The centromeric heterochromatin of Costus spiralis: poorly methylated and transiently acetylated during meiosis

The centromeric region of Costus spiralis is characteristically composed of a small heterochromatic DAPI(+) band flanked by a discrete decondensed region. High concentrations of serine 10 of histone H3 (H3S10ph) around the DAPI(+) band in pachytene chromosomes and the location of this heterochromatin at the chromosome region directed towards the poles during metaphase-anaphase I confirm its integration into the centromeric region. Antibodies against both typical components of euchromatin histones (histone H4 acetylated at lysine 5 (H4K5ac) and histone H3 dimethylated at lysine 4 (H3K4me2)) and heterochromatin (dimethylated lysine 9 of H3 (H3K9me2) and anti-5-methylcytosine (5-mC)) were used to characterize the centromeric chromatin of this species during meiosis. In pachytene chromosomes, the decondensed terminal euchromatin of the chromosome arms were seen to be richer in H4K5ac and H3K4me2 histones, while the more condensed proximal region was relatively stronger labeled with anti-H3K9me2 and anti-5-methylcytosine (5-mC). The centromeric region itself, including the DAPI(+) band, was poor in all of these chromatin modifications, but it was highly enriched in H4K5ac at pachytene. Before and after this stage, the centromeric region was poorly labeled with anti-H4K5ac. Hypomethylation and hyperacetylation of any kind of heterochromatin has rarely been reported, and it may be related to the dominant role of the centromere domain over the heterochromatin repeats.     

Histone H3 methylation patterns in Brassica nigra, Brassica juncea, and Brassica carinata species

Core histones are subjected to various post-translational modifications, and one of them, most intensively studied in plants, is the methylation of histone H3. In the majority of analyzed plant species, dimethylation of H3 at lysine 9 (H3K9me2) is detected in heterochromatin domains, whereas methylation of H3 at lysine 4 (H3K4me2) is detected in euchromatin domains. The distribution of H3K9me2 in the interphase nucleus seems to be correlated with genome size, chromatin organization, but also with tissue specificity. In this paper, we present the analysis of the pattern and level of histone H3 methylation for two allotetraploid and one diploid Brassica species. We have found that the pattern of H3K9me2 in interphase nuclei from root meristematic tissue is comparable within the analyzed species and includes both heterochromatin and euchromatin, but the level of modification differs not only among species but even among nuclei in the same phase of the cell cycle within one species. Moreover, the differences in the level of H3K9me2 are not directly coupled with DNA content in the nuclei and are probably tissue specific.     

Histones H1 and H4 of surface-spread meiotic chromosomes

The chromatin conformation of somatic and meiotic chromosomes is, at least in part, a function of electrostatic nucleosome interactions that are mediated by transient acetylation of the histone H4 N-terminal domain and phosphorylation of histone H1. The distribution of those histones in the chromatin of meiotic chromosomes is reported here. Antibodies to testis-specific histone 1, H1t, detect H1t in the chromatin of mouse meiotic prophase chromosomes only after synapsis and synaptonemal complex (SC) assembly is completed and before core separation is initiated. The H1t protein is evenly distributed over euchromatin, heterochromatin and the SC. Antibodies to acetylated lysine residues 5, 12 or 16 of histone H4, indicate that the euchromatin is more acetylated than the centromeric heterochromatin. The pattern is most pronounced for acetylated residue 5 and least for 16. Antibodies to phosphorylated H1 epitopes do not react with chromatin but, instead, recognize the chromosome cores and SCs. Possibly these are not phosphorylated histone H1 epitopes, but SC proteins with similar potentially phosphorylatable sequences such as KTPTK of the synaptic protein Syn1.     

Cytogenetic mapping with centromeric bacterial artificial chromosomes contigs shows that this recombination‐poor region comprises more than half of barley chromosome 3H

Genetic maps are based on the frequency of recombination and often show different positions of molecular markers in comparison to physical maps, particularly in the centromere that is generally poor in meiotic recombinations. To decipher the position and order of DNA sequences genetically mapped to the centromere of barley (Hordeum vulgare) chromosome 3H, fluorescence in situ hybridization with mitotic metaphase and meiotic pachytene chromosomes was performed with 70 genomic single‐copy probes derived from 65 fingerprinted bacterial artificial chromosomes (BAC) contigs genetically assigned to this recombination cold spot. The total physical distribution of the centromeric 5.5 cM bin of 3H comprises 58% of the mitotic metaphase chromosome length. Mitotic and meiotic chromatin of this recombination‐poor region is preferentially marked by a heterochromatin‐typical histone mark (H3K9me2), while recombination enriched subterminal chromosome regions are enriched in euchromatin‐typical histone marks (H3K4me2, H3K4me3, H3K27me3) suggesting that the meiotic recombination rate could be influenced by the chromatin landscape.     

H3S10 phosphorylation by the JIL-1 kinase regulates H3K9 dimethylation and gene expression at the white locus in Drosophila

The JIL-1 kinase is a multidomain protein that localizes specifically to euchromatin interband regions of polytene chromosomes and is the kinase responsible for histone H3S10 phosphorylation at interphase. Genetic interaction assays have suggested that the function of the epigenetic histone H3S10ph mark is to antagonize heterochromatization by participating in a dynamic balance between factors promoting repression and activation of gene expression as measured by position-effect variegation (PEV) assays. Interestingly, JIL-1 loss-of-function alleles can act either as an enhancer or indirectly as a suppressor of w(m4) PEV depending on the precise levels of JIL-1 kinase activity. In this study, we have explored the relationship between PEV and the relative levels of the H3S10ph and H3K9me2 marks at the white gene in both wild-type and w(m4) backgrounds by ChIP analysis. Our results indicate that H3K9me2 levels at the white gene directly correlate with its level of expression and that H3K9me2 levels in turn are regulated by H3S10 phosphorylation.     

Histone H3S10 phosphorylation by the JIL-1 kinase in pericentric heterochromatin and on the fourth chromosome creates a composite H3S10phK9me2 epigenetic mark

The JIL-1 kinase mainly localizes to euchromatic interband regions of polytene chromosomes and is the kinase responsible for histone H3S10 phosphorylation at interphase in Drosophila. However, recent findings raised the possibility that the binding of some H3S10ph antibodies may be occluded by the H3K9me2 mark obscuring some H3S10 phosphorylation sites. Therefore, we have characterized an antibody to the epigenetic H3S10phK9me2 double mark as well as three commercially available H3S10ph antibodies. The results showed that for some H3S10ph antibodies their labeling indeed can be occluded by the concomitant presence of the H3K9me2 mark. Furthermore, we demonstrate that the double H3S10phK9me2 mark is present in pericentric heterochromatin as well as on the fourth chromosome of wild-type polytene chromosomes but not in preparations from JIL-1 or Su(var)3-9 null larvae. Su(var)3-9 is a methyltransferase mediating H3K9 dimethylation. Furthermore, the H3S10phK9me2 labeling overlapped with that of the non-occluded H3S10ph antibodies as well as with H3K9me2 antibody labeling. Interestingly, when a Lac-I-Su(var)3-9 transgene is overexpressed, it upregulates H3K9me2 dimethylation on the chromosome arms creating extensive ectopic H3S10phK9me2 marks suggesting that the H3K9 dimethylation occurred at euchromatic H3S10ph sites. This is further supported by the finding that under these conditions euchromatic H3S10ph labeling by the occluded antibodies was abolished. Thus, our findings indicate a novel role for the JIL-1 kinase in epigenetic regulation of heterochromatin in the context of the chromocenter and fourth chromosome by creating a composite H3S10phK9me2 mark together with the Su(var)3-9 methyltransferase.     

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.     

Cell-cycle-dependent dissociation of histone H1 from chromatin in nuclei of P. polycephalum.

The dissociation curves of histone H1 from chromatin in interphase and metaphase nuclei from Physarum polycephalum have been determined using CaCl2 as dissociating agent. H1 is less strongly bound to metaphase chromosomes than to interphase chromatin. However, no differences could be detected in the binding of Hl to early S, late S or G2 phase chromatin. The number of CaCl2 molecules involved in binding one H1 molecule to chromatin was reduced from 5 in interphase to 4 in metaphase. The non-electrostatic contribution to the free-energy of binding was small in both cases. A comparison of the binding properties of H1 to sheared chromatin, native chromatin and metaphase chromosomes suggests that the electrostatic binding functions of H1 are completely satisfied within the nucleosome and that further electrostatic interactions are not involved in folding the nucleosomal fibre into the 300 A "solenoid" or the more tightly folded metaphase chromosome.     

Histone H3 tails containing dimethylated lysine and adjacent phosphorylated serine modifications adopt a specific conformation during mitosis and meiosis

Condensation of chromatin, mediated in part by posttranslational modifications of histones, is essential for cell division during mitosis. Histone H3 tails are dimethylated on lysine (Kme2) and become phosphorylated on serine (Sp) residues during mitosis. We have explored the possibility that these double modifications are involved in the establishment of H3 tail conformations during the cell cycle. Here we describe a specific chromatin conformation occurring at Kme2 and adjacently phosphorylated S of H3 tails upon formation of a hydrogen bond. This conformation appears exclusively between early prophase and early anaphase of the mitosis, when chromatin condensation is highest. Moreover, we observed that the conformed H3Kme2Sp tail is present at the diplotene and metaphase stages in spermatocytes and oocytes. Our data together with results obtained by cryoelectron microscopy suggest that the conformation of Kme2Sp-modified H3 tails changes during mitosis and meiosis. This is supported by biostructural modeling of a modified histone H3 tail bound by an antibody, indicating that Kme2Sp-modified H3 tails can adopt at least two different conformations. Thus, the H3K9me2S10p and the H3K27me2S28p sites are involved in the acquisition of specific chromatin conformations during chromatin condensation for cell division.     

Characterisation of pericentrometric and sticky intercalary heterochromatin in Ornithogalum longibracteatum (Hyacinthaceae)

The hexaploid liliaceous plant Ornithogalum longibracteatum (2n=6x=54) has a heterochromatin-rich bimodal karyotype with large (L) and small (S) chromosomes. The composition and subgenomic distribution of heterochromatin was studied using molecular and cytological methods. The major component of centromeric heterochromatin in all chromosomes is Satl, an abundant satellite DNA with a basic repeat unit of 155 bp and an average A+T content (54%). The major component of the large blocks of intercalary heterochromatin in L chromosomes is Sat2, an abundant satellite DNA with a basic repeat unit of 115 bp and a high A+T content (76%). Additionally, traces of Sat2 can be detected at the centromeric regions of S chromosomes, while minor amounts of Satl are discernible in intercalary heterochromatin of L chromosomes. The chromosomal localisation pattern of Sat2 is consistent with the fluorescent staining pattern obtained with the A+T-specific DNA ligand 4'-6-diamidino-2-phenylindole (DAPI). A+T-rich intercalary heterochromatin is sticky and tends to associate ectopically during mitosis. Sister chromatid exchange clustering was found at the junctions between euchromatin and heterochromatin and at the centromeres. The pattern of mitosis-specific phosphorylation of histone H3 was not uniform along the length of the chromosomes. In all L and S chromosomes, from early prophase to ana-/telophase, there is hyperphosphorylation of histone H3 in the pericentromeric chromatin and a slightly elevated phosphorylated histone H3 level at the intercalary heterochromatin of L chromosomes. Consequently, the overall phosphorylated histone H3 metaphase labelling resembles the distribution of Satl in the karyotype of O. longibracteatum.     

Phosphorylation of H3S10 blocks the access of H3K9 by specific antibodies and histone methyltransferase. Implication in regulating chromatin dynamics and epigenetic inheritance during mitosis

Post-translational modifications of histones play a critical role in regulating genome structures and integrity. We have focused on the regulatory relationship between covalent modifications of histone H3 lysine 9 (H3K9) and H3S10 during the cell cycle. Immunofluorescence microscopy revealed that H3S10 phosphorylation in HeLa, A549, and HCT116 cells was high during prophase, prometaphase, and metaphase, whereas H3K9 monomethylation (H3K9me1) and dimethylation (H3K9me2), but not H3K9 trimethylation (H3K9me3), were significantly suppressed. When H3S10 phosphorylation started to diminish during anaphase, H3K9me1 and H3K9me2 signals reemerged. Western blot analyses confirmed that mitotic histones, extracted in an SDS-containing buffer, had little H3K9me1 and H3K9me2 signals but abundant H3K9me3 signals. However, when mitotic histones were extracted in the same buffer without SDS, the difference in H3K9me1 and H3K9me2 signals between interphase and mitotic cells disappeared. Removal of H3S10 phosphorylation by pretreatment with lambda-phosphatase unmasked mitotic H3K9me1 and H3K9me2 signals detected by both fluorescence microscopy and Western blotting. Further, H3S10 phosphorylation completely blocked methylation of H3K9 but not demethylation of the same residue in vitro. Given that several conserved motifs consisting of a Lys residue immediately followed by a Ser residue are present in histone tails, our studies reveal a potential new mechanism by which phosphorylation not only regulates selective access of methylated lysines by cellular factors but also serves to preserve methylation patterns and epigenetic programs during cell division.     

H3S10 phosphorylation by the JIL-1 kinase regulates H3K9 dimethylation and gene expression at the white locus in Drosophila

The JIL-1 kinase is a multidomain protein that localizes specifically to euchromatin interband regions of polytene chromosomes and is the kinase responsible for histone H3S10 phosphorylation at interphase. Genetic interaction assays have suggested that the function of the epigenetic histone H3S10ph mark is to antagonize heterochromatization by participating in a dynamic balance between factors promoting repression and activation of gene expression as measured by position-effect variegation (PEV) assays. Interestingly, JIL-1 loss-of-function alleles can act either as an enhancer or indirectly as a suppressor of w^m4 PEV depending on the precise levels of JIL-1 kinase activity. In this study, we have explored the relationship between PEV and the relative levels of the H3S10ph and H3K9me2 marks at the white gene in both wild-type and w^m4 backgrounds by ChIP analysis. Our results indicate that H3K9me2 levels at the white gene directly correlate with its level of expression and that H3K9me2 levels in turn are regulated by H3S10 phosphorylation.     

A cytochemical study of the nonhistone protein content of condensed and extended chromatin

Cytochemical techniques have been used to study the distribution of nonhistone proteins in sections of interphase nuclei and mitotic chromosomes. Condensed chromatin, including the heterochromatin of interphase nuclei from frog liver, and mitotic metaphase and anaphase chromosomes from bovine kidney, show little or no staining for nonhistone protein. Regions of frog liver nuclei which contain extended chromatin (euchromatin) stain intensely for nonhistone protein. These differences in nonhistone staining of condensed and extended chromatin support the suggestion that regions of condensed chromatin contain considerably less nonhistone protein than regions of extended chromatin. The results suggest further that there may be considerably less nonhistone protein associated with chromosomes and interphase heterochromatin than has been reported in most previous analyses of isolated chromatin and chromosome preparations.     

Chromatin state analysis of the barley epigenome reveals a higher‐order structure defined by H3K27me1 and H3K27me3 abundance

Combinations of histones carrying different covalent modifications are a major component of epigenetic variation. We have mapped nine modified histones in the barley seedling epigenome by chromatin immunoprecipitation next‐generation sequencing (ChIP‐seq). The chromosomal distributions of the modifications group them into four different classes, and members of a given class also tend to coincide at the local DNA level, suggesting that global distribution patterns reflect local epigenetic environments. We used this peak sharing to define 10 chromatin states representing local epigenetic environments in the barley genome. Five states map mainly to genes and five to intergenic regions. Two genic states involving H3K36me3 are preferentially associated with constitutive gene expression, while an H3K27me3‐containing genic state is associated with differentially expressed genes. The 10 states display striking distribution patterns that divide barley chromosomes into three distinct global environments. First, telomere‐proximal regions contain high densities of H3K27me3 covering both genes and intergenic DNA, together with very low levels of the repressive H3K27me1 modification. Flanking these are gene‐rich interior regions that are rich in active chromatin states and have greatly decreased levels of H3K27me3 and increasing amounts of H3K27me1 and H3K9me2. Lastly, H3K27me3‐depleted pericentromeric regions contain gene islands with active chromatin states separated by extensive retrotransposon‐rich regions that are associated with abundant H3K27me1 and H3K9me2 modifications. We propose an epigenomic framework for barley whereby intergenic H3K27me3 specifies facultative heterochromatin in the telomere‐proximal regions and H3K27me1 is diagnostic for constitutive heterochromatin elsewhere in the barley genome.     

Lid2 is required for coordinating H3K4 and H3K9 methylation of heterochromatin and euchromatin

In most eukaryotes, histone methylation patterns regulate chromatin architecture and function: methylation of histone H3 lysine-9 (H3K9) demarcates heterochromatin, whereas H3K4 methylation demarcates euchromatin. We show here that the S. pombe JmjC-domain protein Lid2 is a trimethyl H3K4 demethylase responsible for H3K4 hypomethylation in heterochromatin. Lid2 interacts with the histone lysine-9 methyltransferase, Clr4, through the Dos1/Clr8-Rik1 complex, which also functions in the RNA interference pathway. Disruption of the JmjC domain alone results in severe heterochromatin defects and depletion of siRNA, whereas overexpressing Lid2 enhances heterochromatin silencing. The physical and functional link between H3K4 demethylation and H3K9 methylation suggests that the two reactions act in a coordinated manner. Surprisingly, crossregulation of H3K4 and H3K9 methylation in euchromatin also requires Lid2. We suggest that Lid2 enzymatic activity in euchromatin is regulated through a dynamic interplay with other histone-modification enzymes. Our findings provide mechanistic insight into the coordination of H3K4 and H3K9 methylation.     

A novel role for the Aurora B kinase in epigenetic marking of silent chromatin in differentiated postmitotic cells

Combinatorial modifications of the core histones have the potential to fine-tune the epigenetic regulation of chromatin states. The Aurora B kinase is responsible for generating the double histone H3 modification tri-methylated K9/phosphorylated S10 (H3K9me3/S10ph), which has been implicated in chromosome condensation during mitosis. In this study, we have identified a novel role for Aurora B in epigenetic marking of silent chromatin during cell differentiation. We find that phosphorylation of H3 S10 by Aurora B generates high levels of the double H3K9me3/S10ph modification in differentiated postmitotic cells and also results in delocalisation of HP1beta away from heterochromatin in terminally differentiated plasma cells. Microarray analysis of the H3K9me3/S10ph modification shows a striking increase in the modification across repressed genes during differentiation of mesenchymal stem cells. Our results provide evidence that the Aurora B kinase has a role in marking silent chromatin independently of the cell cycle and suggest that targeting of Aurora B-mediated phosphorylation of H3 S10 to repressed genes could be a mechanism for epigenetic silencing of gene expression.     

Trimethylation of histone H3 lysine 4 impairs methylation of histone H3 lysine 9

Chromatin is broadly compartmentalized in two defined states: euchromatin and heterochromatin. Generally, euchromatin is trimethylated on histone H3 lysine 4 (H3K4^me3) while heterochromatin contains the H3K9^me3 marks. The H3K9^me3 modification is added by lysine methyltransferases (KMTs) such as SETDB1. Herein, we show that SETDB1 interacts with its substrate H3, but only in the absence of the euchromatic mark H3K4^me3. In addition, we show that SETDB1 fails to methylate substrates containing the H3K4^me3 mark. Likewise, the functionally related H3K9 KMTs G9A, GLP, and SUV39H1 also fail to bind and to methylate H3K4^me3 substrates. Accordingly, we provide in vivo evidence that H3K9^me2-enriched histones are devoid of H3K4^me2/3 and that histones depleted of H3K4^me2/3 have elevated H3K9^me2/3. The correlation between the loss of interaction of these KMTs with H3K4^me3 and concomitant methylation impairment leads to the postulate that, at least these four KMTs, require stable interaction with their respective substrates for optimal activity. Thus, novel substrates could be discovered via the identification of KMT interacting proteins. Indeed, we find that SETDB1 binds to and methylates a novel substrate, the inhibitor of growth protein ING2, while SUV39H1 binds to and methylates the heterochromatin protein HP1α. Thus, our observations suggest a mechanism of post-translational regulation of lysine methylation and propose a potential mechanism for the segregation of the biologically opposing marks, H3K4^me3 and H3K9^me3. Furthermore, the correlation between H3-KMTs interaction and substrate methylation highlights that the identification of novel KMT substrates may be facilitated by the identification of interaction partners.