RESUMO
Cytosine methylation is involved in various biological processes such as silencing of transposable elements (TEs) and imprinting. Multiple pathways regulate DNA methylation in different sequence contexts, but the factors that regulate DNA methylation at a given site in the genome largely remain unknown. Here we have surveyed the methylomes of a comprehensive list of 86 Arabidopsis gene silencing mutants by generating single-nucleotide resolution maps of DNA methylation. We find that DNA methylation is site specifically regulated by different factors. Furthermore, we have identified additional regulators of DNA methylation. These data and analyses will serve as a comprehensive community resource for further understanding the control of DNA methylation patterning.
Assuntos
Arabidopsis/genética , Metilação de DNA , Genoma de Planta , Proteínas de Arabidopsis/metabolismo , Ilhas de CpG , Inativação Gênica , Estudo de Associação Genômica Ampla , Histonas/metabolismo , Interferência de RNA , RNA Polimerase II/metabolismo , Fatores de Processamento de RNARESUMO
DNA methylation and histone modification exert epigenetic control over gene expression. CHG methylation by CHROMOMETHYLASE3 (CMT3) depends on histone H3K9 dimethylation (H3K9me2), but the mechanism underlying this relationship is poorly understood. Here, we report multiple lines of evidence that CMT3 interacts with H3K9me2-containing nucleosomes. CMT3 genome locations nearly perfectly correlated with H3K9me2, and CMT3 stably associated with H3K9me2-containing nucleosomes. Crystal structures of maize CMT3 homolog ZMET2, in complex with H3K9me2 peptides, showed that ZMET2 binds H3K9me2 via both bromo adjacent homology (BAH) and chromo domains. The structures reveal an aromatic cage within both BAH and chromo domains as interaction interfaces that capture H3K9me2. Mutations that abolish either interaction disrupt CMT3 binding to nucleosomes and show a complete loss of CMT3 activity in vivo. Our study establishes dual recognition of H3K9me2 marks by BAH and chromo domains and reveals a distinct mechanism of interplay between DNA methylation and histone modification.
Assuntos
Arabidopsis/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , DNA de Plantas/metabolismo , Nucleossomos/metabolismo , Zea mays/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Cristalografia por Raios X , DNA (Citosina-5-)-Metiltransferases/química , Heterocromatina/metabolismo , Histonas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Alinhamento de Sequência , Zea mays/genéticaRESUMO
DNA methylation and H3K9me are hallmarks of heterochromatin in plants and mammals, and are successfully maintained across generations. The biochemical and structural basis for this maintenance is poorly understood. The maintenance DNA methyltransferase from Zea mays, ZMET2, recognizes dimethylation of H3K9 via a chromodomain (CD) and a bromo adjacent homology (BAH) domain, which flank the catalytic domain. Here, we show that dinucleosomes are the preferred ZMET2 substrate, with DNA methylation preferentially targeted to linker DNA. Electron microscopy shows one ZMET2 molecule bridging two nucleosomes within a dinucleosome. We find that the CD stabilizes binding, whereas the BAH domain enables allosteric activation by the H3K9me mark. ZMET2 further couples recognition of H3K9me to an increase in the specificity for hemimethylated versus unmethylated DNA. We propose a model in which synergistic coupling between recognition of nucleosome spacing, H3K9 methylation, and DNA modification allows ZMET2 to maintain DNA methylation in heterochromatin with high fidelity.
Assuntos
Metilação de DNA , Metilases de Modificação do DNA/metabolismo , Nucleossomos/enzimologia , Proteínas de Plantas/metabolismo , Animais , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/ultraestrutura , Ativação Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Microscopia Eletrônica , Modelos Moleculares , Conformação de Ácido Nucleico , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/ultraestrutura , Proteínas de Plantas/genética , Proteínas de Plantas/ultraestrutura , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade , Especificidade por Substrato , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
Nucleosomes compact and regulate access to DNA in the nucleus, and are composed of approximately 147 bases of DNA wrapped around a histone octamer. Here we report a genome-wide nucleosome positioning analysis of Arabidopsis thaliana using massively parallel sequencing of mononucleosomes. By combining this data with profiles of DNA methylation at single base resolution, we identified 10-base periodicities in the DNA methylation status of nucleosome-bound DNA and found that nucleosomal DNA was more highly methylated than flanking DNA. These results indicate that nucleosome positioning influences DNA methylation patterning throughout the genome and that DNA methyltransferases preferentially target nucleosome-bound DNA. We also observed similar trends in human nucleosomal DNA, indicating that the relationships between nucleosomes and DNA methyltransferases are conserved. Finally, as has been observed in animals, nucleosomes were highly enriched on exons, and preferentially positioned at intron-exon and exon-intron boundaries. RNA polymerase II (Pol II) was also enriched on exons relative to introns, consistent with the hypothesis that nucleosome positioning regulates Pol II processivity. DNA methylation is also enriched on exons, consistent with the targeting of DNA methylation to nucleosomes, and suggesting a role for DNA methylation in exon definition.
Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Metilação de DNA/fisiologia , Nucleossomos/metabolismo , Arabidopsis/enzimologia , Montagem e Desmontagem da Cromatina/genética , Imunoprecipitação da Cromatina , Metilação de DNA/genética , DNA Polimerase II/análise , DNA Polimerase II/metabolismo , DNA de Plantas/genética , DNA de Plantas/metabolismo , Éxons/genética , Genes de Plantas/genética , Genoma de Planta/genética , Humanos , Nuclease do Micrococo/metabolismo , Nucleossomos/genética , Análise de Sequência de DNARESUMO
Dimethylation of histone H3 lysine 9 (H3K9m2) and trimethylation of histone H3 lysine 27 (H3K27m3) are two hallmarks of transcriptional repression in many organisms. In Arabidopsis thaliana, H3K27m3 is targeted by Polycomb Group (PcG) proteins and is associated with silent protein-coding genes, while H3K9m2 is correlated with DNA methylation and is associated with transposons and repetitive sequences. Recently, ectopic genic DNA methylation in the CHG context (where H is any base except G) has been observed in globally DNA hypomethylated mutants such as met1, but neither the nature of the hypermethylated loci nor the biological significance of this epigenetic phenomenon have been investigated. Here, we generated high-resolution, genome-wide maps of both H3K9m2 and H3K27m3 in wild-type and met1 plants, which we integrated with transcriptional data, to explore the relationships between these two marks. We found that ectopic H3K9m2 observed in met1 can be due to defects in IBM1-mediated H3K9m2 demethylation at some sites, but most importantly targets H3K27m3-marked genes, suggesting an interplay between these two silencing marks. Furthermore, H3K9m2/DNA-hypermethylation at these PcG targets in met1 is coupled with a decrease in H3K27m3 marks, whereas CG/H3K9m2 hypomethylated transposons become ectopically H3K27m3 hypermethylated. Our results bear interesting similarities with cancer cells, which show global losses of DNA methylation but ectopic hypermethylation of genes previously marked by H3K27m3.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , DNA (Citosina-5-)-Metiltransferases , Metilação de DNA , Histona-Lisina N-Metiltransferase , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Elementos de DNA Transponíveis/genética , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Heterocromatina/genética , Histonas/genética , Sequências Repetitivas Dispersas , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Lisina/genética , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismoRESUMO
In eukaryotic cells, environmental and developmental signals alter chromatin structure and modulate gene expression. Heterochromatin constitutes the transcriptionally inactive state of the genome and in plants and mammals is generally characterized by DNA methylation and histone modifications such as histone H3 lysine 9 (H3K9) methylation. In Arabidopsis thaliana, DNA methylation and H3K9 methylation are usually colocated and set up a mutually self-reinforcing and stable state. Here, in contrast, we found that SUVR5, a plant Su(var)3-9 homolog with a SET histone methyltransferase domain, mediates H3K9me2 deposition and regulates gene expression in a DNA methylation-independent manner. SUVR5 binds DNA through its zinc fingers and represses the expression of a subset of stimulus response genes. This represents a novel mechanism for plants to regulate their chromatin and transcriptional state, which may allow for the adaptability and modulation necessary to rapidly respond to extracellular cues.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Metilação de DNA , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Proteínas de Arabidopsis/química , Sequência de Bases , Sítios de Ligação , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Histona Desmetilases/metabolismo , Histona-Lisina N-Metiltransferase/química , Motivos de Nucleotídeos , Regiões Promotoras Genéticas , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Dedos de ZincoRESUMO
Trimethylation of histone H3 lysine 27 (H3K27me3) plays critical roles in regulating animal development, and in several cases, H3K27me3 is also required for the proper expression of developmentally important genes in plants. However, the extent to which H3K27me3 regulates plant genes on a genome-wide scale remains unknown. In addition, it is not clear whether the establishment and spreading of H3K27me3 occur through the same mechanisms in plants and animals. We identified regions containing H3K27me3 in the genome of the flowering plant Arabidopsis thaliana using a high-density whole-genome tiling microarray. The results suggest that H3K27me3 is a major silencing mechanism in plants that regulates an unexpectedly large number of genes in Arabidopsis (~4,400), and that the maintenance of H3K27me3 is largely independent of other epigenetic pathways, such as DNA methylation or RNA interference. Unlike in animals, where H3K27m3 occupies large genomic regions, in Arabidopsis, we found that H3K27m3 domains were largely restricted to the transcribed regions of single genes. Furthermore, unlike in animals systems, H3K27m3 domains were not preferentially associated with low-nucleosome density regions. The results suggest that different mechanisms may underlie the establishment and spreading of H3K27me3 in plants and animals.
Assuntos
Arabidopsis/genética , Genoma de Planta/fisiologia , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Sequência de Aminoácidos , Inativação Gênica , Genes de Plantas , MetilaçãoRESUMO
Tandem repeat sequences are frequently associated with gene silencing phenomena. The Arabidopsis thaliana FWA gene contains two tandem repeats and is an efficient target for RNA-directed de novo DNA methylation when it is transformed into plants. We showed that the FWA tandem repeats are necessary and sufficient for de novo DNA methylation and that repeated character rather than intrinsic sequence is likely important. Endogenous FWA can adopt either of two stable epigenetic states: methylated and silenced or unmethylated and active. Surprisingly, we found small interfering RNAs (siRNAs) associated with FWA in both states. Despite this, only the methylated form of endogenous FWA could recruit further RNA-directed DNA methylation or cause efficient de novo methylation of transgenic FWA. This suggests that RNA-directed DNA methylation occurs in two steps: first, the initial recruitment of the siRNA-producing machinery, and second, siRNA-directed DNA methylation either in cis or in trans. The efficiency of this second step varies depending on the nature of the siRNA-producing locus, and at some loci, it may require pre-existing chromatin modifications such as DNA methylation itself. Enhancement of RNA-directed DNA methylation by pre-existing DNA methylation could create a self-reinforcing system to enhance the stability of silencing. Tandem repeats throughout the Arabidopsis genome produce siRNAs, suggesting that repeat acquisition may be a general mechanism for the evolution of gene silencing.
Assuntos
Arabidopsis/genética , Metilação de DNA , Inativação Gênica , RNA de Plantas/fisiologia , RNA Interferente Pequeno/genética , Sequências de Repetição em Tandem/genética , Genoma de Planta , Mutação , TransgenesRESUMO
BACKGROUND: Post-translational modifications of histones play important roles in maintaining normal transcription patterns by directly or indirectly affecting the structural properties of the chromatin. In plants, methylation of histone H3 lysine 4 (H3K4me) is associated with genes and required for normal plant development. RESULTS: We have characterized the genome-wide distribution patterns of mono-, di- and trimethylation of H3K4 (H3K4me1, H3K4me2 and H3K4me3, respectively) in Arabidopsis thaliana seedlings using chromatin immunoprecipitation and high-resolution whole-genome tiling microarrays (ChIP-chip). All three types of H3K4me are found to be almost exclusively genic, and two-thirds of Arabidopsis genes contain at least one type of H3K4me. H3K4me2 and H3K4me3 accumulate predominantly in promoters and 5' genic regions, whereas H3K4me1 is distributed within transcribed regions. In addition, H3K4me3-containing genes are highly expressed with low levels of tissue specificity, but H3K4me1 or H3K4me2 may not be directly involved in transcriptional activation. Furthermore, the preferential co-localization of H3K4me3 and H3K27me3 found in mammals does not appear to occur in plants at a genome-wide level, but H3K4me2 and H3K27me3 co-localize at a higher-than-expected frequency. Finally, we found that H3K4me2/3 and DNA methylation appear to be mutually exclusive, but surprisingly, H3K4me1 is highly correlated with CG DNA methylation in the transcribed regions of genes. CONCLUSIONS: H3K4me plays widespread roles in regulating gene expression in plants. Although many aspects of the mechanisms and functions of H3K4me appear to be conserved among all three kingdoms, we observed significant differences in the relationship between H3K4me and transcription or other epigenetic pathways in plants and mammals.
Assuntos
Arabidopsis/genética , Genoma de Planta/genética , Histonas/metabolismo , Lisina/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Citosina/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Metilação , Transcrição GênicaRESUMO
Constitutive heterochromatin in Arabidopsis thaliana is marked by repressive chromatin modifications, including DNA methylation, histone H3 dimethylation at Lys9 (H3K9me2) and monomethylation at Lys27 (H3K27me1). The enzymes catalyzing DNA methylation and H3K9me2 have been identified; alterations in these proteins lead to reactivation of silenced heterochromatic elements. The enzymes responsible for heterochromatic H3K27me1, in contrast, remain unknown. Here we show that the divergent SET-domain proteins ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) and ATXR6 have H3K27 monomethyltransferase activity, and atxr5 atxr6 double mutants have reduced H3K27me1 in vivo and show partial heterochromatin decondensation. Mutations in atxr5 and atxr6 also lead to transcriptional activation of repressed heterochromatic elements. Notably, H3K9me2 and DNA methylation are unaffected in double mutants. These results indicate that ATXR5 and ATXR6 form a new class of H3K27 methyltransferases and that H3K27me1 represents a previously uncharacterized pathway required for transcriptional repression in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Inativação Gênica , Heterocromatina/química , Histonas/metabolismo , Metiltransferases/metabolismo , Animais , Arabidopsis/anatomia & histologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Lisina/metabolismo , Metiltransferases/genética , MutaçãoRESUMO
Methylation of histone H3 lysine 9 (H3K9) is a hallmark of transcriptional silencing in many organisms. In Arabidopsis thaliana, dimethylation of H3K9 (H3K9m2) is important in the silencing of transposons and in the control of DNA methylation. We constructed a high-resolution genome-wide map of H3K9m2 methylation by using chromatin immunoprecipitation coupled with whole genome Roche Nimblegen microarrays (ChIP-chip). We observed a very high coincidence between H3K9m2 and CHG methylation (where H is either A,T or C) throughout the genome. The coding regions of genes that are associated exclusively with methylation in a CG context did not contain H3K9m2. In addition, we observed two distinct patterns of H3K9m2. Transposons and other repeat elements present in the euchromatic arms contained small islands of H3K9m2 present at relatively low levels. In contrast, pericentromeric/centromeric regions of Arabidopsis chromosomes contained long, rarely interrupted blocks of H3K9m2 present at much higher average levels than seen in the chromosome arms. These results suggest a complex interplay between H3K9m2 and different types of DNA methylation and suggest that distinct mechanisms control H3K9m2 in different compartments of the genome.
Assuntos
Arabidopsis/metabolismo , Genoma de Planta , Histonas/química , Lisina/química , Proteínas de Arabidopsis/metabolismo , Cromatina/química , Imunoprecipitação da Cromatina , Metilação de DNA , Elementos de DNA Transponíveis , Epigênese Genética , Genes de Plantas , Estudo de Associação Genômica Ampla , Heterocromatina/metabolismo , Metilação , RNA Interferente Pequeno/metabolismo , Transcrição GênicaRESUMO
Vernalization is the acceleration of flowering by prolonged cold that aligns the onset of reproductive development with spring conditions. A key step of vernalization in Arabidopsis is the epigenetic silencing of FLOWERING LOCUS C (FLC), which encodes a repressor of flowering. The vernalization-induced epigenetic silencing of FLC is associated with histone deacetylation and H3K27me2 and H3K9me2 methylation mediated by VRN/VIN proteins. We have analyzed whether different histone methyltransferases and the chromodomain protein LIKE HETEROCHROMATIN PROTEIN (LHP)1 might play a role in vernalization. No single loss-of-function mutation in the histone methyltransferases studied disrupted the vernalization response; however, lhp1 mutants revealed a role for LHP1 in maintaining epigenetic silencing of FLC. Like LHP1, VRN1 functions in both flowering-time control and vernalization. We explored the localization of VRN1 and found it to be associated generally with Arabidopsis chromosomes but not the heterochromatic chromocenters. This association did not depend on vernalization or VRN2 function and was maintained during mitosis but was lost in meiotic chromosomes, suggesting that VRN1 may contribute to chromatin silencing that is not meiotically stable.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Epistasia Genética , Inativação Gênica , Proteínas de Domínio MADS/genética , Arabidopsis/citologia , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Proteínas Cromossômicas não Histona/genética , Cromossomos/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Flores/genética , Flores/crescimento & desenvolvimento , Flores/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Genótipo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas de Domínio MADS/metabolismo , Meiose , Mutação/genética , Proteínas do Grupo Polycomb , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de TempoRESUMO
ARGONAUTE4 (AGO4) and RNA polymerase IV (Pol IV) are required for DNA methylation guided by 24 nucleotide small interfering RNAs (siRNAs) in Arabidopsis thaliana. Here we show that AGO4 localizes to nucleolus-associated bodies along with the Pol IV subunit NRPD1b; the small nuclear RNA (snRNA) binding protein SmD3; and two markers of Cajal bodies, trimethylguanosine-capped snRNAs and the U2 snRNA binding protein U2B''. AGO4 interacts with the C-terminal domain of NRPD1b, and AGO4 protein stability depends on upstream factors that synthesize siRNAs. AGO4 is also found, along with the DNA methyltransferase DRM2, throughout the nucleus at presumed DNA methylation target sites. Cajal bodies are conserved sites for the maturation of ribonucleoprotein complexes. Our results suggest a function for Cajal bodies as a center for the assembly of an AGO4/NRPD1b/siRNA complex, facilitating its function in RNA-directed gene silencing at target loci.