Epigenetic characterization of chromatin in cycling cells of pedunculate oak, Quercus robur L.
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.
KeywordsInterphase 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
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).
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
Distribution of 5-methylcytosine in interphase nucleus and metaphase chromosomes of Q. robur cycling cells
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).
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).
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.
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