number of different modifications, imposed on histone tails of nucleosomes in a particular chromosome region, are mostly read in combination with other epigenetic marks leading to a certain complexity of this indexing system (Jenuwein and Allis 2001; Allis et al. 2007; Roudier et al. 2011). The combinatorial nature of various histone modifications could result in fine transitory states between activity and repression (Jenuwein and Allis 2001), arguing that some of the oak chromocenters, associated with two antagonistic histone marks, H3K9me1 and H3K4me3, could represent the so-called intermediate heterochromatin, which under appropriate conditions might be transcriptionally active. For instance, in A. thaliana, some transposable elements associate with both H3K9me2 and H3K4me2, marks of repressive and permissive chromatin state, respectively (Lippman et al. 2003). In addition, a small proportion of Arabidopsis genes encoding transcriptional factors (Berr et al. 2010) and some key regulatory genes in mammals (Wang et al. 2009) are showing bivalent marking. It has been suggested that intermediate heterochromatin is heritably maintained in plants, providing a particular epigenetic flexibility and contributing to epigenetic defense mechanisms (Habu et al. 2006; Veiseth et al. 2011).

The most surprising finding in our experiments was the lack of detectable amounts of IF signal corresponding to H3K9me2 in Q. robur cycling cells. IF experiments with H3K9me2 antibodies from two different suppliers failed to produce signals in oak interphase chromatin, while clearly labeling chromocenters of A. thaliana as previously reported (Fransz et al. 2003; Jasencakova et al. 2003; Fuchs et al. 2006). We thus hypothesize that H3K9me2 is probably present at low levels and discontinuous dispersed regions in pedunculate oak genome, making it undetectable at a cytogenetic level. Even though epigenetic landscape is tissue-specific, the distributional pattern and signal intensity of H3K9me2 observed in mature pollen cells of the closely related cork oak (Q. suber) could be in favor of our hypothesis. Ribeiro et al. (2009) showed prominently different intensities of H3K9me2 labeling in vegetative and generative nuclei; the vegetative nucleus was very weakly labeled indicating a reduced level of this histone modification mark in, at least, some of the oak cell types.

Studies in Neurospora have revealed that the status of histone methylation determines the patterns of DNA methylation (Tamaru and Selker 2001), and in A. thaliana, CpG methylation plays the role of a central scaffold directing H3K9 dimethylation in the formation of silent heterochromatin (Tariq et al. 2003). The chromocenters of pedunculate oak were not heavily marked by 5-mC, indicating a low level of DNA methylation and were enriched in H3K9 monomethylation instead of H3K9 dimethylation. This finding, although preliminary, raises an interesting possibility of other plausible epigenetic modifications involved in heterochromatin formation in pedunculate oak.


Epigenetic regulation of gene activity in plants reflects their evolutionary history, mode of development, and lifestyle. This first insight into the epigenetic chromatin landscape of very long-lived oak species Q. robur revealed several specificities compared to other angiosperm plants and more similarities to gymnosperm long-lived trees such as Pinus and Picea (Fuchs et al. 2008). A recent study of the oak genome revealed significant differences between oak, grapevine (Vitis vinifera), and poplar (Populus trichocarpa) genomes (Faivre Rampant et al. 2011). Apparently, oak is to some extent different from other plant species in both genome structure and epigenetic landscape, and further studies of the oak genome and epigenome are expected to provide new insights into the crosstalk between sequence composition, interphase chromatin organization, coupled with epigenetic hallmarks, and function of this peculiar plant genome.



This work was supported by the Croatian Ministry of Science, Education and Sport Grant 119-1191196-1224 and by AUF (Agence Universitaire de Francophonie) PSCI Grant. We thank the anonymous reviewers who contributed to the improvement of this manuscript.

Supplementary material

11295_2013_632_Fig6_ESM.jpg (298 kb)
Supplementary Fig. S1

Immunolocalization of histone modification marks in A. thaliana interphase nuclei. Scale bar is 5 μm (JPEG 297 kb)

11295_2013_632_MOESM1_ESM.eps (6.8 mb)
High resolution image (EPS 6967 kb)


  1. Allis CD, Jenuwein T, Reinberg D (2007) Epigenetics. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  2. Berr A, McCallum EJ, Menard R, Meyer D, Fuchs J, Dong A, Shen WH (2010) Arabidopsis SET DOMAIN GROUP2 is required for H3K4 trimethylation and is crucial for both sporophyte and gametophyte development. Plant Cell 22(10):3232–3248. doi:10.1105/tpc.110.079962 PubMedCrossRefGoogle Scholar
  3. Bolte S, Cordelieres FP (2006) A guided tour into subcellular colocalization analysis in light microscopy. J Microsc 224:213–232. doi:10.1111/j.1365-2818.2006.01706.x PubMedCrossRefGoogle Scholar
  4. Boyko A, Blevins T, Yao Y, Golubov A, Bilichak A, Ilnytskyy Y, Hollunder J, Meins F Jr, Kovalchuk I (2010) Transgenerational adaptation of Arabidopsis to stress requires DNA methylation and the function of dicer-like proteins. PLoS One 5(3):e9514. doi:10.1371/journal.pone.0009514 PubMedCrossRefGoogle Scholar
  5. Braszewska-Zalewska A, Bernas T, Maluszynska J (2010) Epigenetic chromatin modifications in Brassica genomes. Genome 53(3):203–210. doi:10.1139/G09-088 PubMedCrossRefGoogle Scholar
  6. Carchilan M, Delgado M, Ribeiro T, Costa-Nunes PD, Caperta A, Morais-Cecilio L, Jones RN, Viegas W, Houben A (2007) Transcriptionally active heterochromatin in rye B chromosomes. Plant Cell 19(6):1738–49. doi:10.1105/tpc.106.046946 PubMedCrossRefGoogle Scholar
  7. Cokus SJ, Feng S, Zhang X, Chen Z, Merriman B, Haudenschild CD, Pradhan S, Nelson SF, Pellegrini M, Jacobsen SE (2008) Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452:215–219. doi:10.1038/nature06745 PubMedCrossRefGoogle Scholar
  8. Faivre Rampant P, Lesur I, Boussardon C, Bitton F, Martin-Magniette ML, Bodenes C, le Provost G, Berges H, Fluch S, Kremer A, Plomion C (2011) Analysis of BAC end sequences in oak, a keystone forest tree species, providing insight into the composition of its genome. BMC Genomics 12:292. doi:10.1186/1471-2164-12-292 PubMedCrossRefGoogle Scholar
  9. Feng S, Jacobsen SE (2011) Epigenetic modifications in plants: an evolutionary perspective. Curr Opin Plant Biol 14(2):179–86. doi:10.1016/j.pbi.2010.12.002 PubMedCrossRefGoogle Scholar
  10. Filion GJ, van Bemmel JG, Braunschweig U, Talhout W, Kind J, Ward LD, Brugman W, de Castro IJ, Kerkhoven RM, Bussemaker HJ, van Steensel B (2010) Systematic protein location mapping reveals five principal chromatin types in Drosophila cells. Cell 143(2):212–224. doi:10.1016/j.cell.2010.09.009 PubMedCrossRefGoogle Scholar
  11. Fransz P, Soppe W, Schubert I (2003) Heterochromatin in interphase nuclei of Arabidopsis thaliana. Chromosome Res 11(3):227–240. doi:10.1023/A:1022835825899 PubMedCrossRefGoogle Scholar
  12. Fuchs J, Schubert I (2012) Chromosomal distribution and functional interpretation of epigenetic histone marks in plants. In: Bass H, Birchler JA (eds) Plant cytogenetics, vol. 4: plant genetics and genomics: crops and models. Springer, New York, pp 231–253.Google Scholar
  13. Fuchs J, Demidov D, Houben A, Schubert I (2006) Chromosomal histone modification patterns—from conservation to diversity. Trends Plant Sci 11(4):199–208. doi:10.1016/j.tplants.2006.02.008 PubMedCrossRefGoogle Scholar
  14. Fuchs J, Jovtchev G, Schubert I (2008) The chromosomal distribution of histone methylation marks in gymnosperms differs from that of angiosperms. Chromosome Res 16:891–898. doi:10.1007/s10577-008-1252-4 PubMedCrossRefGoogle Scholar
  15. Habu Y, Mathieu O, Tariq M, Probst AV, Smathajitt C, Zhu T, Paszkowski J (2006) Epigenetic regulation of transcription in intermediate heterochromatin. EMBO Rep 7:1279–1284. doi:10.1038/sj.embor.7400835 PubMedCrossRefGoogle Scholar
  16. Hauser MT, Aufsatz W, Joank C, Luschnig C (2011) Transgenerational epigenetic inheritance in plants. Biochim Biophys Acta 1809(8):459–68. doi:10.1016/j.bbagrm.2011.03.007 PubMedCrossRefGoogle Scholar
  17. Houben A, Demidov D, Gernand D, Meister A, Leach CR, Schubert I (2003) Methylation of histone H3 in euchromatin of plant chromosomes depends on basic nuclear DNA content. Plant J 33(6):967–973. doi:10.1046/j.1365-313X.2003.01681.x PubMedCrossRefGoogle Scholar
  18. Jackson JP, Johnson L, Jasencakova Z, Zhang X, PerezBurgos L, Singh PB, Cheng X, Schubert I, Jenuwein T, Jacobsen SE (2004) Dimethylation of histone H3 lysine 9 is a critical mark for DNA methylation and gene silencing in Arabidopsis thaliana. Chromosoma 112:308–315. doi:10.1007/s00412-004-0275-7 PubMedCrossRefGoogle Scholar
  19. Jasencakova Z, Soppe WJ, Meister A, Gernand D, Turner BM, Schubert I (2003) Histone modifications in Arabidopsis: high methylation of H3 lysine 9 is dispensable for constitutive heterochromatin. Plant J 33:471–480. doi:10.1046/j.1365-313X.2003.01638.x PubMedCrossRefGoogle Scholar
  20. Jenuwein T, Allis CD (2001) Translating the histone code. Science 293:1074–1080. doi:10.1126/science.1063127 PubMedCrossRefGoogle Scholar
  21. Johnson LM, Bostick M, Zhang X, Kraft E, Henderson I, Callis J, Jacobsen SE (2007) The SRA methyl-cytosine-binding domain links DNA and histone methylation. Curr Biol 17:379–384. doi:10.1016/j.cub.2007.01.009 PubMedCrossRefGoogle Scholar
  22. Jones JH (1986) Evolution of the Fagaceae: the implications of foliar features. Ann Missouri Bot Gard 73:228–275. doi:10.2307/2399112 CrossRefGoogle Scholar
  23. Leitch IJ, Chase MW, Bennett MD (1998) Phylogenetic analysis of DNA C-values provides evidence for a small ancestral genome size in flowering plants. Ann Bot 82:85–94. doi:10.1006/anbo.1998.0783 CrossRefGoogle Scholar
  24. Li X, Wang X, He K, Ma Y, Su N, He H, Stolc V, Tongprsit W, Jin W, Jiang J, Terzaghi W, Li S, Deng XW (2008) High-resolution mapping of epigenetic modifications of the rice genome uncovers interplay between DNA methylation, histone methylation, and gene expression. Plant Cell 20:259–276. doi:10.1105/tpc.107.056879 PubMedCrossRefGoogle Scholar
  25. Lippman Z, May B, Yordan C, Singer T, Martienssen RA (2003) Distinct mechanisms determine transposon inheritance and methylation via small interfering RNA and histone modifications. PLoS Biol 1:420–428. doi:10.1371/journal.pbio.0000067 CrossRefGoogle Scholar
  26. Lippman Z, Gendrel AV, Black M, Vaughn MW, Dedhia N, McCombie WR, Lavine K, Mittal V, May B, Kasschau KD, Carrington JC, Doerge RW, Colot V, Martienssen R (2004) Role of transposable elements in heterochromatin and epigenetic control. Nature 430:471–476. doi:10.1038/nature02651 PubMedCrossRefGoogle Scholar
  27. Loidl P (2004) A plant dialect of the histone language. Trends Plant Sci 9(2):84–90. doi:10.1016/j.tplants.2003.12.007 PubMedCrossRefGoogle Scholar
  28. Marques A, Fuchs J, Ma L, Heckmann S, Guerra M, Houben A (2010) Characterization of eu- and heterochromatin of citrus with a focus on the condensation behavior of 45S rDNA chromatin. Cytogenet Genome Res 134(1):72–82. doi:10.1159/000323971 CrossRefGoogle Scholar
  29. Ohri D, Ahuja MR (1990) Giemsa C-banding in Quercus. Silvae Genet 39:216–219Google Scholar
  30. Pfluger J, Wagner D (2007) Histone modifications and dynamic regulation of genome accessibility in plants. Curr Opin Plant Biol 10(6):645–652. doi:10.1016/j.pbi.2007.07.013 PubMedCrossRefGoogle Scholar
  31. Ribeiro T, Viegas W, Morais-Cecilio L (2009) Epigenetic marks in the mature pollen of Quercus suber L. (Fagaceae). Sex Plant Reprod 22:1–7. doi:10.1007/s00497-008-0083-y PubMedCrossRefGoogle Scholar
  32. Roudier F, Ahmed I, Berard C, Sarazin A, Mary-Huard T, Cortijo S, Bouyer D, Caillieux E, Duvernois-Berthet E, Al-Shikhley L, Giraut L, Despres B, Drevensek S, Barneche F, Derozier S, Brunaud V, Aubourg S, Schnittger A, Bowler C, Martin-Magniette M, Robin S, Caboche M, Colot V (2011) Integrative epigenomic mapping defines four main chromatin states in Arabidopsis. EMBO J 30(10):1928–38. doi:10.1038/emboj.2011.103 PubMedCrossRefGoogle Scholar
  33. Schnable SS, Ware D, Fulton RS et al (2009) The B73 maize genome: complexity, diversity, and dynamics. Science 326(5956):1112–1115. doi:10.1126/science.1178534 PubMedCrossRefGoogle Scholar
  34. Shi J, Dawe RK (2006) Partitioning of the maize epigenome by the number of methyl groups on histone H3 lysines 9 and 27. Genetics 173(3):1571–1583. doi:10.1534/genetics.106.056853 PubMedCrossRefGoogle Scholar
  35. Soppe WJ, Jasencakova Z, Houben A, Kakutani T, Meister A, Huang MS, Jacobsen SE, Schubert I, Fransz PF (2002) DNA methylation controls histone H3 lysine 9 methylation and heterochromatin assembly in Arabidopsis. EMBO J 21(23):6549–59. doi:10.1093/emboj/cdf657 PubMedCrossRefGoogle Scholar
  36. Suzuki G, Shiomi M, Morihana S, Yamamoto M, Mukai Y (2010) DNA methylation and histone modification in onion chromosomes. Genes Genet Syst 85(6):377–382. doi:10.1266/ggs.85.377 PubMedCrossRefGoogle Scholar
  37. Tamaru H, Selker EU (2001) A histone H3 methyltransferase controls DNA methylation in Neurospora crassa. Nature 414:277–283. doi:10.1038/35104508 PubMedCrossRefGoogle Scholar
  38. Tariq M, Saze H, Probst A, Lichota J, Habu Y, Paszkowski JK (2003) Erasure of CpG methylation in Arabidopsis alters patterns of histone H3 methylation in heterochromatin. Proc Natl Acad Sci U S A 100:8823–8827. doi:10.1073/pnas.1432939100 PubMedCrossRefGoogle Scholar
  39. Toress-Ruiz RA, Hemleben V (1994) Pattern and degree of methylation in ribosomal RNA genes of Cucurbita pepo L. Plant Mol Biol 26:1167–1179. doi:10.1007/BF00040697 CrossRefGoogle Scholar
  40. Veiseth SV, Rahman MA, Yap KL, Fischer A, Egge-Jacobsen W, Ruter G, Zhou MM, Aalen RB, Thorstensen T (2011) The SUVR4 histon lysine methyltransferase binds ubiquitin and converts H3K9me1 to H3K9me3 on transposon chromatin in Arabidopsis. PloS Genetics 7(3):e1001325. doi:10.1371/journal.pgen.1001325 PubMedCrossRefGoogle Scholar
  41. Wang Z, Schones DE, Zhao K (2009) Characterization of human epigenomes. Curr Opin Genet Dev 19(2):127–134. doi:10.1016/j.gde.2009.02.001 PubMedCrossRefGoogle Scholar
  42. Wicker T, Stein N, Albar L, Feuillet C, Schlagenhauf E, Keller B (2001) Analysis of a contiguous 211 kb sequence in diploid wheat (Triticum monococcum L.) reveals multiple mechanisms of genome evolution. Plant J 26(3):307–16. doi:10.1046/j.1365-313X.2001.01028.x PubMedCrossRefGoogle Scholar
  43. Wicker T, Zimmermann W, Perovic D, Paterson AH, Ganal M, Graner A, Stein N (2005) A detailed look at 7 million years of genome evolution in a 439 kb contiguous sequence at the barley Hv-eIF4E locus: recombination, rearrangements and repeats. Plant J 41(2):189–94. doi:10.1111/j.1365-313X.2004.02285.x Google Scholar
  44. Yoder JA, Walsh CP, Bestor TH (1997) Cytosine methylation and the ecology of intragenomic parasites. Trends Genet 13:335–340. doi:10.1016/S0168-9525(97)01181-5 PubMedCrossRefGoogle Scholar
  45. Zhang XY, Yazaki J, Sundaresan A, Cokus S, Chan SWL, Chen HM, Chen H, Henderson IR, Shinn P, Pellegrini M, Jacobsen SE, Ecker JR (2006) Genome-wide high-resolution mapping and functional analysis of DNA methylation in Arabidopsis. Cell 126(6):1189–1201. doi:10.1016/j.cell.2006.08.003 PubMedCrossRefGoogle Scholar
  46. Zhou J, Wang X, He K, Charron JB, Elling AA, Deng XW (2010) Genome-wide profiling of histone H3 lysine 9 acetylation and dimethylation in Arabidopsis reveals correlation between multiple histone marks and gene expression. Plant Mol Biol 72:585–595. doi:10.1007/s11103-009-9594-7 PubMedCrossRefGoogle Scholar
  47. Zoldos Pecnik V (2008) Genome organization and evolution in genus Quercus (Fagaceae): special attention to two European white oaks Quercus petraea (Matt.) Liebl. and Q. robur L. In: A.K. Sharma AK, Sharma A (eds) Plant genome: biodiversity and evolution. Science, Enfield, pp 43–78.Google Scholar
  48. Zoldos V, Papes D, Brown SC, Panaud O, Siljak-Yakovlev S (1998) Genome size and base composition of seven Quercus species: inter- and intra-population variation. Genome 41:162–168. doi:10.1139/g98-006 Google Scholar
  49. Zoldos V, Papes D, Cerbah M, Panaud O, Besendorfer V, Siljak-Yakovlev S (1999) Molecular-cytogenetic studies of ribosomal genes and heterochromatin reveal conserved genome organization among 11 Quercus species. Theor Appl Genet 99:969–977. doi:10.1007/s001220051404 CrossRefGoogle Scholar
  50. Zoldos V, Siljak-Yakovlev S, Papes D, Sarr A, Panaud O (2001) Representational difference analysis reveals genomic differences between Q. robur and Q. suber: implications for the study of genome evolution in the genus Quercus. Mol Genet Genomics 265(2):234–241. doi:10.1007/s004380000420 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • V. Vičić
    • 1
  • D. Barišić
    • 1
  • T. Horvat
    • 1
  • I. Biruš
    • 1
  • Vlatka Zoldos
    • 1
  1. 1.Department of Molecular Biology, Faculty of ScienceUniversity of ZagrebZagrebCroatia

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