RESUMEN
The RNA-directed DNA methylation (RdDM) pathway in plants controls gene expression via cytosine DNA methylation. The ability to manipulate RdDM would shed light on the mechanisms and applications of DNA methylation to control gene expression. Here, we identified diverse RdDM proteins that are capable of targeting methylation and silencing in Arabidopsis when tethered to an artificial zinc finger (ZF-RdDM). We studied their order of action within the RdDM pathway by testing their ability to target methylation in different mutants. We also evaluated ectopic siRNA biogenesis, RNA polymerase V (Pol V) recruitment, targeted DNA methylation, and gene-expression changes at thousands of ZF-RdDM targets. We found that co-targeting both arms of the RdDM pathway, siRNA biogenesis and Pol V recruitment, dramatically enhanced targeted methylation. This work defines how RdDM components establish DNA methylation and enables new strategies for epigenetic gene regulation via targeted DNA methylation.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Metilación de ADN/fisiología , ARN Polimerasas Dirigidas por ADN/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Citosina/metabolismo , ADN/metabolismo , Metilación de ADN/genética , ARN Polimerasas Dirigidas por ADN/genética , Regulación de la Expresión Génica de las Plantas/genética , ARN Polimerasa II/metabolismo , ARN de Planta/genética , ARN Interferente Pequeño/metabolismoRESUMEN
RNA-directed DNA methylation in Arabidopsis thaliana is driven by the plant-specific RNA Polymerase IV (Pol IV). It has been assumed that a Pol IV transcript can give rise to multiple 24-nt small interfering RNAs (siRNAs) that target DNA methylation. Here, we demonstrate that Pol IV-dependent RNAs (P4RNAs) from wild-type Arabidopsis are surprisingly short in length (30 to 40 nt) and mirror 24-nt siRNAs in distribution, abundance, strand bias, and 5'-adenine preference. P4RNAs exhibit transcription start sites similar to Pol II products and are featured with 5'-monophosphates and 3'-misincorporated nucleotides. The 3'-misincorporation preferentially occurs at methylated cytosines on the template DNA strand, suggesting a co-transcriptional feedback to siRNA biogenesis by DNA methylation to reinforce silencing locally. These results highlight an unusual mechanism of Pol IV transcription and suggest a "one precursor, one siRNA" model for the biogenesis of 24-nt siRNAs in Arabidopsis.
Asunto(s)
Arabidopsis/metabolismo , ARN de Planta/genética , ARN Interferente Pequeño/genética , Adenina/metabolismo , Arabidopsis/enzimología , Arabidopsis/genética , Metilación de ADN , ARN Polimerasas Dirigidas por ADN/metabolismo , Modelos Biológicos , Sitio de Iniciación de la TranscripciónRESUMEN
DNA methylation is a conserved epigenetic gene-regulation mechanism. DOMAINS REARRANGED METHYLTRANSFERASE (DRM) is a key de novo methyltransferase in plants, but how DRM acts mechanistically is poorly understood. Here, we report the crystal structure of the methyltransferase domain of tobacco DRM (NtDRM) and reveal a molecular basis for its rearranged structure. NtDRM forms a functional homodimer critical for catalytic activity. We also show that Arabidopsis DRM2 exists in complex with the small interfering RNA (siRNA) effector ARGONAUTE4 (AGO4) and preferentially methylates one DNA strand, likely the strand acting as the template for RNA polymerase V-mediated noncoding RNA transcripts. This strand-biased DNA methylation is also positively correlated with strand-biased siRNA accumulation. These data suggest a model in which DRM2 is guided to target loci by AGO4-siRNA and involves base-pairing of associated siRNAs with nascent RNA transcripts.
Asunto(s)
Arabidopsis/enzimología , Metiltransferasas/metabolismo , Nicotiana/enzimología , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas/metabolismo , Dominio Catalítico , Metiltransferasas/química , Modelos Moleculares , Datos de Secuencia Molecular , Nicotiana/metabolismoRESUMEN
Histone variants play crucial roles in gene expression, genome integrity, and chromosome segregation. We report that the four H2A variants in Arabidopsis define different genomic features, contributing to overall genomic organization. The histone variant H2A.W marks heterochromatin specifically and acts in synergy with heterochromatic marks H3K9me2 and DNA methylation to maintain transposon silencing. In vitro, H2A.W enhances chromatin condensation by promoting fiber-to-fiber interactions via its conserved C-terminal motif. In vivo, H2A.W is required for heterochromatin condensation, demonstrating that H2A.W plays critical roles in heterochromatin organization. Similarities in conserved motifs between H2A.W and another H2A variant in metazoans suggest that plants and animals share common mechanisms for heterochromatin condensation.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ensamble y Desensamble de Cromatina , Heterocromatina/metabolismo , Histonas/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Metilación de ADN , Elementos Transponibles de ADN , Estudio de Asociación del Genoma Completo , Histonas/química , Histonas/genética , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
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.
Asunto(s)
Arabidopsis/genética , Metilación de ADN , Genoma de Planta , Proteínas de Arabidopsis/metabolismo , Islas de CpG , Silenciador del Gen , Estudio de Asociación del Genoma Completo , Histonas/metabolismo , Interferencia de ARN , ARN Polimerasa II/metabolismo , Factores de Empalme de ARNRESUMEN
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.
Asunto(s)
Arabidopsis/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , ADN de Plantas/metabolismo , Nucleosomas/metabolismo , Zea mays/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Cristalografía por Rayos X , ADN (Citosina-5-)-Metiltransferasas/química , Heterocromatina/metabolismo , Histonas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de Secuencia , Zea mays/genéticaRESUMEN
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.
Asunto(s)
Metilación de ADN , Metilasas de Modificación del ADN/metabolismo , Nucleosomas/enzimología , Proteínas de Plantas/metabolismo , Animales , Metilasas de Modificación del ADN/genética , Metilasas de Modificación del ADN/ultraestructura , Activación Enzimática , Escherichia coli/enzimología , Escherichia coli/genética , Microscopía Electrónica , Modelos Moleculares , Conformación de Ácido Nucleico , Nucleosomas/química , Nucleosomas/genética , Nucleosomas/ultraestructura , Proteínas de Plantas/genética , Proteínas de Plantas/ultraestructura , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Relación Estructura-Actividad , Especificidad por Sustrato , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) AND ATXR6 are required for the deposition of H3K27me1 and for maintaining genomic stability in Arabidopsis Reduction of ATXR5/6 activity results in activation of DNA damage response genes, along with tissue-specific derepression of transposable elements (TEs), chromocenter decompaction, and genomic instability characterized by accumulation of excess DNA from heterochromatin. How loss of ATXR5/6 and H3K27me1 leads to these phenotypes remains unclear. Here we provide extensive characterization of the atxr5/6 hypomorphic mutant by comprehensively examining gene expression and epigenetic changes in the mutant. We found that the tissue-specific phenotypes of TE derepression and excessive DNA in this atxr5/6 mutant correlated with residual ATXR6 expression from the hypomorphic ATXR6 allele. However, up-regulation of DNA damage genes occurred regardless of ATXR6 levels and thus appears to be a separable process. We also isolated an atxr6-null allele which showed that ATXR5 and ATXR6 are required for female germline development. Finally, we characterize three previously reported suppressors of the hypomorphic atxr5/6 mutant and show that these rescue atxr5/6 via distinct mechanisms, two of which involve increasing H3K27me1 levels.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Elementos Transponibles de ADN , Regulación de la Expresión Génica de las Plantas , Inestabilidad Genómica , Metiltransferasas/genética , Alelos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Epigénesis Genética , Heterocromatina/metabolismo , Histonas/metabolismo , Metiltransferasas/metabolismo , Mutación , Fenotipo , TranscriptomaRESUMEN
CRISPR-based targeted modification of epigenetic marks such as DNA cytosine methylation is an important strategy to regulate the expression of genes and their associated phenotypes. Although plants have DNA methylation in all sequence contexts (CG, CHG, CHH, where H = A, T, C), methylation in the symmetric CG context is particularly important for gene silencing and is very efficiently maintained through mitotic and meiotic cell divisions. Tools that can directly add CG methylation to specific loci are therefore highly desirable but are currently lacking in plants. Here we have developed two CRISPR-based CG-specific targeted DNA methylation systems for plants using a variant of the bacterial CG-specific DNA methyltransferase MQ1 with reduced activity but high specificity. We demonstrate that the methylation added by MQ1 is highly target specific and can be heritably maintained in the absence of the effector. These tools should be valuable both in crop engineering and in plant genetic research.
Asunto(s)
Arabidopsis , Proteínas Bacterianas , Sistemas CRISPR-Cas , Metilación de ADN , ADN de Plantas/metabolismo , ADN-Citosina Metilasas , Plantas Modificadas Genéticamente , Tenericutes/genética , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN de Plantas/genética , ADN-Citosina Metilasas/genética , ADN-Citosina Metilasas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Tenericutes/enzimologíaRESUMEN
DNA methylation is a major epigenetic modification found across species and has a profound impact on many biological processes. However, its influence on chromatin accessibility and higher-order genome organization remains unclear, particularly in plants. Here, we present genome-wide chromatin accessibility profiles of 18 Arabidopsis mutants that are deficient in CG, CHG, or CHH DNA methylation. We find that DNA methylation in all three sequence contexts impacts chromatin accessibility in heterochromatin. Many chromatin regions maintain inaccessibility when DNA methylation is lost in only one or two sequence contexts, and signatures of accessibility are particularly affected when DNA methylation is reduced in all contexts, suggesting an interplay between different types of DNA methylation. In addition, we found that increased chromatin accessibility was not always accompanied by increased transcription, suggesting that DNA methylation can directly impact chromatin structure by other mechanisms. We also observed that an increase in chromatin accessibility was accompanied by enhanced long-range chromatin interactions. Together, these results provide a valuable resource for chromatin architecture and DNA methylation analyses and uncover a pivotal role for methylation in the maintenance of heterochromatin inaccessibility.
Asunto(s)
Arabidopsis/genética , Cromatina/genética , Metilación de ADN/genética , Genoma de Planta , Mutación/genética , Transcripción GenéticaRESUMEN
RNA polymerase V (Pol V) long noncoding RNAs (lncRNAs) have been proposed to guide ARGONAUTE4 (AGO4) to chromatin in RNA-directed DNA methylation (RdDM) in plants. Here, we provide evidence, based on laser UV-assisted zero-length cross-linking, for functionally relevant AGO4-DNA interaction at RdDM targets. We further demonstrate that Pol V lncRNAs or the act of their transcription are required to lock Pol V holoenzyme into a stable DNA-bound state that allows AGO4 recruitment via redundant glycine-tryptophan/tryptophan-glycine AGO hook motifs present on both Pol V and its associated factor, SPT5L. We propose a model in which AGO4-DNA interaction could be responsible for the unique specificities of RdDM.
Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Metilación de ADN , ADN de Plantas/metabolismo , ARN de Planta/metabolismo , Secuencias de Aminoácidos/genética , Proteínas de Arabidopsis/química , Proteínas Argonautas/química , Cromatina/metabolismo , Metilación de ADN/genética , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Silenciador del Gen , Modelos Biológicos , Interferencia de ARN , Transcripción Genética/genéticaRESUMEN
Plant RNA viruses are used as delivery vectors for their high level of accumulation and efficient spread during virus multiplication and movement. Utilizing this concept, several viral-based guide RNA delivery platforms for CRISPR-Cas9 genome editing have been developed. The CRISPR-Cas9 system has also been adapted for epigenome editing. While systems have been developed for CRISPR-Cas9 based gene activation or site-specific DNA demethylation, viral delivery of guide RNAs remains to be developed for these purposes. To address this gap we have developed a tobacco rattle virus (TRV)-based single guide RNA delivery system for epigenome editing in Arabidopsis thaliana. Because tRNA-like sequences have been shown to facilitate the cell-to-cell movement of RNAs in plants, we used the tRNA-guide RNA expression system to express guide RNAs from the viral genome to promote heritable epigenome editing. We demonstrate that the tRNA-gRNA system with TRV can be used for both transcriptional activation and targeted DNA demethylation of the FLOWERING WAGENINGEN gene in Arabidopsis. We achieved up to ~8% heritability of the induced demethylation phenotype in the progeny of virus inoculated plants. We did not detect the virus in the next generation, indicating effective clearance of the virus from plant tissues. Thus, TRV delivery, combined with a specific tRNA-gRNA architecture, provides for fast and effective epigenome editing.
Asunto(s)
Proteínas de Arabidopsis/genética , Sistemas CRISPR-Cas , Metilación de ADN , Edición Génica/métodos , Marcación de Gen/métodos , Virus de Plantas/genética , ARN Guía de Kinetoplastida/genética , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Epigenoma , ARN de Transferencia/genética , Activación TranscripcionalRESUMEN
The piRNA pathway protects germline genomes from selfish genetic elements (e.g. transposons) through their transcript cleavage in the cytoplasm and/or their transcriptional silencing in the nucleus. Here, we describe a mechanism by which the nuclear and cytoplasmic arms of the piRNA pathway are linked. We find that during mitosis of Drosophila spermatogonia, nuclear Piwi interacts with nuage, the compartment that mediates the cytoplasmic arm of the piRNA pathway. At the end of mitosis, Piwi leaves nuage to return to the nucleus. Dissociation of Piwi from nuage occurs at the depolymerizing microtubules of the central spindle, mediated by a microtubule-depolymerizing kinesin, Klp10A. Depletion of klp10A delays the return of Piwi to the nucleus and affects piRNA production, suggesting the role of nuclear-cytoplasmic communication in piRNA biogenesis. We propose that cell cycle-dependent communication between the nuclear and cytoplasmic arms of the piRNA pathway may play a previously unappreciated role in piRNA regulation.
Asunto(s)
Proteínas Argonautas/metabolismo , Proteínas de Drosophila/metabolismo , Cinesinas/metabolismo , ARN Interferente Pequeño/genética , Animales , Proteínas Argonautas/genética , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , División Celular/fisiología , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Elementos Transponibles de ADN , Proteínas de Drosophila/genética , Drosophila melanogaster , Femenino , Silenciador del Gen , Células Germinativas , Cinesinas/genética , Masculino , Ovario/metabolismo , ARN Interferente Pequeño/metabolismoRESUMEN
Transposable elements (TEs) are DNA repeats that must remain silenced to ensure cell integrity. Several epigenetic pathways including DNA methylation and histone modifications are involved in the silencing of TEs, and in the regulation of gene expression. In Arabidopsis thaliana, the TE-derived plant mobile domain (PMD) proteins have been involved in TE silencing, genome stability, and control of developmental processes. Using a forward genetic screen, we found that the PMD protein MAINTENANCE OF MERISTEMS (MAIN) acts synergistically and redundantly with DNA methylation to silence TEs. We found that MAIN and its close homolog MAIN-LIKE 1 (MAIL1) interact together, as well as with the phosphoprotein phosphatase (PPP) PP7-like (PP7L). Remarkably, main, mail1, pp7l single and mail1 pp7l double mutants display similar developmental phenotypes, and share common subsets of upregulated TEs and misregulated genes. Finally, phylogenetic analyses of PMD and PP7-type PPP domains among the Eudicot lineage suggest neo-association processes between the two protein domains to potentially generate new protein function. We propose that, through this interaction, the PMD and PPP domains may constitute a functional protein module required for the proper expression of a common set of genes, and for silencing of TEs.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Elementos Transponibles de ADN/genética , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Metilación de ADN , Epigénesis Genética , Heterocromatina/metabolismo , Mutación , Proteínas Nucleares/genética , Fosfoproteínas Fosfatasas/genética , Unión Proteica , Dominios Proteicos , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
Chromosomes form 3D structures that are critical to the regulation of cellular and genetic processes. Here, we present a study of global chromatin interaction patterns in Arabidopsis thaliana. Our genome-wide approach confirmed interactions that were previously observed by other methods as well as uncovered long-range interactions such as those among small heterochromatic regions embedded in euchromatic arms. We also found that interactions are correlated with various epigenetic marks that are localized in active or silenced chromatin. Arabidopsis chromosomes do not contain large local interactive domains that resemble the topological domains described in animals but, instead, contain relatively small interactive regions scattered around the genome that contain H3K27me3 or H3K9me2. We generated interaction maps in mutants that are defective in specific epigenetic pathways and found altered interaction patterns that correlate with changes in the epigenome. These analyses provide further insights into molecular mechanisms of epigenetic regulation of the genome.
Asunto(s)
Arabidopsis/genética , Cromatina/metabolismo , Cromosomas de las Plantas/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiología , Cromatina/ultraestructura , Cromosomas de las Plantas/química , ADN de Plantas/química , Epigénesis Genética/genética , Genoma de Planta , Genómica/métodos , Mutación , Conformación de Ácido NucleicoRESUMEN
In Arabidopsis, CHG DNA methylation is controlled by the H3K9 methylation mark through a self-reinforcing loop between DNA methyltransferase CHROMOMETHYLASE3 (CMT3) and H3K9 histone methyltransferase KRYPTONITE/SUVH4 (KYP). We report on the structure of KYP in complex with methylated DNA, substrate H3 peptide, and cofactor SAH, thereby defining the spatial positioning of the SRA domain relative to the SET domain. The methylated DNA is bound by the SRA domain with the 5mC flipped out of the DNA, while the H3(1-15) peptide substrate binds between the SET and post-SET domains, with the ε-ammonium of K9 positioned adjacent to bound SAH. These structural insights, complemented by functional data on key mutants of residues lining the 5mC and H3K9-binding pockets within KYP, establish how methylated DNA recruits KYP to the histone substrate. Together, the structures of KYP and previously reported CMT3 complexes provide insights into molecular mechanisms linking DNA and histone methylation.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Metilación de ADN , ADN de Plantas/química , ADN de Plantas/genética , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/fisiología , Arabidopsis/química , Arabidopsis/metabolismo , Sitios de Unión/genética , Epigénesis Genética , Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Modelos Moleculares , S-Adenosilhomocisteína/metabolismo , Difracción de Rayos XRESUMEN
Genome-wide characterization by next-generation sequencing has greatly improved our understanding of the landscape of epigenetic modifications. Since 2008, whole-genome bisulfite sequencing (WGBS) has become the gold standard for DNA methylation analysis, and a tremendous amount of WGBS data has been generated by the research community. However, the systematic comparison of DNA methylation profiles to identify regulatory mechanisms has yet to be fully explored. Here we reprocessed the raw data of over 500 publicly available Arabidopsis WGBS libraries from various mutant backgrounds, tissue types, and stress treatments and also filtered them based on sequencing depth and efficiency of bisulfite conversion. This enabled us to identify high-confidence differentially methylated regions (hcDMRs) by comparing each test library to over 50 high-quality wild-type controls. We developed statistical and quantitative measurements to analyze the overlapping of DMRs and to cluster libraries based on their effect on DNA methylation. In addition to confirming existing relationships, we revealed unanticipated connections between well-known genes. For instance, MET1 and CMT3 were found to be required for the maintenance of asymmetric CHH methylation at nonoverlapping regions of CMT2 targeted heterochromatin. Our comparative methylome approach has established a framework for extracting biological insights via large-scale comparison of methylomes and can also be adopted for other genomics datasets.
Asunto(s)
Arabidopsis/genética , Metilación de ADN , Epigenómica , Regulación de la Expresión Génica de las Plantas , Análisis por Conglomerados , Biología Computacional , Islas de CpG , Epigénesis Genética , Biblioteca de Genes , Genoma de Planta , Heterocromatina/química , Secuenciación de Nucleótidos de Alto Rendimiento , Plantas Modificadas Genéticamente , Análisis de Secuencia de ADN , Análisis de Secuencia de ARN , Programas InformáticosRESUMEN
RNA-directed DNA methylation in Arabidopsis thaliana depends on the upstream synthesis of 24-nucleotide small interfering RNAs (siRNAs) by RNA POLYMERASE IV (Pol IV) and downstream synthesis of non-coding transcripts by Pol V. Pol V transcripts are thought to interact with siRNAs which then recruit DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) to methylate DNA. The SU(VAR)3-9 homologues SUVH2 and SUVH9 act in this downstream step but the mechanism of their action is unknown. Here we show that genome-wide Pol V association with chromatin redundantly requires SUVH2 and SUVH9. Although SUVH2 and SUVH9 resemble histone methyltransferases, a crystal structure reveals that SUVH9 lacks a peptide-substrate binding cleft and lacks a properly formed S-adenosyl methionine (SAM)-binding pocket necessary for normal catalysis, consistent with a lack of methyltransferase activity for these proteins. SUVH2 and SUVH9 both contain SRA (SET- and RING-ASSOCIATED) domains capable of binding methylated DNA, suggesting that they function to recruit Pol V through DNA methylation. Consistent with this model, mutation of DNA METHYLTRANSFERASE 1 (MET1) causes loss of DNA methylation, a nearly complete loss of Pol V at its normal locations, and redistribution of Pol V to sites that become hypermethylated. Furthermore, tethering SUVH9 [corrected] with a zinc finger to an unmethylated site is sufficient to recruit Pol V and establish DNA methylation and gene silencing. These results indicate that Pol V is recruited to DNA methylation through the methyl-DNA binding SUVH2 and SUVH9 proteins, and our mechanistic findings suggest a means for selectively targeting regions of plant genomes for epigenetic silencing.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis , Metilación de ADN , ARN Polimerasas Dirigidas por ADN/metabolismo , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/metabolismo , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sitios de Unión/genética , Biocatálisis , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Cristalografía por Rayos X , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Flores/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Genoma de Planta/genética , Modelos Moleculares , Mutación/genética , Fenotipo , Estructura Terciaria de Proteína , Transporte de Proteínas , ARN de Planta/biosíntesis , ARN de Planta/genética , ARN de Planta/metabolismo , ARN Interferente Pequeño/biosíntesis , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transcripción Genética , Dedos de ZincRESUMEN
DNA methylation is an epigenetic modification that has critical roles in gene silencing, development and genome integrity. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) and targeted by 24-nucleotide small interfering RNAs (siRNAs) through a pathway termed RNA-directed DNA methylation (RdDM). This pathway requires two plant-specific RNA polymerases: Pol-IV, which functions to initiate siRNA biogenesis, and Pol-V, which functions to generate scaffold transcripts that recruit downstream RdDM factors. To understand the mechanisms controlling Pol-IV targeting we investigated the function of SAWADEE HOMEODOMAIN HOMOLOG 1 (SHH1), a Pol-IV-interacting protein. Here we show that SHH1 acts upstream in the RdDM pathway to enable siRNA production from a large subset of the most active RdDM targets, and that SHH1 is required for Pol-IV occupancy at these same loci. We also show that the SHH1 SAWADEE domain is a novel chromatin-binding module that adopts a unique tandem Tudor-like fold and functions as a dual lysine reader, probing for both unmethylated K4 and methylated K9 modifications on the histone 3 (H3) tail. Finally, we show that key residues within both lysine-binding pockets of SHH1 are required in vivo to maintain siRNA and DNA methylation levels as well as Pol-IV occupancy at RdDM targets, demonstrating a central role for methylated H3K9 binding in SHH1 function and providing the first insights into the mechanism of Pol-IV targeting. Given the parallels between methylation systems in plants and mammals, a further understanding of this early targeting step may aid our ability to control the expression of endogenous and newly introduced genes, which has broad implications for agriculture and gene therapy.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Arabidopsis/metabolismo , Metilación de ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sitios de Unión/genética , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Cristalografía por Rayos X , ARN Polimerasas Dirigidas por ADN/genética , Epigénesis Genética/genética , Histonas/química , Histonas/metabolismo , Proteínas de Homeodominio/química , Lisina/química , Lisina/metabolismo , Metiltransferasas/genética , Metiltransferasas/metabolismo , Modelos Moleculares , Mutación , Pliegue de Proteína , Estructura Terciaria de Proteína , ARN Interferente Pequeño/biosíntesis , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
DNA methylation plays important roles in many biological processes, such as silencing of transposable elements, imprinting, and regulating gene expression. Many studies of DNA methylation have shown its essential roles in angiosperms (flowering plants). However, few studies have examined the roles and patterns of DNA methylation in gymnosperms. Here, we present genome-wide high coverage single-base resolution methylation maps of Norway spruce (Picea abies) from both needles and somatic embryogenesis culture cells via whole genome bisulfite sequencing. On average, DNA methylation levels of CG and CHG of Norway spruce were higher than most other plants studied. CHH methylation was found at a relatively low level; however, at least one copy of most of the RNA-directed DNA methylation pathway genes was found in Norway spruce, and CHH methylation was correlated with levels of siRNAs. In comparison with needles, somatic embryogenesis culture cells that are used for clonally propagating spruce trees showed lower levels of CG and CHG methylation but higher level of CHH methylation, suggesting that like in other species, these culture cells show abnormal methylation patterns.