RESUMO
Over millions of years, eukaryotes evolved from unicellular to multicellular organisms with increasingly complex genomes and sophisticated gene expression networks. Consequently, chromatin regulators evolved to support this increased complexity. The ATP-dependent chromatin remodelers of the SWI/SNF family are multiprotein complexes that modulate nucleosome positioning and appear under different configurations, which perform distinct functions. While the composition, architecture, and activity of these subclasses are well understood in a limited number of fungal and animal model organisms, the lack of comprehensive information in other eukaryotic organisms precludes the identification of a reliable evolutionary model of SWI/SNF complexes. Here, we performed a systematic analysis using 36 species from animal, fungal, and plant lineages to assess the conservation of known SWI/SNF subunits across eukaryotes. We identified evolutionary relationships that allowed us to propose the composition of a hypothetical ancestral SWI/SNF complex in the last eukaryotic common ancestor. This last common ancestor appears to have undergone several rounds of lineage-specific subunit gains and losses, shaping the current conformation of the known subclasses in animals and fungi. In addition, our results unravel a plant SWI/SNF complex, reminiscent of the animal BAF subclass, which incorporates a set of plant-specific subunits of still unknown function.
Assuntos
Proteínas Cromossômicas não Histona , Fatores de Transcrição , Animais , Cromatina , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Eucariotos/genética , Eucariotos/metabolismo , Estruturas Vegetais/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The ability to target epigenetic marks like DNA methylation to specific loci is important in both basic research and in crop plant engineering. However, heritability of targeted DNA methylation, how it impacts gene expression, and which epigenetic features are required for proper establishment are mostly unknown. Here, we show that targeting the CG-specific methyltransferase M.SssI with an artificial zinc finger protein can establish heritable CG methylation and silencing of a targeted locus in Arabidopsis. In addition, we observe highly heritable widespread ectopic CG methylation mainly over euchromatic regions. This hypermethylation shows little effect on transcription while it triggers a mild but significant reduction in the accumulation of H2A.Z and H3K27me3. Moreover, ectopic methylation occurs preferentially at less open chromatin that lacks positive histone marks. These results outline general principles of the heritability and interaction of CG methylation with other epigenomic features that should help guide future efforts to engineer epigenomes.
Assuntos
Arabidopsis/genética , Proteínas de Bactérias/genética , Metilação de DNA , DNA-Citosina Metilases/genética , Regulação da Expressão Gênica de Plantas , Spiroplasma/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Bactérias/metabolismo , Cromatina/genética , Cromatina/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação/métodos , DNA-Citosina Metilases/metabolismo , Histonas/metabolismo , Plantas Geneticamente Modificadas , RNA-Seq/métodos , Spiroplasma/enzimologiaRESUMO
Transcription by RNA polymerase V (Pol V) in plants is required for RNA-directed DNA methylation, leading to transcriptional gene silencing. Global chromatin association of Pol V requires components of the DDR complex DRD1, DMS3 and RDM1, but the assembly process of this complex and the underlying mechanism for Pol V recruitment remain unknown. Here we show that all DDR complex components co-localize with Pol V, and we report the cryoEM structures of two complexes associated with Pol V recruitment-DR (DMS3-RDM1) and DDR' (DMS3-RDM1-DRD1 peptide), at 3.6 Å and 3.5 Å resolution, respectively. RDM1 dimerization at the center frames the assembly of the entire complex and mediates interactions between DMS3 and DRD1 with a stoichiometry of 1 DRD1:4 DMS3:2 RDM1. DRD1 binding to the DR complex induces a drastic movement of a DMS3 coiled-coil helix bundle. We hypothesize that both complexes are functional intermediates that mediate Pol V recruitment.
Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , RNA de Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/ultraestrutura , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/ultraestrutura , Microscopia Crioeletrônica , DNA de Plantas/genética , DNA de Plantas/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/ultraestrutura , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/ultraestrutura , Regulação da Expressão Gênica de Plantas , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Conformação Proteica , RNA de Plantas/química , RNA de Plantas/genéticaRESUMO
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.
