On the chromatin structure of eukaryotic telomeres

Telomeres prevent chromosome fusions and degradation by exonucleases and are implicated in DNA repair, homologous recombination, chromosome pairing and segregation. All these functions of telomeres require the integrity of their chromatin structure, which has been traditionally considered as heterochromatic. In agreement with this idea, different studies have reported that telomeres associate with heterochromatic marks. However, these studies addressed simultaneously the chromatin structures of telomeres and subtelomeric regions or the chromatin structure of telomeres and Interstitial Telomeric Sequences (ITSs). The independent analysis of Arabidopsis telomeres, subtelomeric regions and ITSs has allowed the discovery of euchromatic telomeres. In Arabidopsis, whereas subtelomeric regions and ITSs associate with heterochromatic marks, telomeres exhibit euchromatic features. We think that this scenario could be found in other model systems if the chromatin organizations of telomeres, subtelomeric regions and ITSs are independently analyzed.     

On the chromatin structure of eukaryotic telomeres

Telomeres prevent chromosome fusions and degradation by exonucleases and are implicated in DNA repair, homologous recombination, chromosome pairing and segregation. All these functions of telomeres require the integrity of their chromatin structure, which has been traditionally considered as heterochromatic. In agreement with this idea, different studies have reported that telomeres associate with heterochromatic marks. However, these studies addressed simultaneously the chromatin structures of telomeres and subtelomeric regions or the chromatin structure of telomeres and Interstitial Telomeric Sequences (ITSs). The independent analysis of Arabidopsis telomeres, subtelomeric regions and ITSs has allowed the discovery of euchromatic telomeres. In Arabidopsis, whereas subtelomeric regions and ITSs associate with heterochromatic marks, telomeres exhibit euchromatic features. We think that this scenario could be found in other model systems if the chromatin organizations of telomeres, subtelomeric regions and ITSs are independently analyzed.Key words: telomeres, subtelomeres, euchromatin, heterochromatin, ChIP, immunolocalizationTelomeric DNA usually contains tandem repeats of a short GC rich motif. The number of repeats and, therefore, the length of telomeres is subject to regulation and influences relevant biological processes like aging and cancer.^1–3 In situ hybridization studies have revealed that telomeric repeats are also present at interstitial chromosomal loci.^4,5 An analysis of the genome sequence from different eukaryotes indicates that ITSs have a widespread distribution in different model systems including zebrafish, chicken, opossum, mouse, dog, cattle, horse, human, rice, poplar or Arabidopsis (see Fig. 1 for an example; www.ncbi.nlm.nih.gov/mapview). These ITSs have been related to chromosomal aberrations, fragile sites, hot spots for recombination and diseases caused by genomic instability, although their functions remain unknown.⁶Open in a separate windowFigure 1Distribution of the main telomeric repeat arrays in the genome of several model organisms. These representations have been performed by using the megaBLAST program and the all assemblies genomic databases at NCBI (www.ncbi.nlm.nih.gov/mapview). Searches for homology with 100 tandem telomeric repeats were done using the default parameters except that the expected threshold was set to 10 and the filters were turned off. Chromosomes are represented as vertical bars and numbered at the bottom. The horizontal bars represent the telomeric repeat arrays. Colors indicate the BLAST scores (red ≥200; pink 80–200; green 50–80).Telomeres and ITSs have probably cross talk through evolution. In some instances, ITSs could have been generated by telomeric fusions. Pioneering studies performed by Hermann J. Muller in Drosophila and Barbara McClintock in maize showed that newly formed chromosome ends tend to fuse giving rise to the so-called breakage-fusion-bridge cycle.^7,8 This cycle can lead to stable chromosomal reorganizations after healing of the broken ends. In addition, Muller and McClintock found that, unlike these newly formed broken chromosome ends, natural chromosomal ends are quite stable and do not tend to fuse.⁹ It is currently known that telomere dysfunction due to mutations that cause telomeric shortening or abolish the expression of certain telomeric proteins can lead to telomeric fusions, anaphase bridges and genome reorganizations.^1–3,10,11 Therefore, telomeric shortening or alterations of telomeric chromatin structure might be expected to generate ITSs through evolution by promoting telomeric fusions.¹² ITSs might also originate through the activity of telomerase during the repair process of double strand breaks or by recombination.^13–16 In addition, telomerase activity might lead to the formation of new telomeres by healing of chromosome breaks within internal telomeric repeats and even within other sequences.^17–19 This process of healing involves the acquisition of telomeric chromatin structure.DNA folds into two major chromatin organizations inside the cell nucleus: heterochromatin and euchromatin. Heterochromatin is highly condensed in interphase nuclei and is usually associated with repetitive and silent DNA. By contrast, euchromatin has an open conformation and is often related to the capacity to be transcribed. Both kinds of chromatin exhibit defined epigenetic modifications that influence their biochemical behavior. Thus, the study of these epigenetic marks is an issue of major interest.The chromatin structures of telomeres and ITSs might be different. Therefore, they should be studied independently. Chromatin structure analyses are usually performed by immunocytolocalization or by chromatin immunoprecipitation (ChIP).^20–23 Special care should be taken when the epigenetic status of telomeres is analyzed by immunocytolocalization. This technique does not allow differentiating between telomeres and subtelomeric regions. Since subtelomeric regions are known to be heterochromatic in many eukaryotic organisms, heterochromatic marks should be immunolocalized at the chromosome ends of these organisms. However, these marks could correspond to subtelomeric regions and not to telomeres.The ChIP technique implies the immunoprecipitation of chromatin with specific antibodies and the further analysis of the immunoprecipitated DNA. DNA sequences immunoprecipitated by a specific antibody are thought to associate in vivo with the feature recognized by this antibody. Whereas the enrichment of single copy sequences in the immunoprecipitated DNA has been usually analyzed by quantitative PCR, the analyses of repetitive DNA sequences have been often performed by hybridization. Thus, multiple telomeric chromatin structure analyses have been performed by hybridizing immunoprecipitated DNA with a telomeric probe. However, these analyses displayed simultaneously the chromatin structures of telomeres and ITSs. High throughput sequencing analyses of the immunoprecipitated DNA might help overcome this problem. Nevertheless, since the reads obtained with these techniques at present are short, it is still difficult to ascertain whether the enrichment of immunoprecipitated telomeric sequences corresponds to telomeres or to ITSs. Third-generation long-read accurate technologies and new algorithms that discriminate between telomeres and ITSs should solve the problem.In principle, the combination of immunocytolocalization and ChIP experiments should help to differentiate between telomeres and ITSs. However, since subtelomeric regions are known to influence telomere function and contain degenerated ITSs, at least in some organisms like humans or Arabidopsis, this may not be necessarily true.⁶ A specific epigenetic mark might be required for telomere function, found associated with telomeric repeats by ChIP and with the end of chromosomes by immunocytolocalization and still not associate with true telomeres but with subtelomeric regions and ITSs or just with subtelomeric ITSs.An alternative way to analyze the chromatin structure of telomeres by ChIP involves the use of frequently cutting restriction enzymes. The chromatin structures of Arabidopsis telomeres and ITSs have been independently studied by using Tru9I, a restriction enzyme that recognizes the sequence TTAA.²⁴ Since telomeres in Arabidopsis and in other model systems are composed of perfect telomeric repeat arrays, they remain uncut after digestion with Tru9I.²⁵ In contrast, Arabidopsis ITSs are frequently cut because they are composed of short arrays of perfect telomeric repeats interspersed with degenerated repeats.^25–28 Thus, when Arabidopsis genomic DNA is digested with Tru9I and hybridized with a telomeric probe, most of the signals corresponding to ITSs disappear.²⁵ The use of Tru9I has made possible to discover that Arabidopsis telomeres exhibit euchromatic features. In contrast, Arabidopsis ITSs and subtelomeric regions are heterochromatic.²⁴ In Arabidopsis, heterochromatin is characterized by cytosine methylation, which can be targeted at CpG, CpNpG or CpNpN residues (where N is any nucleotide), and by H3K9me1,2, H3K27me1,2 and H4K20me1. In turn, Arabidopsis euchromatin is characterized by H3K4me1,2,3, H3K36me1,2,3, H4K20me2,3 and by histones acetylation.²⁹ ChIP experiments processed with Tru9I have revealed that Arabidopsis telomeres have high levels of euchromatic marks (H3K4me2, H3K9 and H4K16 acetylation) and low levels of heterochromatic marks (H3K9me2, H3K27me1 and DNA methylation).²⁴ Therefore, Arabidopsis telomeres exhibit epigenetic modifications characteristic of euchromatin.Different studies in mice, humans or Arabidopsis have reported that telomeres are heterochromatic based on the existence of siRNAs containing telomeric sequences, on the association of telomeric sequences with telomeric and with heterochromatin proteins, on the methylation of telomeric sequences or on the histones modifications associated with telomeric sequences.^30–34 However, the experiments presented in those studies addressed simultaneously the chromatin organizations of telomeres and subtelomeric regions or of telomeres and ITSs. Telomeres have also been reported to be heterochromatic based on the existence of the so-called TElomeric Repeat containing RNAs (TERRA), which are present in different eukaryotes.³⁵ At telomeric regions, TERRA are transcribed from subtelomeric promoters towards chromosome ends. Since human subtelomeric TERRA are mostly composed of subtelomeric sequences, with only about 200 bp of telomeric sequences at their 3′ ends, they might be related to subtelomeric heterochromatin formation rather than to the formation of telomeric chromatin. Nevertheless, TERRA interact with human telomeric proteins and influence telomere function. In addition, TERRA might also be related to ITSs heterochromatinization.^34,35We believe that the scenario found in Arabidopsis could also be found in other model systems if the chromatin structures of telomeres, subtelomeric regions and ITSs are independently analyzed. Several reports have described the presence of histone H3.3 at mice telomeres.^36–39 Since this histone variant has been previously associated with active chromatin, these studies are compatible with a euchromatic organization of telomeres. However, again in these reports, the experiments shown addressed simultaneously the chromatin organization of telomeres and subtelomeric regions or of telomeres and ITSs. In general terms, we believe that a clear distinction between telomeres and ITSs should be established when future ChIP experiments are analyzed. The use of third generation high throughput sequencing technologies or of frequently cutting restriction enzymes might help in this task.As mentioned above, the epigenetic modifications associated with telomeric regions are known to be important for telomere function. These modifications are required to provide genome stability.³³ In this context, it will be relevant to ascertain how the function of Arabidopsis telomeres is influenced by their euchromatic marks and by the presence of heterochromatin at subtelomeric regions.     

Arabidopsis thaliana telomeres exhibit euchromatic features

Telomere function is influenced by chromatin structure and organization, which usually involves epigenetic modifications. We describe here the chromatin structure of Arabidopsis thaliana telomeres. Based on the study of six different epigenetic marks we show that Arabidopsis telomeres exhibit euchromatic features. In contrast, subtelomeric regions and telomeric sequences present at interstitial chromosomal loci are heterochromatic. Histone methyltransferases and the chromatin remodeling protein DDM1 control subtelomeric heterochromatin formation. Whereas histone methyltransferases are required for histone H3K9(2Me) and non-CpG DNA methylation, DDM1 directs CpG methylation but not H3K9(2Me) or non-CpG methylation. These results argue that both kinds of proteins participate in different pathways to reinforce subtelomeric heterochromatin formation.     

Euchromatic subdomains in rice centromeres are associated with genes and transcription

The presence of the centromere-specific histone H3 variant, CENH3, defines centromeric (CEN) chromatin, but poorly understood epigenetic mechanisms determine its establishment and maintenance. CEN chromatin is embedded within pericentromeric heterochromatin in most higher eukaryotes, but, interestingly, it can show euchromatic characteristics; for example, the euchromatic histone modification mark dimethylated H3 Lys 4 (H3K4me2) is uniquely associated with animal centromeres. To examine the histone marks and chromatin properties of plant centromeres, we developed a genomic tiling array for four fully sequenced rice (Oryza sativa) centromeres and used chromatin immunoprecipitation-chip to study the patterns of four euchromatic histone modification marks: H3K4me2, trimethylated H3 Lys 4, trimethylated H3 Lys 36, and acetylated H3 Lys 4, 9. The vast majority of the four histone marks were associated with genes located in the H3 subdomains within the centromere cores. We demonstrate that H3K4me2 is not a ubiquitous component of rice CEN chromatin, and the euchromatic characteristics of rice CEN chromatin are hallmarks of the transcribed sequences embedded in the centromeric H3 subdomains. We propose that the transcribed sequences located in rice centromeres may provide a barrier preventing loading of CENH3 into the H3 subdomains. The separation of CENH3 and H3 subdomains in the centromere core may be favorable for the formation of three-dimensional centromere structure and for rice centromere function.     

Distinct histone modifications define initiation and repair of meiotic recombination in the mouse

Little is known about the factors determining the location and activity of the rapidly evolving meiotic crossover hotspots that shape genome diversity. Here, we show that several histone modifications are enriched at the active mouse Psmb9 hotspot, and we distinguish those marks that precede from those that follow hotspot recombinational activity. H3K4Me3, H3K4Me2 and H3K9Ac are specifically enriched in the chromatids that carry an active initiation site, and in the absence of DNA double-strand breaks (DSBs) in Spo11^−/− mice. We thus propose that these marks are part of the substrate for recombination initiation at the Psmb9 hotspot. In contrast, hyperacetylation of H4 is increased as a consequence of DSB formation, as shown by its dependency on Spo11 and by the enrichment detected on both recombining chromatids. In addition, the comparison with another hotspot, Hlx1, strongly suggests that H3K4Me3 and H4 hyperacetylation are common features of DSB formation and repair, respectively. Altogether, the chromatin signatures of the Psmb9 and Hlx1 hotspots provide a basis for understanding the distribution of meiotic recombination.     

Mapping global histone methylation patterns in the coding regions of human genes

Histone methylation patterns in the human genome, especially in euchromatin regions, have not been systematically characterized. In this study, we examined the profile of histone H3 methylation (Me) patterns at different lysines (Ks) in the coding regions of human genes by genome-wide location analyses by using chromatin immunoprecipitation linked to cDNA arrays. Specifically, we compared H3-KMe marks known to be associated with active gene expression, namely, H3-K4Me, H3-K36Me, and H3-K79Me, as well as those associated with gene repression, namely, H3-K9Me, H3-K27Me, and H4-K20Me. We further compared these to histone lysine acetylation (H3-K9/14Ac). Our results demonstrated that: first, close correlations are present between active histone marks except between H3-K36Me2 and H3-K4Me2. Notably, histone H3-K79Me2 is closely associated with H3-K4Me2 and H3-K36Me2 in the coding regions. Second, close correlations are present between histone marks associated with gene silencing such as H3-K9Me3, H3-K27Me2, and H4-K20Me2. Third, a poor correlation is observed between euchromatin marks (H3-K9/K14Ac, H3-K4Me2, H3-K36Me2, and H3-K79Me2) and heterochromatin marks (H3-K9Me2, H3-K9Me3, H3-K27Me2, and H4-K20Me2). Fourth, H3-K9Me2 is neither associated with active nor repressive histone methylations. Finally, histone H3-K4Me2, H3-K4Me3, H3-K36Me2, and H3-K79Me2 are associated with hyperacetylation and active genes, whereas H3-K9Me2, H3-K9Me3, H3-K27Me2, and H4-K20Me2 are associated with hypoacetylation. These data provide novel new information regarding histone KMe distribution patterns in the coding regions of human genes.     

Sir2 is required for Clr4 to initiate centromeric heterochromatin assembly in fission yeast

Heterochromatin assembly in fission yeast depends on the Clr4 histone methyltransferase, which targets H3K9. We show that the histone deacetylase Sir2 is required for Clr4 activity at telomeres, but acts redundantly with Clr3 histone deacetylase to maintain centromeric heterochromatin. However, Sir2 is critical for Clr4 function during de novo centromeric heterochromatin assembly. We identified new targets of Sir2 and tested if their deacetylation is necessary for Clr4‐mediated heterochromatin establishment. Sir2 preferentially deacetylates H4K16Ac and H3K4Ac, but mutation of these residues to mimic acetylation did not prevent Clr4‐mediated heterochromatin establishment. Sir2 also deacetylates H3K9Ac and H3K14Ac. Strains bearing H3K9 or H3K14 mutations exhibit heterochromatin defects. H3K9 mutation blocks Clr4 function, but why H3K14 mutation impacts heterochromatin was not known. Here, we demonstrate that recruitment of Clr4 to centromeres is blocked by mutation of H3K14. We suggest that Sir2 deacetylates H3K14 to target Clr4 to centromeres. Further, we demonstrate that Sir2 is critical for de novo accumulation of H3K9me2 in RNAi‐deficient cells. These analyses place Sir2 and H3K14 deacetylation upstream of Clr4 recruitment during heterochromatin assembly.     

Active and repressive chromatin are interspersed without spreading in an imprinted gene cluster in the mammalian genome

The Igf2r imprinted cluster is an epigenetic silencing model in which expression of a ncRNA silences multiple genes in cis. Here, we map a 250 kb region in mouse embryonic fibroblast cells to show that histone modifications associated with expressed and silent genes are mutually exclusive and localized to discrete regions. Expressed genes were modified at promoter regions by H3K4me3 + H3K4me2 + H3K9Ac and on putative regulatory elements flanking active promoters by H3K4me2 + H3K9Ac. Silent genes showed two types of nonoverlapping profile. One type spread over large domains of tissue-specific silent genes and contained H3K27me3 alone. A second type formed localized foci on silent imprinted gene promoters and a nonexpressed pseudogene and contained H3K9me3 + H4K20me3 +/- HP1. Thus, mammalian chromosome arms contain active chromatin interspersed with repressive chromatin resembling the type of heterochromatin previously considered a feature of centromeres, telomeres, and the inactive X chromosome.     

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.     

The visualization of large organized chromatin domains enriched in the H3K9me2 mark within a single chromosome in a single cell

Despite considerable efforts, our understanding of the organization of higher order chromatin conformations in single cells and how these relate to chromatin marks remains poor. We have earlier invented the Chromatin In Situ Proximity (ChrISP) technique to determine proximities between chromatin fibers within a single chromosome. Here we used ChrISP to identify chromosome 11-specific hubs that are enriched in the H3K9me2 mark and that project toward the nuclear membrane in finger-like structures. Conversely, chromosome 11-specfic chromatin hubs, visualized by the presence of either H3K9me1 or H3K9me3 marks, are chromosome-wide and largely absent at the nuclear periphery. As the nuclear periphery-specific chromatin hubs were lost in the induced reduction of H3K9me2 levels, they likely represent Large Organization Chromatin in Lysine Methylation (LOCK) domains, previously identified by ChIP-seq analysis. Strikingly, the downregulation of the H3K9me2/3 marks also led to the chromosome-wide compaction of chromosome 11, suggesting a pleiotropic function of these features not recognized before. The ChrISP-mediated visualization of dynamic chromatin states in single cells thus provides an analysis of chromatin structures with a resolution far exceeding that of any other light microscopic technique.     

Chromatin Organization and Remodeling of Interstitial Telomeric Sites During Meiosis in the Mongolian Gerbil (Meriones unguiculatus)

Telomeric DNA repeats are key features of chromosomes that allow the maintenance of integrity and stability in the telomeres. However, interstitial telomere sites (ITSs) can also be found along the chromosomes, especially near the centromere, where they may appear following chromosomal rearrangements like Robertsonian translocations. There is no defined role for ITSs, but they are linked to DNA damage-prone sites. We were interested in studying the structural organization of ITSs during meiosis, a kind of cell division in which programmed DNA damage events and noticeable chromatin reorganizations occur. Here we describe the presence of highly amplified ITSs in the pericentromeric region of Mongolian gerbil (Meriones unguiculatus) chromosomes. During meiosis, ITSs show a different chromatin conformation than DNA repeats at telomeres, appearing more extended and accumulating heterochromatin markers. Interestingly, ITSs also recruit the telomeric proteins RAP1 and TRF1, but in a stage-dependent manner, appearing mainly at late prophase I stages. We did not find a specific accumulation of DNA repair factors to the ITSs, such as γH2AX or RAD51 at these stages, but we could detect the presence of MLH1, a marker for reciprocal recombination. However, contrary to previous reports, we did not find a specific accumulation of crossovers at ITSs. Intriguingly, some centromeric regions of metacentric chromosomes may bind the nuclear envelope through the association to SUN1 protein, a feature usually performed by telomeres. Therefore, ITSs present a particular and dynamic chromatin configuration in meiosis, which could be involved in maintaining their genetic stability, but they additionally retain some features of distal telomeres, provided by their capability to associate to telomere-binding proteins.     

A complex network of interactions governs DNA methylation at telomeric regions

DNA methylation modulates telomere function. In Arabidopsis thaliana, telomeric regions have a bimodal chromatin organization with unmethylated telomeres and methylated subtelomeres. To gain insight into this organization we have generated TAIR10-Tel, a modified version of the Arabidopsis reference genome with additional sequences at most chromosome ends. TAIR10-Tel has allowed us to analyse DNA methylation at nucleotide resolution level in telomeric regions. We have analysed the wild-type strain and mutants that encode inactive versions of all currently known relevant methyltransferases involved in cytosine methylation. These analyses have revealed that subtelomeric DNA methylation extends 1 to 2 kbp from Interstitial Telomeric Sequences (ITSs) that abut or are very near to telomeres. However, DNA methylation drops at the telomeric side of the telomere-subtelomere boundaries and disappears at the inner part of telomeres. We present a comprehensive and integrative model for subtelomeric DNA methylation that should help to decipher the mechanisms that govern the epigenetic regulation of telomeres. This model involves a complex network of interactions between methyltransferases and subtelomeric DNA sequences.     

The visualization of large organized chromatin domains enriched in the H3K9me2 mark within a single chromosome in a single cell

Despite considerable efforts, our understanding of the organization of higher order chromatin conformations in single cells and how these relate to chromatin marks remains poor. We have earlier invented the Chromatin In Situ Proximity (ChrISP) technique to determine proximities between chromatin fibers within a single chromosome. Here we used ChrISP to identify chromosome 11-specific hubs that are enriched in the H3K9me2 mark and that project toward the nuclear membrane in finger-like structures. Conversely, chromosome 11-specfic chromatin hubs, visualized by the presence of either H3K9me1 or H3K9me3 marks, are chromosome-wide and largely absent at the nuclear periphery. As the nuclear periphery-specific chromatin hubs were lost in the induced reduction of H3K9me2 levels, they likely represent Large Organization Chromatin in Lysine Methylation (LOCK) domains, previously identified by ChIP-seq analysis. Strikingly, the downregulation of the H3K9me2/3 marks also led to the chromosome-wide compaction of chromosome 11, suggesting a pleiotropic function of these features not recognized before. The ChrISP-mediated visualization of dynamic chromatin states in single cells thus provides an analysis of chromatin structures with a resolution far exceeding that of any other light microscopic technique.     

Epigenetic marks for chromosome imprinting during spermatogenesis in coccids

The establishment of sex-specific epigenetic marks during gametogenesis is one of the key feature of genomic imprinting. By immunocytological analysis, we thoroughly characterized the chromatin remodeling events that take place during gametogenesis in the mealybug Planococcus citri, in which an entire haploid set of (imprinted) chromosomes undergoes facultative heterochromatinization in male embryos. Building on our previous work, we have investigated the interplay of several epigenetic marks in the regulation of this genome-wide phenomenon. We characterized the germline patterns of histone modifications, Me(3)K9H3, Me(2)K9H3, and Me(3)K20H4, and of heterochromatic proteins, PCHET2 (HP1-like) and HP2-like during male and female gametogenesis. We found that at all stages in oogenesis chromatin is devoid of any detectable epigenetic marks. On the other hand, spermatogenesis is accompanied by a complex pattern of redistribution of epigenetic marks from euchromatin to heterochromatin, and vice versa. At the end of spermatogenesis, sperm heads are decorated by all the molecules we tested, except for PCHET2. However, only Me(3)K9H3 and Me(2)K9H3 are still detectable in the male pronucleus. We suggest that the histone H3 lysine 9 methylation is the signal used to establish the male-specific imprinting on the paternal genome, thus allowing it to be distinguished from the maternal genome in the developing embryo. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Partitioning of the maize epigenome by the number of methyl groups on histone H3 lysines 9 and 27

We report a detailed analysis of maize chromosome structure with respect to seven histone H3 methylation states (dimethylation at lysine 4 and mono-, di-, and trimethylation at lysines 9 and 27). Three-dimensional light microscopy and the fine cytological resolution of maize pachytene chromosomes made it possible to compare the distribution of individual histone methylation events to each other and to DNA staining intensity. Major conclusions are that (1) H3K27me2 marks classical heterochromatin; (2) H3K4me2 is limited to areas between and around H3K27me2-marked chromomeres, clearly demarcating the euchromatic gene space; (3) H3K9me2 is restricted to the euchromatic gene space; (4) H3K27me3 occurs in a few (roughly seven) focused euchromatic domains; (5) centromeres and CENP-C are closely associated with H3K9me2 and H3K9me3; and (6) histone H4K20 di- and trimethylation are nearly or completely absent in maize. Each methylation state identifies different regions of the epigenome. We discuss the evolutionary lability of histone methylation profiles and draw a distinction between H3K9me2-mediated gene silencing and heterochromatin formation.