RESUMEN
In mammals, the environment plays a critical role in promoting the final steps in neuronal development during the early postnatal period. While epigenetic factors are thought to contribute to this process, the underlying molecular mechanisms remain poorly understood. Here, we show that in the brain during early life, the DNA methyltransferase DNMT3A transiently binds across transcribed regions of lowly expressed genes, and its binding specifies the pattern of DNA methylation at CA sequences (mCA) within these genes. We find that DNMT3A occupancy and mCA deposition within the transcribed regions of genes is negatively regulated by gene transcription and may be modified by early-life experience. Once deposited, mCA is bound by the methyl-DNA-binding protein MECP2 and functions in a rheostat-like manner to fine-tune the cell-type-specific transcription of genes that are critical for brain function.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Epigénesis Genética , Neuronas/metabolismo , Animales , Encéfalo/citología , Encéfalo/metabolismo , ADN Metiltransferasa 3A , Regulación del Desarrollo de la Expresión Génica , Humanos , Proteína 2 de Unión a Metil-CpG , Ratones , Transcripción Genética , Activación TranscripcionalRESUMEN
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
Mutations in the methyl-DNA-binding repressor protein MeCP2 cause the devastating neurodevelopmental disorder Rett syndrome. It has been challenging to understand how MeCP2 regulates transcription because MeCP2 binds broadly across the genome and MeCP2 mutations are associated with widespread small-magnitude changes in neuronal gene expression. We demonstrate here that MeCP2 represses nascent RNA transcription of highly methylated long genes in the brain through its interaction with the NCoR co-repressor complex. By measuring the rates of transcriptional initiation and elongation directly in the brain, we find that MeCP2 has no measurable effect on transcriptional elongation, but instead represses the rate at which Pol II initiates transcription of highly methylated long genes. These findings suggest a new model of MeCP2 function in which MeCP2 binds broadly across highly methylated regions of DNA, but acts at transcription start sites to attenuate transcriptional initiation.
Asunto(s)
Metilación de ADN/genética , Proteína 2 de Unión a Metil-CpG/genética , Proteínas Represoras/genética , Transcripción Genética/genética , Animales , Encéfalo/fisiología , ADN/genética , Masculino , Ratones , Ratones Noqueados , Mutación/genética , Neuronas/fisiología , ARN/genética , Síndrome de Rett/genéticaRESUMEN
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
Disruption of the MECP2 gene leads to Rett syndrome (RTT), a severe neurological disorder with features of autism. MECP2 encodes a methyl-DNA-binding protein that has been proposed to function as a transcriptional repressor, but despite numerous mouse studies examining neuronal gene expression in Mecp2 mutants, no clear model has emerged for how MeCP2 protein regulates transcription. Here we identify a genome-wide length-dependent increase in gene expression in MeCP2 mutant mouse models and human RTT brains. We present evidence that MeCP2 represses gene expression by binding to methylated CA sites within long genes, and that in neurons lacking MeCP2, decreasing the expression of long genes attenuates RTT-associated cellular deficits. In addition, we find that long genes as a population are enriched for neuronal functions and selectively expressed in the brain. These findings suggest that mutations in MeCP2 may cause neurological dysfunction by specifically disrupting long gene expression in the brain.
Asunto(s)
Metilación de ADN/genética , Proteína 2 de Unión a Metil-CpG/genética , Proteína 2 de Unión a Metil-CpG/metabolismo , Mutación/genética , Síndrome de Rett/genética , Animales , Secuencia de Bases , Encéfalo/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Metiltransferasa 3A , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Proteína 2 de Unión a Metil-CpG/deficiencia , Ratones , Datos de Secuencia Molecular , Neuronas/metabolismoRESUMEN
Eukaryotes have hundreds of nearly identical 45S ribosomal RNA (rRNA) genes, each encoding the 18S, 5.8S, and 25S catalytic rRNAs. Because cellular demands for ribosomes and protein synthesis vary during development, the number of active rRNA genes is subject to dosage control. In genetic hybrids, one manifestation of dosage control is nucleolar dominance, an epigenetic phenomenon in which the rRNA genes of one progenitor are repressed. For instance, in Arabidopsis suecica, the allotetraploid hybrid of Arabidopsis thaliana and Arabidopsis arenosa, the A. thaliana-derived rRNA genes are selectively silenced. An analogous phenomenon occurs in nonhybrid A. thaliana, in which specific classes of rRNA gene variants are inactivated. An RNA-mediated knockdown screen identified SUVR4 {SUPPRESSOR OF VARIEGATION 3-9 [SU(VAR)3-9]-RELATED 4} as a histone H3 Lys 9 (H3K9) methyltransferase required for nucleolar dominance in A. suecica. H3K9 methyltransferases are also required for variant-specific silencing in A. thaliana, but SUVH5 [SU(VAR)3-9 HOMOLOG 5] and SUVH6, rather than SUVR4, are the key activities in this genomic context. Mutations disrupting the H3K27 methyltransferases ATXR5 or ATXR6 affect which rRNA gene variants are expressed or silenced, and in atxr5 atxr6 double mutants, dominance relationships among variants are reversed relative to wild type. Interestingly, these changes in gene expression are accompanied by changes in the relative abundance of the rRNA gene variants at the DNA level, including overreplication of the normally silenced class and decreased abundance of the normally dominant class. Collectively, our results indicate that histone methylation can affect both the doses of different variants and their differential silencing through the choice mechanisms that achieve dosage control.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Dosificación de Gen , Regulación de la Expresión Génica de las Plantas , Genes de ARNr , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteínas de Arabidopsis/genética , Nucléolo Celular/enzimología , Metilación de ADN , N-Metiltransferasa de Histona-Lisina/genética , Histonas/metabolismo , Metiltransferasas/genética , Metiltransferasas/metabolismoRESUMEN
In plants, CG DNA methylation is prevalent in the transcribed regions of many constitutively expressed genes (gene body methylation; gbM), but the origin and function of gbM remain unknown. Here we report the discovery that Eutrema salsugineum has lost gbM from its genome, to our knowledge the first instance for an angiosperm. Of all known DNA methyltransferases, only CHROMOMETHYLASE 3 (CMT3) is missing from E. salsugineum Identification of an additional angiosperm, Conringia planisiliqua, which independently lost CMT3 and gbM, supports that CMT3 is required for the establishment of gbM. Detailed analyses of gene expression, the histone variant H2A.Z, and various histone modifications in E. salsugineum and in Arabidopsis thaliana epigenetic recombinant inbred lines found no evidence in support of any role for gbM in regulating transcription or affecting the composition and modification of chromatin over evolutionary timescales.
Asunto(s)
Metilación de ADN , Evolución Molecular , Magnoliopsida/genética , ADN (Citosina-5-)-Metiltransferasas/fisiología , Histonas/metabolismoRESUMEN
Epigenetic gene silencing is of central importance to maintain genome integrity and is mediated by an elaborate interplay between DNA methylation, histone posttranslational modifications, and chromatin remodeling complexes. DNA methylation and repressive histone marks usually correlate with transcriptionally silent heterochromatin, however there are exceptions to this relationship. In Arabidopsis, mutation of Morpheus Molecule 1 (MOM1) causes transcriptional derepression of heterochromatin independently of changes in DNA methylation. More recently, two Arabidopsis homologues of mouse microrchidia (MORC) genes have also been implicated in gene silencing and heterochromatin condensation without altering genome-wide DNA methylation patterns. In this study, we show that Arabidopsis microrchidia (AtMORC6) physically interacts with AtMORC1 and with its close homologue, AtMORC2, in two mutually exclusive protein complexes. RNA-sequencing analyses of high-order mutants indicate that AtMORC1 and AtMORC2 act redundantly to repress a common set of loci. We also examined genetic interactions between AtMORC6 and MOM1 pathways. Although AtMORC6 and MOM1 control the silencing of a very similar set of genomic loci, we observed synergistic transcriptional regulation in the mom1/atmorc6 double mutant, suggesting that these epigenetic regulators act mainly by different silencing mechanisms.
Asunto(s)
Adenosina Trifosfatasas/química , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Adenosina Trifosfatasas/genética , Proteínas de Arabidopsis/química , Metilación de ADN , Elementos Transponibles de ADN , Epigénesis Genética , Genotipo , Heterocromatina/metabolismo , Mutación , Unión ProteicaRESUMEN
DNA methylation in Arabidopsis thaliana is maintained by at least four different enzymes: DNA methyltransferase1 (MET1), chromomethylase3 (CMT3), domains rearranged methyltransferase2 (DRM2), and chromomethylase2 (CMT2). However, DNA methylation is established exclusively by the enzyme DRM2, which acts in the RNA-directed DNA methylation (RdDM) pathway. Some RdDM components belong to gene families and have partially redundant functions, such as the endoribonucleases dicer-like 2, 3, and 4, and involved in de novo2 (IDN2) interactors IDN2-like 1 and 2. Traditional mutagenesis screens usually fail to detect genes if they are redundant, as the loss of one gene can be compensated by a related gene. In an effort to circumvent this issue, we used coexpression data to identify closely related genes that are coregulated with genes in the RdDM pathway. Here we report the discovery of two redundant proteins, SNF2-ring-helicase-like1 and -2 (FRG1 and -2) that are putative chromatin modifiers belonging to the SNF2 family of helicase-like proteins. Analysis of genome-wide bisulfite sequencing shows that simultaneous mutations of FRG1 and -2 cause defects in methylation at specific RdDM targeted loci. We also show that FRG1 physically associates with Su(var)3-9-related SUVR2, a known RdDM component, in vivo. Combined, our results identify FRG1 and FRG2 as previously unidentified components of the RdDM machinery.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Cromatina/química , Proteínas Cromosómicas no Histona/metabolismo , Metilación de ADN , ARN de Planta/química , Arabidopsis/metabolismo , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Epigénesis Genética , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Mutagénesis , Mutación , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
Multiple pathways prevent DNA replication from occurring more than once per cell cycle. These pathways block re-replication by strictly controlling the activity of pre-replication complexes, which assemble at specific sites in the genome called origins. Here we show that mutations in the homologous histone 3 lysine 27 (H3K27) monomethyltransferases, ARABIDOPSIS TRITHORAX-RELATED PROTEIN5 (ATXR5) and ATXR6, lead to re-replication of specific genomic locations. Most of these locations correspond to transposons and other repetitive and silent elements of the Arabidopsis genome. These sites also correspond to high levels of H3K27 monomethylation, and mutation of the catalytic SET domain is sufficient to cause the re-replication defect. Mutation of ATXR5 and ATXR6 also causes upregulation of transposon expression and has pleiotropic effects on plant development. These results uncover a novel pathway that prevents over-replication of heterochromatin in Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis , Replicación del ADN/fisiología , Heterocromatina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Metiltransferasas/metabolismo , Secuencias de Aminoácidos , Arabidopsis/citología , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Dominio Catalítico/genética , Metilación de ADN , Replicación del ADN/genética , Elementos Transponibles de ADN/genética , ADN de Plantas/análisis , ADN de Plantas/biosíntesis , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Genoma de Planta/genética , Heterocromatina/metabolismo , Histonas/química , Lisina/metabolismo , Metilación , Metiltransferasas/química , Metiltransferasas/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación , Origen de RéplicaRESUMEN
Nucleosomes compact and regulate access to DNA in the nucleus, and are composed of approximately 147 bases of DNA wrapped around a histone octamer. Here we report a genome-wide nucleosome positioning analysis of Arabidopsis thaliana using massively parallel sequencing of mononucleosomes. By combining this data with profiles of DNA methylation at single base resolution, we identified 10-base periodicities in the DNA methylation status of nucleosome-bound DNA and found that nucleosomal DNA was more highly methylated than flanking DNA. These results indicate that nucleosome positioning influences DNA methylation patterning throughout the genome and that DNA methyltransferases preferentially target nucleosome-bound DNA. We also observed similar trends in human nucleosomal DNA, indicating that the relationships between nucleosomes and DNA methyltransferases are conserved. Finally, as has been observed in animals, nucleosomes were highly enriched on exons, and preferentially positioned at intron-exon and exon-intron boundaries. RNA polymerase II (Pol II) was also enriched on exons relative to introns, consistent with the hypothesis that nucleosome positioning regulates Pol II processivity. DNA methylation is also enriched on exons, consistent with the targeting of DNA methylation to nucleosomes, and suggesting a role for DNA methylation in exon definition.
Asunto(s)
Arabidopsis/genética , Arabidopsis/metabolismo , Ensamble y Desensamble de Cromatina/fisiología , Metilación de ADN/fisiología , Nucleosomas/metabolismo , Arabidopsis/enzimología , Ensamble y Desensamble de Cromatina/genética , Inmunoprecipitación de Cromatina , Metilación de ADN/genética , ADN Polimerasa II/análisis , ADN Polimerasa II/metabolismo , ADN de Plantas/genética , ADN de Plantas/metabolismo , Exones/genética , Genes de Plantas/genética , Genoma de Planta/genética , Humanos , Nucleasa Microcócica/metabolismo , Nucleosomas/genética , Análisis de Secuencia de ADNRESUMEN
Studies of DNA methylation from fungi, plants, and animals indicate that gene body methylation is ancient and highly conserved in eukaryotic genomes, but its role has not been clearly defined. It has been postulated that regulation of alternative splicing of transcripts was an original function of DNA methylation, but a direct experimental test of the effect of methylation on alternative slicing at the whole genome level has never been performed. To do this, we developed a unique method to administer RNA interference (RNAi) in a high-throughput and noninvasive manner and then used it to knock down the expression of DNA methyl-transferase 3 (dnmt3), which is required for de novo DNA methylation. We chose the honey bee (Apis mellifera) for this test because it has recently emerged as an important model organism for studying the effects of DNA methylation on development and social behavior, and DNA methylation in honey bees is predominantly on gene bodies. Here we show that dnmt3 RNAi decreased global genomic methylation level as expected and in addition caused widespread and diverse changes in alternative splicing in fat tissue. Four different types of splicing events were affected by dnmt3 gene knockdown, and change in two types, exon skipping and intron retention, was directly related to decreased methylation. These results demonstrate that one function of gene body DNA methylation is to regulate alternative splicing.
Asunto(s)
Empalme Alternativo/fisiología , Abejas/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Proteínas de Insectos/metabolismo , Interferencia de ARN , Animales , Abejas/genética , Conducta Animal , ADN (Citosina-5-)-Metiltransferasas/genética , Técnicas de Silenciamiento del Gen , Proteínas de Insectos/genética , Conducta SocialRESUMEN
Dimethylation of histone H3 lysine 9 (H3K9m2) and trimethylation of histone H3 lysine 27 (H3K27m3) are two hallmarks of transcriptional repression in many organisms. In Arabidopsis thaliana, H3K27m3 is targeted by Polycomb Group (PcG) proteins and is associated with silent protein-coding genes, while H3K9m2 is correlated with DNA methylation and is associated with transposons and repetitive sequences. Recently, ectopic genic DNA methylation in the CHG context (where H is any base except G) has been observed in globally DNA hypomethylated mutants such as met1, but neither the nature of the hypermethylated loci nor the biological significance of this epigenetic phenomenon have been investigated. Here, we generated high-resolution, genome-wide maps of both H3K9m2 and H3K27m3 in wild-type and met1 plants, which we integrated with transcriptional data, to explore the relationships between these two marks. We found that ectopic H3K9m2 observed in met1 can be due to defects in IBM1-mediated H3K9m2 demethylation at some sites, but most importantly targets H3K27m3-marked genes, suggesting an interplay between these two silencing marks. Furthermore, H3K9m2/DNA-hypermethylation at these PcG targets in met1 is coupled with a decrease in H3K27m3 marks, whereas CG/H3K9m2 hypomethylated transposons become ectopically H3K27m3 hypermethylated. Our results bear interesting similarities with cancer cells, which show global losses of DNA methylation but ectopic hypermethylation of genes previously marked by H3K27m3.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , ADN (Citosina-5-)-Metiltransferasas , Metilación de ADN , N-Metiltransferasa de Histona-Lisina , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Elementos Transponibles de ADN/genética , Epigénesis Genética , Regulación de la Expresión Génica de las Plantas , Heterocromatina/genética , Histonas/genética , Secuencias Repetitivas Esparcidas , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Lisina/genética , Proteínas del Grupo Polycomb/genética , Proteínas del Grupo Polycomb/metabolismoRESUMEN
Nucleosomes package eukaryotic DNA and are composed of four different histone proteins, designated H3, H4, H2A, and H2B. Histone H3 has two main variants, H3.1 and H3.3, which show different genomic localization patterns in animals. We profiled H3.1 and H3.3 variants in the genome of the plant Arabidopsis thaliana and found that the localization of these variants shows broad similarity in plants and animals, along with some unique features. H3.1 was enriched in silent areas of the genome, including regions containing the repressive chromatin modifications H3 lysine 27 methylation, H3 lysine 9 methylation, and DNA methylation. In contrast, H3.3 was enriched in actively transcribed genes, especially peaking at the 3' end of genes, and correlated with histone modifications associated with gene activation, such as histone H3 lysine 4 methylation and H2B ubiquitylation, as well as RNA Pol II occupancy. Surprisingly, both H3.1 and H3.3 were enriched on defined origins of replication, as was overall nucleosome density, suggesting a novel characteristic of plant origins. Our results are broadly consistent with the hypothesis that H3.1 acts as the canonical histone that is incorporated during DNA replication, whereas H3.3 acts as the replacement histone that can be incorporated outside of S-phase during chromatin-disrupting processes like transcription.
Asunto(s)
Arabidopsis/genética , Genoma de Planta , Histonas/genética , Inmunoprecipitación de Cromatina , Metilación de ADNRESUMEN
The relationship between epigenetic marks on chromatin and the regulation of DNA replication is poorly understood. Mutations of the H3K27 methyltransferase genes, Arabidopsis trithorax-related protein5 (ATXR5) and ATXR6, result in re-replication (repeated origin firing within the same cell cycle). Here we show that mutations that reduce DNA methylation act to suppress the re-replication phenotype of atxr5 atxr6 mutants. This suggests that DNA methylation, a mark enriched at the same heterochromatic regions that re-replicate in atxr5/6 mutants, is required for aberrant re-replication. In contrast, RNA sequencing analyses suggest that ATXR5/6 and DNA methylation cooperatively transcriptionally silence transposable elements (TEs). Hence our results suggest a complex relationship between ATXR5/6 and DNA methylation in the regulation of DNA replication and transcription of TEs.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/genética , Metilación de ADN/genética , Replicación del ADN , Heterocromatina , Metiltransferasas , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ciclo Celular/genética , Replicación del ADN/genética , Elementos Transponibles de ADN/genética , Epigénesis Genética/genética , Expresión Génica , Heterocromatina/genética , N-Metiltransferasa de Histona-Lisina/genética , Recombinación Homóloga , Metiltransferasas/genética , Metiltransferasas/metabolismo , Mutación , Origen de Réplica/genética , Análisis de Secuencia de ARNRESUMEN
In eukaryotic cells, environmental and developmental signals alter chromatin structure and modulate gene expression. Heterochromatin constitutes the transcriptionally inactive state of the genome and in plants and mammals is generally characterized by DNA methylation and histone modifications such as histone H3 lysine 9 (H3K9) methylation. In Arabidopsis thaliana, DNA methylation and H3K9 methylation are usually colocated and set up a mutually self-reinforcing and stable state. Here, in contrast, we found that SUVR5, a plant Su(var)3-9 homolog with a SET histone methyltransferase domain, mediates H3K9me2 deposition and regulates gene expression in a DNA methylation-independent manner. SUVR5 binds DNA through its zinc fingers and represses the expression of a subset of stimulus response genes. This represents a novel mechanism for plants to regulate their chromatin and transcriptional state, which may allow for the adaptability and modulation necessary to rapidly respond to extracellular cues.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Metilación de ADN , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Proteínas de Arabidopsis/química , Secuencia de Bases , Sitios de Unión , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Histona Demetilasas/metabolismo , N-Metiltransferasa de Histona-Lisina/química , Motivos de Nucleótidos , Regiones Promotoras Genéticas , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Dedos de ZincRESUMEN
Cytosine residues in the vertebrate genome are enzymatically modified to 5-methylcytosine, which participates in transcriptional repression of genes during development and disease progression. 5-Methylcytosine can be further enzymatically modified to 5-hydroxymethylcytosine by the TET family of methylcytosine dioxygenases. Analysis of 5-methylcytosine and 5-hydroxymethylcytosine is confounded, as these modifications are indistinguishable by traditional sequencing methods even when supplemented by bisulfite conversion. Here we demonstrate a simple enzymatic approach that involves cloning, identification, and quantification of 5-hydroxymethylcytosine in various CCGG loci within murine and human genomes. 5-Hydroxymethylcytosine was prevalent in human and murine brain and heart genomic DNAs at several regions. The cultured cell lines NIH3T3 and HeLa both displayed very low or undetectable amounts of 5-hydroxymethylcytosine at the examined loci. Interestingly, 5-hydroxymethylcytosine levels in mouse embryonic stem cell DNA first increased then slowly decreased upon differentiation to embryoid bodies, whereas 5-methylcytosine levels increased gradually over time. Finally, using a quantitative PCR approach, we established that a portion of VANGL1 and EGFR gene body methylation in human tissue DNA samples is indeed hydroxymethylation.
Asunto(s)
Islas de CpG/fisiología , Citosina/análogos & derivados , Metilación de ADN/fisiología , Genoma Humano/fisiología , 5-Metilcitosina/análogos & derivados , Animales , Encéfalo/metabolismo , Citosina/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células HeLa , Humanos , Ratones , Miocardio/metabolismo , Células 3T3 NIH , Especificidad de ÓrganosRESUMEN
Although embryonic brain development and neurodegeneration have received considerable attention, the events that govern postnatal brain maturation are less understood. Here, we identify the miR-29 family to be strikingly induced during the late stages of brain maturation. Brain maturation is associated with a transient, postnatal period of de novo non-CG (CH) DNA methylation mediated by DNMT3A. We examine whether an important function of miR-29 during brain maturation is to restrict the period of CH methylation via its targeting of Dnmt3a. Deletion of miR-29 in the brain, or knockin mutations preventing miR-29 to specifically target Dnmt3a, result in increased DNMT3A expression, higher CH methylation, and repression of genes associated with neuronal activity and neuropsychiatric disorders. These mouse models also develop neurological deficits and premature lethality. Our results identify an essential role for miR-29 in restricting CH methylation in the brain and illustrate the importance of CH methylation regulation for normal brain maturation.
Asunto(s)
Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Metilación de ADN/genética , MicroARNs/metabolismo , Regiones no Traducidas 3'/genética , Animales , Animales Recién Nacidos , Secuencia de Bases , Conducta Animal , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Regulación hacia Abajo/genética , Regulación del Desarrollo de la Expresión Génica , Ratones Endogámicos C57BL , MicroARNs/genética , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/patología , Neuronas/metabolismo , Neuronas/patología , Convulsiones/genética , Convulsiones/patología , Transducción de Señal , Sinapsis/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
The maturation of the mammalian brain occurs after birth, and this stage of neuronal development is frequently impaired in neurological disorders, such as autism and schizophrenia. However, the mechanisms that regulate postnatal brain maturation are poorly defined. By purifying neuronal subpopulations across brain development in mice, we identify a postnatal switch in the transcriptional regulatory circuits that operates in the maturing mammalian brain. We show that this developmental transition includes the formation of hundreds of cell-type-specific neuronal enhancers that appear to be modulated by neuronal activity. Once selected, these enhancers are active throughout adulthood, suggesting that their formation in early life shapes neuronal identity and regulates mature brain function.