Histone methyltransferases direct different degrees of methylation to define distinct chromatin domains

The functional significance of mono-, di-, and trimethylation of lysine residues within histone proteins remains unclear. Antibodies developed to selectively recognize each of these methylated states at histone H3 lysine 9 (H3 Lys9) demonstrated that mono- and dimethylation localized specifically to silent domains within euchromatin. In contrast, trimethylated H3 Lys9 was enriched at pericentric heterochromatin. Enzymes known to methylate H3 Lys9 displayed remarkably different enzymatic properties in vivo. G9a was responsible for all detectable H3 Lys9 dimethylation and a significant amount of monomethylation within silent euchromatin. In contrast, Suv39h1 and Suv39h2 directed H3 Lys9 trimethylation specifically at pericentric heterochromatin. Thus, different methylated states of H3 Lys9 are directed by specific histone methyltransferases to "mark" distinct domains of silent chromatin.     

Different types of plant chromatin associated with modified histones H3 and H4 and methylated DNA

Eukaryotic chromosomes are organized into two large and distinct domains, euchromatin and heterochromatin, which are cytologically characterized by different degrees of chromatin compaction during interphase/prophase and by post-synthesis modifications of histones and DNA methylation. Typically, heterochromatin remains condensed during the entire cell cycle whereas euchromatin is decondensed at interphase. However, a fraction of the euchromatin can also remain condensed during interphase and appears as early condensing prophase chromatin. 5S and 45S rDNA sites and telomere DNA were used to characterize these regions in metaphase and interphase nuclei. We investigated the chromosomal distribution of modified histones and methylated DNA in the early and late condensing prophase chromatin of two species with clear differentiation between these domains. Both species, Costus spiralis and Eleutherine bulbosa, additionally have a small amount of classical heterochromatin detected by CMA/DAPI staining. The distribution of H4 acetylated at lysine 5 (H4K5ac), H3 phosphorylated at serine 10 (H3S10ph), H3 dimethylated at lysine 4 or 9 (H3K4me2, H3K9me2), and 5-methylcytosine was compared in metaphase, prophase, and interphase cells by immunostaining with specific antibodies. In both species, the late condensing prophase chromatin was highly enriched in H4K5ac and H3K4me2 whereas the early condensing chromatin was very poor in these marks. H3K9me2 was apparently uniformly distributed along the chromosomes whereas the early condensing chromatin was slightly enriched in 5-methylcytosine. Signals of H3S10ph were restricted to the pericentromeric region of all chromosomes. Notably, none of these marks distinguished classical heterochromatin from the early condensing euchromatin. It is suggested that the early condensing chromatin is an intermediate type between classical heterochromatin and euchromatin.     

Two distinct methods analyzing chromatin structure using centrifugation and antibodies to modified histone H3: both provide similar chromatin states of the Rit1/Bcl11b gene

Chromatin state of a 2-Mb region harboring Rit1/Bcl11b on mouse chromosome 12 was examined using two distinct methods. One is ChIP assay examining the degree of enrichment with histone H3 methylated at lysine 9 (H3-mLys9) in chromatin and the other is H/E (heterochromatin/euchromatin) assay that measures a chromatin condensation state by using centrifugation. The ChIP assay showed that a 50-kb interval covering the gene and an upstream region constituted chromatin enriched with unmethylated H3-mLys9 in cells expressing Rit1 compared to cells not expressing Rit1. In contrast, regions other than the 50-kb interval did not show much difference in the enrichment between the two different types of cells. On the other hand, H/E assay of two expressing and two non-expressing tissues provided compatible fractionation patterns, suggesting that the chromatin condensation state detected by H/E assay is correlated with the chromatin state controlled by histone H3 tail modification linked to gene expression. These results indicate that the centrifugation-based H/E assay should provide a new approach to the regulation of chromatin structure with respect to its condensation state, complementing ChIP assays.     

Histone modifications in Arabidopsis- high methylation of H3 lysine 9 is dispensable for constitutive heterochromatin

N-terminal modifications of nucleosomal core histones are involved in gene regulation, DNA repair and recombination as well as in chromatin modeling. The degree of individual histone modifications may vary between specific chromatin domains and throughout the cell cycle. We have studied the nuclear patterns of histone H3 and H4 acetylation and of H3 methylation in Arabidopsis. A replication-linked increase of acetylation only occurred at H4 lysine 16 (not for lysines 5 and 12) and at H3 lysine 18. The last was not observed in other plants. Strong methylation at H3 lysine 4 was restricted to euchromatin, while strong methylation at H3 lysine 9 occurred preferentially in heterochromatic chromocenters of Arabidopsis nuclei. Chromocenter appearance, DNA methylation and histone modification patterns were similar in nuclei of wild-type and kryptonite mutant (which lacks H3 lysine 9-specific histone methyltransferase), except that methylation at H3 lysine 9 in heterochromatic chromocenters was reduced to the same low level as in euchromatin. Thus, a high level of H3methylK9 is apparently not necessary to maintain chromocenter structure and does not prevent methylation of H3 lysine 4 within Arabidopsis chromocenters.     

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.     

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.     

Histone modifications and nuclear architecture: a review.

Epigenetic modifications, such as acetylation, phosphorylation, methylation, ubiquitination, and ADP ribosylation, of the highly conserved core histones, H2A, H2B, H3, and H4, influence the genetic potential of DNA. The enormous regulatory potential of histone modification is illustrated in the vast array of epigenetic markers found throughout the genome. More than the other types of histone modification, acetylation and methylation of specific lysine residues on N-terminal histone tails are fundamental for the formation of chromatin domains, such as euchromatin, and facultative and constitutive heterochromatin. In addition, the modification of histones can cause a region of chromatin to undergo nuclear compartmentalization and, as such, specific epigenetic markers are non-randomly distributed within interphase nuclei. In this review, we summarize the principles behind epigenetic compartmentalization and the functional consequences of chromatin arrangement within interphase nuclei.     

The Paf1 complex factors Leo1 and Paf1 promote local histone turnover to modulate chromatin states in fission yeast

The maintenance of open and repressed chromatin states is crucial for the regulation of gene expression. To study the genes involved in maintaining chromatin states, we generated a random mutant library in Schizosaccharomyces pombe and monitored the silencing of reporter genes inserted into the euchromatic region adjacent to the heterochromatic mating type locus. We show that Leo1–Paf1 [a subcomplex of the RNA polymerase II‐associated factor 1 complex (Paf1C)] is required to prevent the spreading of heterochromatin into euchromatin by mapping the heterochromatin mark H3K9me2 using high‐resolution genomewide ChIP (ChIP–exo). Loss of Leo1–Paf1 increases heterochromatin stability at several facultative heterochromatin loci in an RNAi‐independent manner. Instead, deletion of Leo1 decreases nucleosome turnover, leading to heterochromatin stabilization. Our data reveal that Leo1–Paf1 promotes chromatin state fluctuations by enhancing histone turnover.     

Three dimensional analysis of histone methylation patterns in normal and tumor cell nuclei

Histone modifications represent an important epigenetic mechanism for the organization of higher order chromatin structure and gene regulation. Methylation of position-specific lysine residues in the histone H3 and H4 amino termini has linked with the formation of constitutive and facultative heterochromatin as well as with specifically repressed single gene loci. Using an antibody, directed against dimethylated lysine 9 of histone H3 and several other lysine methylation sites, we visualized the nuclear distribution pattern of chromatin flagged by these methylated lysines in 3D preserved nuclei of normal and malignant cell types. Optical confocal serial sections were used for a quantitative evaluation. We demonstrate distinct differences of these histone methylation patterns among nuclei of different cell types after exit of the cell cycle. Changes in the pattern formation were also observed during the cell cycle. Our data suggest an important role of methylated histones in the reestablishment of higher order chromatin arrangements during telophase/early G1. Cell type specific histone methylation patterns are possibly casually involved in the formation of cell type specific heterochromatin compartments, composed of (peri)centromeric regions and chromosomal subregions from neighboring chromosomes territories, which contain silent genes.     

The profile of repeat-associated histone lysine methylation states in the mouse epigenome

Histone lysine methylation has been shown to index silenced chromatin regions at, for example, pericentric heterochromatin or of the inactive X chromosome. Here, we examined the distribution of repressive histone lysine methylation states over the entire family of DNA repeats in the mouse genome. Using chromatin immunoprecipitation in a cluster analysis representing repetitive elements, our data demonstrate the selective enrichment of distinct H3-K9, H3-K27 and H4-K20 methylation marks across tandem repeats (e.g. major and minor satellites), DNA transposons, retrotransposons, long interspersed nucleotide elements and short interspersed nucleotide elements. Tandem repeats, but not the other repetitive elements, give rise to double-stranded (ds) RNAs that are further elevated in embryonic stem (ES) cells lacking the H3-K9-specific Suv39h histone methyltransferases. Importantly, although H3-K9 tri- and H4-K20 trimethylation appear stable at the satellite repeats, many of the other repeat-associated repressive marks vary in chromatin of differentiated ES cells or of embryonic trophoblasts and fibroblasts. Our data define a profile of repressive histone lysine methylation states for the repetitive complement of four distinct mouse epigenomes and suggest tandem repeats and dsRNA as primary triggers for more stable chromatin imprints.