RESUMO
MAIN CONCLUSION: In ddm1 mutants, the DNA methylation is primarily affected in the heterochromatic region of the chromosomes, which is associated with the segregation distortion of SNPs in the F2 progenies. Segregation distortion (SD) is common in most genetic mapping experiments and a valuable resource to determine how gene loci induce deviation. Meiotic DNA crossing over and SD are under the control of several types of epigenetic modifications. DNA methylation is an important regulatory epigenetic modification that is inherited across generations. In the present study, we investigated the relationship between SD and DNA methylation. The ecotypes Col-0/C24 and chromatin remodeler mutants ddm1-10/Col and ddm1-15/C24 were reciprocally crossed to obtain F2 generations. A total of 300 plants for each reciprocally crossed plant in the F2 generations were subjected to next-generation sequencing to detect the single-nucleotide polymorphisms (SNPs) as DNA markers. All SNPs were analyzed using the Chi-square test method to determine their segregation ratio in F2 generations. Through the segregation ratio, whole-genome SNPs were classified into 16 classes. In class 10, the SNPs in the reciprocal crosses of wild type showed the expected Mendelian ratio of 1:2:1, while those in the reciprocal crosses of ddm1 mutants showed distortion. In contrast, all SNPs in class 16 displayed a normal 1:2:1 ratio, and class 1 showed SD, regardless of wild type or mutants, as assessed using CAPS (cleaved amplified polymorphic sequences) marker analysis to confirm the next-generation sequencing. In ddm1 mutants, the DNA methylation is highly reduced throughout the whole genome and more significantly in the heterochromatic regions of chromosomes. Our results showed that the ddm1 mutants exhibit low levels of DNA methylation, which facilitates the SD of SNPs primarily located in the heterochromatic region of chromosomes by reducing the heterozygous ratio. The present study will provide a strong base for future research focusing on the impact of DNA methylation on trait segregation and plant evolution.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cromatina , Metilação de DNA/genética , Proteínas de Ligação a DNA/metabolismo , Mutação , Fatores de Transcrição/genéticaRESUMO
Trees are long-lived organisms that continuously adapt to their environments, a process in which epigenetic mechanisms are likely to play a key role. Via downregulation of the chromatin remodeler DECREASED IN DNA METHYLATION 1 (DDM1) in poplar (Populus tremula × Populus alba) RNAi lines, we examined how DNA methylation coordinates genomic and physiological responses to moderate water deficit. We compared the growth and drought response of two RNAi-ddm1 lines to wild-type (WT) trees under well-watered and water deficit/rewatering conditions, and analyzed their methylomes, transcriptomes, mobilomes and phytohormone contents in the shoot apical meristem. The RNAi-ddm1 lines were more tolerant to drought-induced cavitation but did not differ in height or stem diameter growth. About 5000 differentially methylated regions were consistently detected in both RNAi-ddm1 lines, colocalizing with 910 genes and 89 active transposable elements. Under water deficit conditions, 136 differentially expressed genes were found, including many involved in phytohormone pathways; changes in phytohormone concentrations were also detected. Finally, the combination of hypomethylation and drought led to the mobility of two transposable elements. Our findings suggest major roles for DNA methylation in regulation of genes involved in hormone-related stress responses, and the maintenance of genome integrity through repression of transposable elements.
Assuntos
Populus , Metilação de DNA/genética , Secas , Regulação da Expressão Gênica de Plantas , Meristema , Populus/genética , Interferência de RNARESUMO
Hybrid vigor or heterosis refers to the superior performance of F1 hybrid plants over their parents. Heterosis is particularly important in the production systems of major crops. Recent studies have suggested that epigenetic regulation such as DNA methylation is involved in heterosis, but the molecular mechanism of heterosis is still unclear. To address the epigenetic contribution to heterosis in Arabidopsis thaliana, we used mutant genes that have roles in DNA methylation. Hybrids between C24 and Columbia-0 (Col) without RNA polymerase IV (Pol IV) or methyltransferase I (MET1) function did not reduce the level of biomass heterosis (as evaluated by rosette diameter). Hybrids with a mutation in decrease in dna methylation 1 (ddm1) showed a decreased heterosis level. Vegetative heterosis in the ddm1 mutant hybrid was reduced but not eliminated; a complete reduction could result if there was a change in methylation at all loci critical for generating the level of heterosis, whereas if only a proportion of the loci have methylation changes there may only be a partial reduction in heterosis.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Metilação de DNA , Proteínas de Ligação a DNA/genética , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Fatores de Transcrição/genética , Arabidopsis/metabolismo , Biomassa , Cruzamentos Genéticos , DNA (Citosina-5-)-Metiltransferases/deficiência , DNA (Citosina-5-)-Metiltransferases/genética , Proteínas de Ligação a DNA/deficiência , RNA Polimerases Dirigidas por DNA/deficiência , RNA Polimerases Dirigidas por DNA/genética , Vigor Híbrido , Mutação , Fatores de Transcrição/deficiênciaRESUMO
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
Clubroot caused by the protist Plasmodiophora brassicae is a major disease affecting cultivated Brassicaceae. Using a combination of quantitative trait locus (QTL) fine mapping, CRISPR-Cas9 validation, and extensive analyses of DNA sequence and methylation patterns, we revealed that the two adjacent neighboring NLR (nucleotide-binding and leucine-rich repeat) genes AT5G47260 and AT5G47280 cooperate in controlling broad-spectrum quantitative partial resistance to the root pathogen P. brassicae in Arabidopsis and that they are epigenetically regulated. The variation in DNA methylation is not associated with any nucleotide variation or any transposable element presence/absence variants and is stably inherited. Variations in DNA methylation at the Pb-At5.2 QTL are widespread across Arabidopsis accessions and correlate negatively with variations in expression of the two genes. Our study demonstrates that natural, stable, and transgenerationally inherited epigenetic variations can play an important role in shaping resistance to plant pathogens by modulating the expression of immune receptors.
Assuntos
Arabidopsis , Resistência à Doença , Doenças das Plantas , Arabidopsis/genética , Arabidopsis/imunologia , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Doenças das Plantas/parasitologia , Resistência à Doença/genética , Proteínas NLR/genética , Proteínas NLR/metabolismo , Metilação de DNA , Plasmodioforídeos/fisiologia , Locos de Características Quantitativas/genética , Proteínas de Arabidopsis/genética , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , AlelosRESUMO
BACKGROUND: Plants memorize previous pathogen attacks and are "primed" to produce a faster and stronger defense response, which is critical for defense against pathogens. In plants, cytosines in transposons and gene bodies are reported to be frequently methylated. Demethylation of transposons can affect disease resistance by regulating the transcription of nearby genes during defense response, but the role of gene body methylation (GBM) in defense responses remains unclear. RESULTS: Here, we find that loss of the chromatin remodeler decrease in DNA methylation 1 (ddm1) synergistically enhances resistance to a biotrophic pathogen under mild chemical priming. DDM1 mediates gene body methylation at a subset of stress-responsive genes with distinct chromatin properties from conventional gene body methylated genes. Decreased gene body methylation in loss of ddm1 mutant is associated with hyperactivation of these gene body methylated genes. Knockout of glyoxysomal protein kinase 1 (gpk1), a hypomethylated gene in ddm1 loss-of-function mutant, impairs priming of defense response to pathogen infection in Arabidopsis. We also find that DDM1-mediated gene body methylation is prone to epigenetic variation among natural Arabidopsis populations, and GPK1 expression is hyperactivated in natural variants with demethylated GPK1. CONCLUSIONS: Based on our collective results, we propose that DDM1-mediated GBM provides a possible regulatory axis for plants to modulate the inducibility of the immune response.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Metilação de DNA , Fatores de Transcrição/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cromatina/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
How different intrinsic sequence variations and regulatory modifications of histones combine in nucleosomes remain unclear. To test the importance of histone variants in the organization of chromatin we investigated how histone variants and histone modifications assemble in the Arabidopsis thaliana genome. We showed that a limited number of chromatin states divide euchromatin and heterochromatin into several subdomains. We found that histone variants are as significant as histone modifications in determining the composition of chromatin states. Particularly strong associations were observed between H2A variants and specific combinations of histone modifications. To study the role of H2A variants in organizing chromatin states we determined the role of the chromatin remodeler DECREASED IN DNA METHYLATION (DDM1) in the organization of chromatin states. We showed that the loss of DDM1 prevented the exchange of the histone variant H2A.Z to H2A.W in constitutive heterochromatin, resulting in significant effects on the definition and distribution of chromatin states in and outside of constitutive heterochromatin. We thus propose that dynamic exchanges of histone variants control the organization of histone modifications into chromatin states, acting as molecular landmarks.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Cromatina/genética , Histonas/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Heterocromatina/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Nucleossomos/genéticaRESUMO
Cytosine methylation within CG dinucleotides (mCG) can be epigenetically inherited over many generations. Such inheritance is thought to be mediated by a semiconservative mechanism that produces binary present/absent methylation patterns. However, we show here that, in Arabidopsis thaliana h1ddm1 mutants, intermediate heterochromatic mCG is stably inherited across many generations and is quantitatively associated with transposon expression. We develop a mathematical model that estimates the rates of semiconservative maintenance failure and de novo methylation at each transposon, demonstrating that mCG can be stably inherited at any level via a dynamic balance of these activities. We find that DRM2-the core methyltransferase of the RNA-directed DNA methylation pathway-catalyzes most of the heterochromatic de novo mCG, with de novo rates orders of magnitude higher than previously thought, whereas chromomethylases make smaller contributions. Our results demonstrate that stable epigenetic inheritance of mCG in plant heterochromatin is enabled by extensive de novo methylation.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Histonas/metabolismo , Metilação de DNA/genética , Epigênese Genética , Regulação da Expressão Gênica de PlantasRESUMO
Epigenetic modifications are indispensable for regulating gene bodies and TE silencing. DECREASE IN DNA METHYLATION 1 (DDM1) is a chromatin remodeller involved in histone modifications and DNA methylation. Apart from maintaining the epigenome, DDM1 also maintains key plant traits such as flowering time and heterosis. The role of DDM1 in epigenetic regulation is best characterised in plants, especially arabidopsis, rice, maize and tomato. The epigenetic changes induced by DDM1 establish the stable inheritance of many plant traits for at least eight generations, yet DDM1 does not methylate protein-coding genes. The DDM1 TE silencing mechanism is distinct and has evolved independently of other silencing pathways. Unlike the RNA-directed DNA Methylation (RdDM) pathway, DDM1 does not depend on siRNAs to enforce the heterochromatic state of TEs. Here, we review DDM1 TE silencing activity in the RdDM and non-RdDM contexts. The DDM1 TE silencing machinery is strongly associated with the histone linker H1 and histone H2A.W. While the linker histone H1 excludes the RdDM factors from methylating the heterochromatin, the histone H2A.W variant prevents TE mobility. The DDM1-H2A.W strategy alone silences nearly all the mobile TEs in the arabidopsis genome. Thus, the DDM1-directed TE silencing essentially preserves heterochromatic features and abolishes mobile threats to genome stability.
RESUMO
RNA granules (RGs) are membraneless intracellular compartments that play important roles in the post-transcriptional control of gene expression. Stress granules (SGs) are a type of RGs that form under environmental challenges and/or internal cellular stresses. Stress treatments lead to strong mRNAs translational inhibition and storage in SGs until the normal growth conditions are restored. Intriguingly, we recently showed that plant stress granules are associated with siRNA bodies, where the RDR6-mediated and transposon-derived siRNA biogenesis occurs ( Kim et al., 2021 ). This protocol provides a technical workflow for the enrichment of cytoplasmic RGs from Arabidopsis seedlings. We used the DNA methylation-deficient ddm1 mutant in our study, but the method can be applied to any other plant samples with strong RG formation. The resulting RG fractions can be further tested for either RNAs or proteins using RNA-seq and mass spectrometry-based proteomics.
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Long-read data is a great tool to discover new active transposable elements (TEs). However, no ready-to-use tools were available to gather this information from low coverage ONT datasets. Here, we developed a novel pipeline, nanotei, that allows detection of TE-contained structural variants, including individual TE transpositions. We exploited this pipeline to identify TE insertion in the Arabidopsis thaliana genome. Using nanotei, we identified tens of TE copies, including ones for the well-characterized ONSEN retrotransposon family that were hidden in genome assembly gaps. The results demonstrate that some TEs are inaccessible for analysis with the current A. thaliana (TAIR10.1) genome assembly. We further explored the mobilome of the ddm1 mutant with elevated TE activity. Nanotei captured all TEs previously known to be active in ddm1 and also identified transposition of non-autonomous TEs. Of them, one non-autonomous TE derived from (AT5TE33540) belongs to TR-GAG retrotransposons with a single open reading frame (ORF) encoding the GAG protein. These results provide the first direct evidence that TR-GAGs and other non-autonomous LTR retrotransposons can transpose in the plant genome, albeit in the absence of most of the encoded proteins. In summary, nanotei is a useful tool to detect active TEs and their insertions in plant genomes using low-coverage data from Nanopore genome sequencing.
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eIF4A is a DEAD box containing RNA helicase that plays crucial roles in regulating translation initiation, growth and abiotic stress tolerance in plants. It also functions as an ATP-dependent RNA binding protein to curb granule formation by limiting RNA-RNA interactions that promote RNA condensation and formation of ribonucleoprotein particles in vivo. Helicase activity of eIF4A is known to be dictated by its binding partners. Proteins interacting with eIF4A have been identified across land plants. In monocots a close link between eIF4A regulated processes and DNA methylation in epigenetic regulation of plant development is inferred from interaction between OseIF4A and the de novo methyltransferase OsDRM2 and loss-of-function studies of these genes in Oryza sativa and Brachypodium distachyon. In the moss Physcomitrella patens, eIF4A1 encoded by Pp3c6_1080V3.1 interacts with the heterogeneous nuclear ribonucleoprotein (hnRNP) PpLIF2L1, homolog of which in Arabidopsis regulates transcription of stress-responsive genes. In this study, using different protein-protein interaction methods, targeted gene knockout strategy and quantitative expression analysis we show genetic interaction between PpeIF4A1 and the putative nucleosome remodeler protein PpDDM1 and between PpDDM1 and PpLIF2L1 in vivo. Stress-induced co-expression of PpeIF4A1, PpDDM1 and PpLIF2L1, their roles in salt stress tolerance and differences in subnuclear distribution of PpLIF2L1 in ppeif4a1 cells in comparison to wild type suggest existence of a regulatory network comprising of RNA helicases, chromatin remodelling proteins and hnRNP active in stress-responsive biological processes in P. patens.
Assuntos
Adenosina Trifosfatases/metabolismo , Bryopsida/metabolismo , Montagem e Desmontagem da Cromatina , Fator de Iniciação 4A em Eucariotos/metabolismo , Fatores de Transcrição/metabolismo , Metilação de DNA , Ligação ProteicaRESUMO
Meiotic recombination is the main driver of genetic diversity in wheat breeding. The rate and location of crossover (CO) events are regulated by genetic and epigenetic factors. In wheat, most COs occur in subtelomeric regions but are rare in centromeric and pericentric areas. The aim of this work was to increase COs in both "hot" and "cold" chromosomal locations. We used Virus-Induced gene Silencing (VIGS) to downregulate the expression of recombination-suppressing genes XRCC2 and FANCM and of epigenetic maintenance genes MET1 and DDM1 during meiosis. VIGS suppresses genes in a dominant, transient and non-transgenic manner, which is convenient in wheat, a hard-to-transform polyploid. F1 hybrids of a cross between two tetraploid lines whose genome was fully sequenced (wild emmer and durum wheat), were infected with a VIGS vector â¼ 2 weeks before meiosis. Recombination was measured in F2 seedlings derived from F1-infected plants and non-infected controls. We found significant up and down-regulation of CO rates along subtelomeric regions as a result of silencing either MET1, DDM1 or XRCC2 during meiosis. In addition, we found up to 93% increase in COs in XRCC2-VIGS treatment in the pericentric regions of some chromosomes. Silencing FANCM showed no effect on CO. Overall, we show that CO distribution was affected by VIGS treatments rather than the total number of COs which did not change. We conclude that transient silencing of specific genes during meiosis can be used as a simple, fast and non-transgenic strategy to improve breeding abilities in specific chromosomal regions.
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The nucleosome remodeling protein decrease in DNA methylation 1 (DDM1)/Lsh maintains normal levels of DNA methylation. Direct interaction between Lsh and DNA methyltransferase 1 (Dnmt1) and their localization to heterochromatin in the presence of heterochromatin protein-1α (HP1α) is a mechanism by which the concentration of DNMTs is increased at heterochromatin, and chromosome structures are stabilized in metazoans. In plants, however, it is unclear how DDM1 cooperates with methyltransferases and like heterochromatin protein 1 (LHP1). In this study, we provide evidence for a novel interaction between moss DDM1 (PpDDM1) and the chromomethylase PpCMT, that has not been reported in any plant, and between PpDDM1 and PpLHP1, that has not been reported before in any organism. Our protein-protein interaction studies may provide mechanistic insight into heterochromatin regulation.
Assuntos
Bryopsida/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Proteínas Cromossômicas não Histona/química , Ligação Proteica , Domínios ProteicosRESUMO
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
OBJECTIVES: It is a common practice in Arabidopsis to transfer a mutation generated in one genetic background to other genetic background via crossing. However, the drawback of this methodology is unavoidable presence of genomic fragments from the donor parent being often replacing desirable genomic fragments of the recurrent parent. Here, we highlighted problem of Arabidopsis mutants being recombinant introgression lines that can lead to unreliable and misinterpreted results. RESULTS: We studied the regulation of low copy number transposable elements Tag1 and Evelknievel (EK), located at the end of the bottom arm of chromosome 1 and both are present in the Arabidopsis Landsberg erecta (Ler) but not in Columbia (Col) ecotype. Using various epigenetic mutants (cmt3, ddm1, kyp2, ago4, rdr2 hen1 etc.), we found that certain mutants in the Ler background are deficient of Tag1 or EK or both and represent recombinant introgression lines whereby chromosomal regions from Col have been recombined into the Ler genome. Our data support a recent proposal calling for formulating standards for authentication of plant lines that are used in plant research. Most important is to verify that a given trait or genomic locus under study is correctly identified, particularly when using mutants generated by crossing.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Ecótipo , Arabidopsis/classificaçãoRESUMO
Cytosine methylation regulates essential genome functions across eukaryotes, but the fundamental question of whether nucleosomal or naked DNA is the preferred substrate of plant and animal methyltransferases remains unresolved. Here, we show that genetic inactivation of a single DDM1/Lsh family nucleosome remodeler biases methylation toward inter-nucleosomal linker DNA in Arabidopsis thaliana and mouse. We find that DDM1 enables methylation of DNA bound to the nucleosome, suggesting that nucleosome-free DNA is the preferred substrate of eukaryotic methyltransferases in vivo. Furthermore, we show that simultaneous mutation of DDM1 and linker histone H1 in Arabidopsis reproduces the strong linker-specific methylation patterns of species that diverged from flowering plants and animals over a billion years ago. Our results indicate that in the absence of remodeling, nucleosomes are strong barriers to DNA methyltransferases. Linker-specific methylation can evolve simply by breaking the connection between nucleosome remodeling and DNA methylation.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , DNA Helicases/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Nucleossomos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Arabidopsis/genética , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Camundongos , Fatores de Transcrição/genéticaRESUMO
In plants, hybrid vigor is influenced by genetic and epigenetic mechanisms; however, the molecular pathways are poorly understood. We investigated the potential contributions of epigenetic regulators to heterosis in Arabidposis and found that the chromatin remodeler DECREASED DNA METHYLATION 1 (DDM1) affects early seedling growth heterosis in Col/C24 hybrids. ddm1 mutants showed impaired heterosis and increased expression of non-additively expressed genes related to salicylic acid metabolism. Interestingly, our data suggest that salicylic acid is a hormetic regulator of seedling growth heterosis, and that hybrid vigor arises from crosses that produce optimal salicylic acid levels. Although DNA methylation failed to correlate with differential non-additively expressed gene expression, we uncovered DDM1 as an epigenetic link between salicylic acid metabolism and heterosis, and propose that the endogenous salicylic acid levels of parental plants can be used to predict the heterotic outcome. Salicylic acid protects plants from pathogens and abiotic stress. Thus, our findings suggest that stress-induced hormesis, which has been associated with increased longevity in other organisms, may underlie specific hybrid vigor traits.
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The strict and efficient transcription termination is required to prevent production of aberrant read-through transcripts that may cause transcriptional interference at neighboring genes. However, the exact regulatory mechanism remains poorly understood. Through a genetic screening of a LUCIFERASE (LUC) reporter system, we found that Arabidopsis nucleosome remodeler DECREASE IN DNA METHYLATION1 (DDM1) is a key component of this regulatory machinery and plays an important role in transcription termination, thus limiting transcriptional read-through (TRT). By whole-genome strand-specific RNA sequencing, we identified and confirmed 43 endogenous TRTs between genes, transposable elements (TEs), or genes and TEs in the ddm1-10 mutant, which mainly occurred at heterochromatin regions. The DNA methylation analysis of these TRT regions revealed that TRT occurred frequently at the intergenic regions with a higher methylation level in wild type comparing to the regions where TRT did not occur. Our results suggest that the level of intergenic DNA methylation may involve in preventing aberrant gene TRT or producing new gene during evolution.
Assuntos
Proteínas de Arabidopsis/metabolismo , Montagem e Desmontagem da Cromatina , Metilação de DNA , DNA Intergênico/genética , Proteínas de Ligação a DNA/metabolismo , Nucleossomos/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/genética , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Mutação , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Chromatin modifications such as DNA methylation are targeted to transposable elements by small RNAs in a process termed RNA-directed DNA methylation (RdDM). In plants, canonical RdDM functions through RNA polymerase IV to reinforce pre-existing transposable element silencing. Recent investigations have identified a "non-canonical" form of RdDM dependent on RNA polymerase II expression to initiate and re-establish silencing of active transposable elements. This expression-dependent RdDM mechanism functions through RNAi degradation of transposable element mRNAs into small RNAs guided by the RNA-dependent RNA polymerase 6 (RDR6) protein and is therefore referred to as RDR6-RdDM. RESULTS: We performed whole-genome MethylC-seq in 20 mutants that distinguish RdDM mechanisms when transposable elements are either transcriptionally silent or active. We identified a new mechanism of expression-dependent RdDM, which functions through DICER-LIKE3 (DCL3) but bypasses the requirement of both RNA polymerase IV and RDR6 (termed DCL3-RdDM). We found that RNA polymerase II expression-dependent forms of RdDM function on over 20 % of transcribed transposable elements, including the majority of full-length elements with all of the domains required for autonomous transposition. Lastly, we find that RDR6-RdDM preferentially targets long transposable elements due to the specificity of primary small RNAs to cleave full-length mRNAs. CONCLUSIONS: Expression-dependent forms of RdDM function to critically target DNA methylation to full-length and transcriptionally active transposable elements, suggesting that these pathways are key to suppressing mobilization. This targeting specificity is initiated on the mRNA cleavage-level, yet manifested as chromatin-level silencing that in plants is epigenetically inherited from generation to generation.