RESUMO
Nucleosome remodelers of the DDM1/Lsh family are required for DNA methylation of transposable elements, but the reason for this is unknown. How DDM1 interacts with other methylation pathways, such as small-RNA-directed DNA methylation (RdDM), which is thought to mediate plant asymmetric methylation through DRM enzymes, is also unclear. Here, we show that most asymmetric methylation is facilitated by DDM1 and mediated by the methyltransferase CMT2 separately from RdDM. We find that heterochromatic sequences preferentially require DDM1 for DNA methylation and that this preference depends on linker histone H1. RdDM is instead inhibited by heterochromatin and absolutely requires the nucleosome remodeler DRD1. Together, DDM1 and RdDM mediate nearly all transposon methylation and collaborate to repress transposition and regulate the methylation and expression of genes. Our results indicate that DDM1 provides DNA methyltransferases access to H1-containing heterochromatin to allow stable silencing of transposable elements in cooperation with the RdDM pathway.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Elementos de DNA Transponíveis , Proteínas de Ligação a DNA/metabolismo , Heterocromatina , Histonas/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Regulação da Expressão Gênica de Plantas , Nucleossomos/metabolismoRESUMO
Cytosine (DNA) methylation in plants regulates the expression of genes and transposons. While methylation in plant genomes occurs at CG, CHG, and CHH sequence contexts, the comparative roles of the individual methylation contexts remain elusive. Here, we present Physcomitrella patens as the second plant system, besides Arabidopsis thaliana, with viable mutants with an essentially complete loss of methylation in the CG and non-CG contexts. In contrast to A. thaliana, P. patens has more robust CHH methylation, similar CG and CHG methylation levels, and minimal cross-talk between CG and non-CG methylation, making it possible to study context-specific effects independently. Our data found CHH methylation to act in redundancy with symmetric methylation in silencing transposons and to regulate the expression of CG/CHG-depleted transposons. Specific elimination of CG methylation did not dysregulate transposons or genes. In contrast, exclusive removal of non-CG methylation massively up-regulated transposons and genes. In addition, comparing two exclusively but equally CG- or CHG-methylated genomes, we show that CHG methylation acts as a greater transcriptional regulator than CG methylation. These results disentangle the transcriptional roles of CG and non-CG, as well as symmetric and asymmetric methylation in a plant genome, and point to the crucial role of non-CG methylation in genome regulation.
Assuntos
Bryopsida/genética , Metilação de DNA/genética , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Mutação/genética , Elementos de DNA Transponíveis/genética , Epigenoma , Inativação Gênica , Modelos Genéticos , Regulação para Cima/genéticaRESUMO
MOTIVATION: The COP9 signalosome is a highly conserved multi-protein complex consisting of eight subunits, which influences key developmental pathways through its regulation of protein stability and transcription. In Arabidopsis thaliana, mutations in the COP9 signalosome exhibit a number of diverse pleiotropic phenotypes. Total or partial loss of COP9 signalosome function in Arabidopsis leads to misregulation of a number of genes involved in DNA methylation, suggesting that part of the pleiotropic phenotype is due to global effects on DNA methylation. RESULTS: We determined and analyzed the methylomes and transcriptomes of both partial- and total-loss-of-function Arabidopsis mutants of the COP9 signalosome. Our results support the hypothesis that the COP9 signalosome has a global genome-wide effect on methylation and that this effect is at least partially encoded in the DNA. Our analyses suggest that COP9 signalosome-dependent methylation is related to gene expression regulation in various ways. Differentially methylated regions tend to be closer in the 3D conformation of the genome to differentially expressed genes. These results suggest that the COP9 signalosome has a more comprehensive effect on gene expression than thought before, and this is partially related to regulation of methylation. The high level of COP9 signalosome conservation among eukaryotes may also suggest that COP9 signalosome regulates methylation not only in plants but also in other eukaryotes, including humans. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Arabidopsis , Arabidopsis/genética , Proteínas de Arabidopsis , Complexo do Signalossomo COP9 , Epigênese GenéticaRESUMO
Dedifferentiation, that is, the acquisition of stem cell-like state, commonly induced by stress (e.g., protoplasting), is characterized by open chromatin conformation, a chromatin state that could lead to activation of transposable elements (TEs). Here, we studied the activation of the Arabidopsis class II TE Tag1, in which two copies, situated close to each other (near genes) on chromosome 1 are found in Landsberg erecta (Ler) but not in Columbia (Col). We first transformed protoplasts with a construct in which a truncated Tag1 (ΔTag1 non-autonomous) blocks the expression of a reporter gene AtMBD5-GFP and found a relatively high ectopic excision of ΔTag1 accompanied by expression of AtMBD5-GFP in protoplasts derived from Ler compared to Col; further increase was observed in ddm1 (decrease in DNA methylation1) protoplasts (Ler background). Ectopic excision was associated with transcription of the endogenous Tag1 and changes in histone H3 methylation at the promoter region. Focusing on the endogenous Tag1 elements we found low level of excision in Ler protoplasts, which was slightly and strongly enhanced in ddm1 and cmt3 (chromomethylase3) protoplasts, respectively, concomitantly with reduction in Tag1 gene body (GB) CHG methylation and increased Tag1 transcription; strong activation of Tag1 was also observed in cmt3 leaves. Notably, in cmt3, but not in ddm1, Tag1 elements were excised out from their original sites and transposed elsewhere in the genome. Our results suggest that dedifferentiation is associated with Tag1 activation and that CMT3 rather than DDM1 plays a central role in restraining Tag1 activation via inducing GB CHG methylation.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Elementos de DNA Transponíveis , Proteínas de Ligação a DNA/genética , DNA-Citosina Metilases/genética , Regulação da Expressão Gênica de Plantas , Fatores de Transcrição/genética , Transposases/genética , Arabidopsis/citologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Desdiferenciação Celular , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , DNA-Citosina Metilases/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histonas/genética , Histonas/metabolismo , Células Vegetais/metabolismo , Folhas de Planta/citologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Protoplastos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Transformação Genética , Transposases/metabolismoRESUMO
Cytosine methylation at CG sites ((m)CG) plays critical roles in development, epigenetic inheritance, and genome stability in mammals and plants. In the dicot model plant Arabidopsis thaliana, methyltransferase 1 (MET1), a principal CG methylase, functions to maintain (m)CG during DNA replication, with its null mutation resulting in global hypomethylation and pleiotropic developmental defects. Null mutation of a critical CG methylase has not been characterized at a whole-genome level in other higher eukaryotes, leaving the generality of the Arabidopsis findings largely speculative. Rice is a model plant of monocots, to which many of our important crops belong. Here we have characterized a null mutant of OsMet1-2, the major CG methylase in rice. We found that seeds homozygous for OsMet1-2 gene mutation (OsMET1-2(-/-)), which directly segregated from normal heterozygote plants (OsMET1-2(+/-)), were seriously maldeveloped, and all germinated seedlings underwent swift necrotic death. Compared with wild type, genome-wide loss of (m)CG occurred in the mutant methylome, which was accompanied by a plethora of quantitative molecular phenotypes including dysregulated expression of diverse protein-coding genes, activation and repression of transposable elements, and altered small RNA profiles. Our results have revealed conservation but also distinct functional differences in CG methylases between rice and Arabidopsis.
Assuntos
Metilação de DNA/genética , Regulação da Expressão Gênica de Plantas , Genoma de Planta/genética , Metiltransferases/genética , Mutação/genética , Oryza/genética , Plântula/genética , Citosina/metabolismo , Elementos de DNA Transponíveis/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Genes de Plantas/genética , Fases de Leitura Aberta/genética , Oryza/enzimologia , Oryza/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Plântula/crescimento & desenvolvimentoRESUMO
Cytosine methylation silences transposable elements in plants, vertebrates, and fungi but also regulates gene expression. Plant methylation is catalyzed by three families of enzymes, each with a preferred sequence context: CG, CHG (H = A, C, or T), and CHH, with CHH methylation targeted by the RNAi pathway. Arabidopsis thaliana endosperm, a placenta-like tissue that nourishes the embryo, is globally hypomethylated in the CG context while retaining high non-CG methylation. Global methylation dynamics in seeds of cereal crops that provide the bulk of human nutrition remain unknown. Here, we show that rice endosperm DNA is hypomethylated in all sequence contexts. Non-CG methylation is reduced evenly across the genome, whereas CG hypomethylation is localized. CHH methylation of small transposable elements is increased in embryos, suggesting that endosperm demethylation enhances transposon silencing. Genes preferentially expressed in endosperm, including those coding for major storage proteins and starch synthesizing enzymes, are frequently hypomethylated in endosperm, indicating that DNA methylation is a crucial regulator of rice endosperm biogenesis. Our data show that genome-wide reshaping of seed DNA methylation is conserved among angiosperms and has a profound effect on gene expression in cereal crops.
Assuntos
Metilação de DNA , DNA de Plantas/genética , Oryza/genética , DNA Glicosilases/genética , DNA de Plantas/metabolismo , Endosperma/genética , Endosperma/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Oryza/metabolismo , FilogeniaRESUMO
The Snf2 chromatin remodeler, DECREASE IN DNA METHYLATION 1 (DDM1) facilitates DNA methylation. In flowering plants, DDM1 mediates methylation in heterochromatin, which is targeted primarily by MET1 and CMT methylases and is necessary for silencing transposons and for proper development. DNA methylation mechanisms evolved throughout plant evolution, whereas the role of DDM1 in early terrestrial plants remains elusive. Here, we studied the function of DDM1 in the moss, Physcomitrium (Physcomitrella) patens, which has robust DNA methylation that suppresses transposons and is mediated by a MET1, a CMT, and a DNMT3 methylases. To elucidate the role of DDM1 in P. patens, we have generated a knockout mutant and found DNA methylation to be strongly disrupted at any of its sequence contexts. Symmetric CG and CHG sequences were affected stronger than asymmetric CHH sites. Furthermore, despite their separate targeting mechanisms, CG (MET) and CHG (CMT) methylation were similarly depleted by about 75%. CHH (DNMT3) methylation was overall reduced by about 25%, with an evident hyper-methylation activity within lowly-methylated euchromatic transposon sequences. Despite the strong hypomethylation effect, only a minute number of transposons were transcriptionally activated in Ppddm1. Finally, Ppddm1 was found to develop normally throughout the plant life cycle. These results demonstrate that DNA methylation is strongly dependent on DDM1 in a non-flowering plant; that DDM1 is required for plant-DNMT3 (CHH) methylases, though to a lower extent than for MET1 and CMT enzymes; and that distinct and separate methylation pathways (e.g. MET1-CG and CMT-CHG), can be equally regulated by the chromatin and that DDM1 plays a role in it. Finally, our data suggest that the biological significance of DDM1 in terms of transposon regulation and plant development, is species dependent.
Assuntos
Proteínas de Arabidopsis , Bryopsida , Metilação de DNA , Bryopsida/metabolismo , Cromatina/metabolismo , Metiltransferases/genética , Heterocromatina/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Arabidopsis/genéticaRESUMO
The Arabidopsis MBD7 (AtMBD7) - a naturally occurring poly MBD protein - was previously found to be functional in binding methylated-CpG dinucleotides in vitro and localized to highly methylated chromocenters in vivo. Furthermore, AtMBD7 has significantly lower mobility within the nucleus conferred by cooperative activity of its three MBD motifs. Here we show that besides the MBD motifs, AtMBD7 possesses a strong chromatin binding domain located at its C-terminus designated sticky-C (StkC). Mutational analysis showed that a glutamic acid residue near the C-terminus is essential though not sufficient for the StkC function. Further analysis demonstrated that this motif can render nuclear proteins highly immobile both in plant and animal cells, without affecting their native subnuclear localization. Thus, the C-terminal, StkC motif plays an important role in fastening AtMBD7 to its chromosomal, CpG-methylated sites. It may be possible to utilize this motif for fastening nuclear proteins to their chromosomal sites both in plant and animal cells for research and gene therapy applications.
Assuntos
Proteínas de Arabidopsis/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Domínios e Motivos de Interação entre Proteínas/fisiologia , Substituição de Aminoácidos/fisiologia , Proteínas de Arabidopsis/genética , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Imunoprecipitação da Cromatina , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Difusão , Recuperação de Fluorescência Após Fotodegradação , Ácido Glutâmico/genética , Células HeLa , Humanos , Repressores Lac/genética , Repressores Lac/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/genética , Protoplastos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Transformação Genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Cytosine methylome data is commonly generated through next-generation sequencing, with analyses averaging methylation states of individual reads. We propose an alternative method of analysing single-read methylome data. Using this method, we identify patterns relating to the mechanism of two plant non-CG-methylating enzymes, CMT2 and DRM2. CMT2-methylated regions show higher stochasticity, while DRM2-methylated regions have higher variation among cells. Based on these patterns, we develop a classifier that predicts enzyme activity in different species and tissues. To facilitate further single-read analyses, we develop a genome browser, SRBrowse, optimised for visualising and analysing sequencing data at single-read resolution.
Assuntos
Metilação de DNA , Epigenoma , Genômica/métodos , Metiltransferases/metabolismo , Software , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , HumanosRESUMO
The effect of DNA methylation on various aspects of plant cellular and developmental processes has been well documented over the past 35 years. However, the underlying molecular mechanism interpreting the methylation signal has only recently been explored with the isolation and characterization of the Arabidopsis methyl-CpG-binding domain (MBD) proteins. In this review, we highlight recent advances and present new models concerning Arabidopsis MBD proteins and their possible role in controlling chromatin structure mediated by CpG methylation.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/fisiologia , Modelos Genéticos , Motivos de Aminoácidos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/química , Sítios de Ligação , Montagem e Desmontagem da Cromatina/fisiologia , Sequência Conservada , Proteínas de Ligação a DNA/química , Inativação Gênica/fisiologia , Genes de RNArRESUMO
To properly regulate the genome, cytosine methylation is established by animal DNA methyltransferase 3 s (DNMT3s). While altered DNMT3 homologs, Domains rearranged methyltransferases (DRMs), have been shown to establish methylation via the RNA directed DNA methylation (RdDM) pathway, the role of true-plant DNMT3 orthologs remains elusive. Here, we profile de novo (RPS transgene) and genomic methylation in the basal plant, Physcomitrella patens, mutated in each of its PpDNMTs. We show that PpDNMT3b mediates CG and CHH de novo methylation, independently of PpDRMs. Complementary de novo CHG methylation is specifically mediated by the CHROMOMETHYLASE, PpCMT. Intragenomically, PpDNMT3b functions preferentially within heterochromatin and is affected by PpCMT. In comparison, PpDRMs target active-euchromatic transposons. Overall, our data resolve how DNA methylation in plants can be established in heterochromatin independently of RdDM; suggest that DRMs have emerged to target euchromatin; and link DNMT3 loss in angiosperms to the initiation of heterochromatic CHH methylation by CMT2.
Assuntos
Bryopsida/fisiologia , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/fisiologia , Heterocromatina/genética , Proteínas de Plantas/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , Evolução Molecular , Redes e Vias Metabólicas/fisiologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Seleção Genética/fisiologiaRESUMO
BACKGROUND: DNA methylation of active genes, also known as gene body methylation, is found in many animal and plant genomes. Despite this, the transcriptional and developmental role of such methylation remains poorly understood. Here, we explore the dynamic range of DNA methylation in honey bee, a model organism for gene body methylation. RESULTS: Our data show that CG methylation in gene bodies globally fluctuates during honey bee development. However, these changes cause no gene expression alterations. Intriguingly, despite the global alterations, tissue-specific CG methylation patterns of complete genes or exons are rare, implying robust maintenance of genic methylation during development. Additionally, we show that CG methylation maintenance fluctuates in somatic cells, while reaching maximum fidelity in sperm cells. Finally, unlike universally present CG methylation, we discovered non-CG methylation specifically in bee heads that resembles such methylation in mammalian brain tissue. CONCLUSIONS: Based on these results, we propose that gene body CG methylation can oscillate during development if it is kept to a level adequate to preserve function. Additionally, our data suggest that heightened non-CG methylation is a conserved regulator of animal nervous systems.
Assuntos
Abelhas/genética , Metilação de DNA , Animais , Abelhas/crescimento & desenvolvimento , Análise por Conglomerados , DNA (Citosina-5-)-Metiltransferases/genética , Éxons , Regulação da Expressão Gênica , Células Germinativas , Proteínas de Insetos/genética , Íntrons , Larva/genética , Masculino , Sistema Nervoso/metabolismo , Análise de Componente Principal , Splicing de RNA , Espermatozoides/citologia , Espermatozoides/metabolismoRESUMO
The original version of this Article contained an error in Fig. 5, in which the evolutionary origin of DRM2 was incorrectly placed prior to the divergence between gymnosperms and angiosperms . The correct evolutionary origin of DRM2 should be in angiosperms. In addition, in the "Percent methylation change" section of the Methods, Equation 1 was incorrect. This has been corrected in both the PDF and HTML versions of the Article.An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Cytosine methylation is the most prevalent epigenetic modification of plant nuclear DNA, which occurs in symmetrical CpG or CpNpG as well as in non-symmetrical contexts. Intensive studies demonstrated the central role played by cytosine methylation in genome organization, gene expression and in plant growth and development. However, the way by which the methyl group is interpreted into a functional state has only recently begun to be explored with the isolation and characterization of methylated DNA binding proteins capable of binding 5-methylcytosine. These proteins belong to an evolutionary conserved protein family initially described in animals termed methyl-CpG-binding domain (MBD) proteins. Here, we highlight recent advances and present new prospects concerning plant MBD proteins and their possible role in controlling chromatin structure mediated by CpG methylation.
Assuntos
DNA de Plantas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Plantas/metabolismo , Plantas/genética , Plantas/metabolismo , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Ilhas de CpG , Metilação de DNA , DNA de Plantas/química , DNA de Plantas/genética , Proteínas de Ligação a DNA/genética , Humanos , Mamíferos , Modelos Biológicos , Proteínas de Plantas/genéticaRESUMO
BACKGROUND: The Chromomethylase 1 (CMT1) has long been considered a nonessential gene because, in certain Arabidopsis ecotypes, the CMT1 gene is disrupted by the Evelknievel (EK) retroelement, inserted within exon 13, or contains frameshift mutations, resulting in a truncated, non-functional protein. In contrast to other transposable elements, no transcriptional activation of EK was observed under stress conditions (e.g., protoplasting). RESULTS: We wanted to explore the regulatory pathway responsible for EK silencing in the Ler ecotype and its effect on CMT1 transcription. Methylome databases confirmed that EK retroelement is heavily methylated and methylation is extended toward CMT1 downstream region. Strong transcriptional activation of EK accompanied by significant reduction in non-CG methylation was found in cmt3 and kyp2, but not in ddm1 or RdDM mutants. EK activation in cmt3 and kyp2 did not interfere with upstream CMT1 expression but abolish transcription through the EK. We identified, in wild-type Ler, three spliced variants in which the entire EK is spliced out; one variant (25% of splicing incidents) facilitates proper reconstitution of an intact CMT1 mRNA. We could recover very low amount of the full-length CMT1 mRNA from WT Ler and Col, but not from cmt3 mutant. CONCLUSIONS: Our findings highlight CMT3-SUVH4/KYP as the major pathway silencing the intragenic EK via inducing non-CG methylation. Furthermore, retroelement insertion within exons (e.g., CMT1) may not lead to a complete abolishment of the gene product when the element is kept silent. Rather the element can be spliced out to bring about reconstruction of an intact, functional mRNA and possibly retrieval of an active protein.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , DNA (Citosina-5-)-Metiltransferases/genética , DNA-Citosina Metilases/genética , Histona-Lisina N-Metiltransferase/genética , Retroelementos , Proteínas de Arabidopsis/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/genética , DNA-Citosina Metilases/metabolismo , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Genoma de Planta , Histona-Lisina N-Metiltransferase/metabolismo , Mutação , RNA Mensageiro/genética , RNA de Plantas/genética , Fatores de Transcrição/genéticaRESUMO
Plants exhibit high capacity to regenerate in three alternative pathways: tissue repair, somatic embryogenesis and de novo organogenesis. For most plants, de novo organ initiation can be easily achieved in tissue culture by exposing explants to auxin and/or cytokinin, yet the competence to regenerate varies among species and within tissues from the same plant. In Arabidopsis, root explants incubated directly on cytokinin-rich shoot inducing medium (SIM-direct), are incapable of regenerating shoots, and a pre-incubation step on auxin-rich callus inducing medium (CIM) is required to acquire competency to regenerate on the SIM. However the mechanism underlying competency acquisition still remains elusive. Here we show that the chromomethylase 3 (cmt3) mutant which exhibits significant reduction in CHG methylation, shows high capacity to regenerate on SIM-direct and that regeneration occurs via direct organogenesis. In WT, WUSCHEL (WUS) promoter, an essential gene for shoot formation, is highly methylated, and its expression on SIM requires pre-incubation on CIM. However, in cmt3, WUS expression induced by SIM-direct. We propose that pre-incubation on CIM is required for the re-activation of cell division. Following the transfer of roots to SIM, the intensive cell division activity continues, and in the presence of cytokinin leads to a dilution in DNA methylation that allows certain genes required for shoot regeneration to respond to SIM, thereby advancing shoot formation.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Metilação de DNA/genética , Raízes de Plantas/fisiologia , Brotos de Planta/fisiologia , Regeneração , Arabidopsis/efeitos dos fármacos , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ciclo Celular/efeitos dos fármacos , Meios de Cultura/farmacologia , Metilação de DNA/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Estudos de Associação Genética , Mutação/genética , Organogênese/efeitos dos fármacos , Organogênese/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/ultraestrutura , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/crescimento & desenvolvimento , Regiões Promotoras Genéticas/genética , Regeneração/efeitos dos fármacosRESUMO
The Arabidopsis thaliana central cell, the companion cell of the egg, undergoes DNA demethylation before fertilization, but the targeting preferences, mechanism, and biological significance of this process remain unclear. Here, we show that active DNA demethylation mediated by the DEMETER DNA glycosylase accounts for all of the demethylation in the central cell and preferentially targets small, AT-rich, and nucleosome-depleted euchromatic transposable elements. The vegetative cell, the companion cell of sperm, also undergoes DEMETER-dependent demethylation of similar sequences, and lack of DEMETER in vegetative cells causes reduced small RNA-directed DNA methylation of transposons in sperm. Our results demonstrate that demethylation in companion cells reinforces transposon methylation in plant gametes and likely contributes to stable silencing of transposable elements across generations.
Assuntos
Arabidopsis/genética , Metilação de DNA , Elementos de DNA Transponíveis/genética , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Células Germinativas Vegetais/metabolismo , Arabidopsis/citologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , DNA de Plantas/metabolismo , Endosperma/citologia , Endosperma/genética , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/metabolismo , RNA de Plantas/metabolismo , Transativadores/genética , Transativadores/metabolismoRESUMO
Cytosine methylation is an ancient process with conserved enzymology but diverse biological functions that include defense against transposable elements and regulation of gene expression. Here we will discuss the evolution and biological significance of eukaryotic DNA methylation, the likely drivers of that evolution, and major remaining mysteries.
Assuntos
Metilação de DNA , Evolução Molecular , Animais , FilogeniaRESUMO
Eukaryotic cytosine methylation represses transcription but also occurs in the bodies of active genes, and the extent of methylation biology conservation is unclear. We quantified DNA methylation in 17 eukaryotic genomes and found that gene body methylation is conserved between plants and animals, whereas selective methylation of transposons is not. We show that methylation of plant transposons in the CHG context extends to green algae and that exclusion of histone H2A.Z from methylated DNA is conserved between plants and animals, and we present evidence for RNA-directed DNA methylation of fungal genes. Our data demonstrate that extant DNA methylation systems are mosaics of conserved and derived features, and indicate that gene body methylation is an ancient property of eukaryotic genomes.
Assuntos
Evolução Biológica , Metilação de DNA , Eucariotos/genética , Genoma Fúngico , Genoma de Planta , Genoma , Animais , Clorófitas/genética , Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases/metabolismo , Elementos de DNA Transponíveis , Eucariotos/metabolismo , Fungos/genética , Fungos/metabolismo , Regulação da Expressão Gênica , Histonas/metabolismo , Filogenia , Plantas/genética , Plantas/metabolismo , Regiões Promotoras Genéticas , Seleção Genética , Transcrição GênicaRESUMO
Parent-of-origin-specific (imprinted) gene expression is regulated in Arabidopsis thaliana endosperm by cytosine demethylation of the maternal genome mediated by the DNA glycosylase DEMETER, but the extent of the methylation changes is not known. Here, we show that virtually the entire endosperm genome is demethylated, coupled with extensive local non-CG hypermethylation of small interfering RNA-targeted sequences. Mutation of DEMETER partially restores endosperm CG methylation to levels found in other tissues, indicating that CG demethylation is specific to maternal sequences. Endosperm demethylation is accompanied by CHH hypermethylation of embryo transposable elements. Our findings demonstrate extensive reconfiguration of the endosperm methylation landscape that likely reinforces transposon silencing in the embryo.