Additive inheritance of histone modifications in Arabidopsis thaliana intra-specific hybrids

Plant genomes are earmarked with defined patterns of chromatin marks. Little is known about the stability of these epigenomes when related, but distinct genomes are brought together by intra‐species hybridization. Arabidopsis thaliana accessions and their reciprocal hybrids were used as a model system to investigate the dynamics of histone modification patterns. The genome‐wide distribution of histone modifications H3K4me2 and H3K27me3 in the inbred parental accessions Col‐0, C24 and Cvi and their hybrid offspring was compared by chromatin immunoprecipitation in combination with genome tiling array hybridization. The analysis revealed that, in addition to DNA sequence polymorphisms, chromatin modification variations exist among accessions of A. thaliana. The range of these variations was higher for H3K27me3 (typically a repressive mark) than for H3K4me2 (typically an active mark). H3K4me2 and H3K27me3 were rather stable in response to intra‐species hybridization, with mainly additive inheritance in hybrid offspring. In conclusion, intra‐species hybridization does not result in gross changes to chromatin modifications.     

The progeny of Arabidopsis thaliana plants exposed to salt exhibit changes in DNA methylation, histone modifications and gene expression

Plants are able to acclimate to new growth conditions on a relatively short time-scale. Recently, we showed that the progeny of plants exposed to various abiotic stresses exhibited changes in genome stability, methylation patterns and stress tolerance. Here, we performed a more detailed analysis of methylation patterns in the progeny of Arabidopsis thaliana (Arabidopsis) plants exposed to 25 and 75 mM sodium chloride. We found that the majority of gene promoters exhibiting changes in methylation were hypermethylated, and this group was overrepresented by regulators of the chromatin structure. The analysis of DNA methylation at gene bodies showed that hypermethylation in the progeny of stressed plants was primarily due to changes in the 5' and 3' ends as well as in exons rather than introns. All but one hypermethylated gene tested had lower gene expression. The analysis of histone modifications in the promoters and coding sequences showed that hypermethylation and lower gene expression correlated with the enrichment of H3K9me2 and depletion of H3K9ac histones. Thus, our work demonstrated a high degree of correlation between changes in DNA methylation, histone modifications and gene expression in the progeny of salt-stressed plants.     

Antibiotic-induced DNA methylation changes in calluses of Arabidopsis thaliana

This study was carried out to determine the involvement of the antibiotic kanamycin, commonly used as a selective agent in transformation protocols, in the phenomenon of somaclonal variation. Both genetic and epigenetic events were looked for. Two complementary approaches were used to evaluate global methylation changes in the genome of callus derived from the leaves of Arabidopsis thaliana L.; immunolabelling using monoclonal-antibodies raised against 5-methylcytosine in order to define the relative abundance of methylated cytosine; the analysis of methylation-sensitive polymorphism technique (MSAP) to assess methylation changes at CCGG sequences. In addition, the same samples were analysed using AFLPs in order to determine the extent of genomic change with respect to callus grown in the absence of kanamycin and plants grown from seed. The use of kanamycin as a selective agent caused extensive methylation changes in the genome with both hyper- and hypomethylation events seen. However, the net result was genome-wide hypomethylation: this effect was dosage dependent; the higher the dose, the greater the effect. At the same time, sequence mutation was detected.     

The role of DNA methylation,nucleosome occupancy and histone modifications in paramutation

Paramutation is the transfer of epigenetic information between alleles that leads to a heritable change in expression of one of these alleles. Paramutation at the tissue‐specifically expressed maize (Zea mays) b1 locus involves the low‐expressing B′ and high‐expressing B‐I allele. Combined in the same nucleus, B′ heritably changes B‐I into B′. A hepta‐repeat located 100‐kb upstream of the b1 coding region is required for paramutation and for high b1 expression. The role of epigenetic modifications in paramutation is currently not well understood. In this study, we show that the B′ hepta‐repeat is DNA‐hypermethylated in all tissues analyzed. Importantly, combining B′ and B‐I in one nucleus results in de novo methylation of the B‐I repeats early in plant development. These findings indicate a role for hepta‐repeat DNA methylation in the establishment and maintenance of the silenced B′ state. In contrast, nucleosome occupancy, H3 acetylation, and H3K9 and H3K27 methylation are mainly involved in tissue‐specific regulation of the hepta‐repeat. Nucleosome depletion and H3 acetylation are tissue‐specifically regulated at the B‐I hepta‐repeat and associated with enhancement of b1 expression. H3K9 and H3K27 methylation are tissue‐specifically localized at the B′ hepta‐repeat and reinforce the silenced B′ chromatin state. The B′ coding region is H3K27 dimethylated in all tissues analyzed, indicating a role in the maintenance of the silenced B′ state. Taken together, these findings provide insight into the mechanisms underlying paramutation and tissue‐specific regulation of b1 at the level of chromatin structure.     

Gardening the genome: DNA methylation in Arabidopsis thaliana

DNA methylation has two essential roles in plants and animals - defending the genome against transposons and regulating gene expression. Recent experiments in Arabidopsis thaliana have begun to address crucial questions about how DNA methylation is established and maintained. One cardinal insight has been the discovery that DNA methylation can be guided by small RNAs produced through RNA-interference pathways. Plants and mammals use a similar suite of DNA methyltransferases to propagate DNA methylation, but plants have also developed a glycosylase-based mechanism for removing DNA methylation, and there are hints that similar processes function in other organisms.     

DNA methylation affects nuclear organization, histone modifications, and linker histone binding but not chromatin compaction

DNA methylation has been implicated in chromatin condensation and nuclear organization, especially at sites of constitutive heterochromatin. How this is mediated has not been clear. In this study, using mutant mouse embryonic stem cells completely lacking in DNA methylation, we show that DNA methylation affects nuclear organization and nucleosome structure but not chromatin compaction. In the absence of DNA methylation, there is increased nuclear clustering of pericentric heterochromatin and extensive changes in primary chromatin structure. Global levels of histone H3 methylation and acetylation are altered, and there is a decrease in the mobility of linker histones. However, the compaction of both bulk chromatin and heterochromatin, as assayed by nuclease digestion and sucrose gradient sedimentation, is unaltered by the loss of DNA methylation. This study shows how the complete loss of a major epigenetic mark can have an impact on unexpected levels of chromatin structure and nuclear organization and provides evidence for a novel link between DNA methylation and linker histones in the regulation of chromatin structure.     

Allele-specific DNA methylation analyses associated with siRNAs in Arabidopsis hybrids

Accumulating evidence has suggested that epigenetic marks including DNA methylation,small RNA and histone modification may involve hybrid vigor in plants.However,knowledge about how epigenetic marks in hybrids regulate gene expression is still limited.Based on genome-wide DNA methylation landscapes of Arabidopsis thaliana Ler and C24 ecotypes and their reciprocal F1 hybrids which were obtained in our previous work,we analyzed allele-specific DNA methylation and distinguished cis-and trans-regulated DNA methylation in hybrids.Our study indicated that both cis-and trans-regulated DNA methylation played roles in hybrids,when cis-regulation played a major role in CG methylation and trans-regulation played major roles in CHG and CHH methylation.In addition,we observed correlations between trans-regulated DNA methylation and siRNA densities.Enriched siRNA regions were significantly concurrent with highly trans-regulated DNA methylation regions.Our results illustrated DNA methylation regulation patterns integrated with siRNAs in Arabidopsis hybrids,and shed light on understanding the mechanism of epigenetic reprogramming for hybrid vigor.     

Characterization of post-translational modifications of histone H2B-variants isolated from Arabidopsis thaliana

Eukaryotic DNA is structurally packed into chromatin by the basic histone proteins H2A, H2B, H3, and H4. There is increasing evidence that incorporation and post-translational modifications of histone variants have a fundamental role in gene regulation. While modifications of H3 and H4 histones are now well-established, considerably less is known about H2B modifications. Here, we present the first detailed characterization of H2B-variants isolated from the model plant Arabidopsis thaliana. We combined reversed-phase chromatography with tandem mass spectrometry to identify post-translational modifications of the H2B-variants HTB1, HTB2, HTB4, HTB9, and HTB11, isolated from total chromatin and euchromatin-enriched fractions. The HTB9-variant has acetylation sites at lysines 6, 11, 27, 32, 38, and 39, while Lys-145 can be ubiquitinated. Analogous modifications and an additional methylation of Lys-3 were identified for HTB11. HTB2 shows similar acetylation and ubiquitination sites and an additional methylation at Lys-11. Furthermore, the N-terminal alanine residues of HTB9 and HTB11 were found to be mono-, di-, or trimethylated or unmodified. No methylation of arginine residues was detected. The data suggest that most of these modification sites are only partially occupied. Our study significantly expands the map of covalent Arabidopsis histone modifications and is the first step to unraveling the histone code in higher plants.     

DNA methylation controls histone H3 lysine 9 methylation and heterochromatin assembly in Arabidopsis

We propose a model for heterochromatin assembly that links DNA methylation with histone methylation and DNA replication. The hypomethylated Arabidopsis mutants ddm1 and met1 were used to investigate the relationship between DNA methylation and chromatin organization. Both mutants show a reduction of heterochromatin due to dispersion of pericentromeric low-copy sequences away from heterochromatic chromocenters. DDM1 and MET1 control heterochromatin assembly at chromocenters by their influence on DNA maintenance (CpG) methylation and subsequent methylation of histone H3 lysine 9. In addition, DDM1 is required for deacetylation of histone H4 lysine 16. Analysis of F(1) hybrids between wild-type and hypomethylated mutants revealed that DNA methylation is epigenetically inherited and represents the genomic imprint that is required to maintain pericentromeric heterochromatin.     

Epigenetic interplay of histone modifications and DNA methylation mediated by HDA6

One of the most fundamental questions in the control of gene expression is how epigenetic patterns of DNA methylation and histone modifications are established. Our recent studies demonstrate that histone deacetylase HDA6 integrates DNA methylation and histone modifications in gene silencing by interacting with DNA methyltransferase MET1 and histone demethylase FLD, suggesting that regulatory crosstalk between histone modifications and DNA methylation could be mediated by the interaction of various epigenetic modification proteins.