RESUMEN
Nucleosomes block access to DNA methyltransferase, unless they are remodeled by DECREASE in DNA METHYLATION 1 (DDM1LSH/HELLS), a Snf2-like master regulator of epigenetic inheritance. We show that DDM1 promotes replacement of histone variant H3.3 by H3.1. In ddm1 mutants, DNA methylation is partly restored by loss of the H3.3 chaperone HIRA, while the H3.1 chaperone CAF-1 becomes essential. The single-particle cryo-EM structure at 3.2 Å of DDM1 with a variant nucleosome reveals engagement with histone H3.3 near residues required for assembly and with the unmodified H4 tail. An N-terminal autoinhibitory domain inhibits activity, while a disulfide bond in the helicase domain supports activity. DDM1 co-localizes with H3.1 and H3.3 during the cell cycle, and with the DNA methyltransferase MET1Dnmt1, but is blocked by H4K16 acetylation. The male germline H3.3 variant MGH3/HTR10 is resistant to remodeling by DDM1 and acts as a placeholder nucleosome in sperm cells for epigenetic inheritance.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Metilación de ADN , Histonas , Nucleosomas , Ensamble y Desensamble de Cromatina , ADN , Metilasas de Modificación del ADN , Epigénesis Genética , Histonas/genética , Nucleosomas/genética , Semen , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismoRESUMEN
Histones serve many purposes in eukaryotic cells in the regulation of diverse genomic processes, including transcription, replication, DNA repair, and chromatin organization. As such, experimental systems to assess histone function are fundamental resources toward elucidating the regulation of activities occurring on chromatin. One set of important tools for investigating histone function are histone replacement systems, in which endogenous histone expression can be partially or completely replaced with a mutant histone. Histone replacement systems allow systematic screens of histone regulatory functions and the direct assessment of functions for histone residues. In this review, we describe existing histone replacement systems in model organisms, the benefits and limitations of these systems, and opportunities for future research with histone replacement strategies.
Asunto(s)
Cromatina , Histonas , Histonas/metabolismo , Ensamble y Desensamble de Cromatina , Células Eucariotas/metabolismo , Reparación del ADNRESUMEN
Attenuated strains of the naturally occurring plant pathogen Agrobacterium tumefaciens can transfer virtually any DNA sequence of interest to model plants and crops. This has made Agrobacterium-mediated transformation (AMT) one of the most commonly used tools in agricultural biotechnology. Understanding AMT, and its functional consequences, is of fundamental importance given that it sits at the intersection of many fundamental fields of study, including plant-microbe interactions, DNA repair/genome stability, and epigenetic regulation of gene expression. Despite extensive research and use of AMT over the last 40 years, the extent of genomic disruption associated with integrating exogenous DNA into plant genomes using this method remains underappreciated. However, new technologies like long-read sequencing make this disruption more apparent, complementing previous findings from multiple research groups that have tackled this question in the past. In this review, we cover progress on the molecular mechanisms involved in Agrobacterium-mediated DNA integration into plant genomes. We also discuss localized mutations at the site of insertion and describe the structure of these DNA insertions, which can range from single copy insertions to large concatemers, consisting of complex DNA originating from different sources. Finally, we discuss the prevalence of large-scale genomic rearrangements associated with the integration of DNA during AMT with examples. Understanding the intended and unintended effects of AMT on genome stability is critical to all plant researchers who use this methodology to generate new genetic variants.
Asunto(s)
Epigénesis Genética , Plantas , Plantas/genética , Plantas/microbiología , Agrobacterium tumefaciens/genética , Genómica , ADN , Inestabilidad Genómica/genética , Transformación Genética , ADN Bacteriano/genética , Plantas Modificadas Genéticamente/genéticaRESUMEN
Despite the broad array of roles for epigenetic mechanisms on regulating diverse processes in eukaryotes, no experimental system is currently available in plants for the direct assessment of histone function. In this work, we present the development of a genetic strategy in Arabidopsis (Arabidopsis thaliana) whereby modified histone H4 transgenes can completely replace the expression of endogenous histone H4 genes. Accordingly, we established a collection of plants expressing different H4 point mutants targeting residues that may be post-translationally modified in vivo. To demonstrate its utility, we screened this new H4 mutant collection to uncover substitutions in H4 that alter flowering time. We identified different mutations in the H4 tail (H4R17A) and the H4 globular domain (H4R36A, H4R39K, H4R39A, and H4K44A) that strongly accelerate the floral transition. Furthermore, we identified a conserved regulatory relationship between H4R17 and the ISWI chromatin remodeling complex in plants: As with other biological systems, H4R17 regulates nucleosome spacing via ISWI. Overall, this work provides a large set of H4 mutants to the plant epigenetics community that can be used to systematically assess histone H4 function in Arabidopsis and a roadmap to replicate this strategy for studying other histone proteins in plants.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Histonas/metabolismo , Nucleosomas/metabolismoRESUMEN
Epigenetic mechanisms play diverse roles in the regulation of genome stability in eukaryotes. In Arabidopsis thaliana, genome stability is maintained during DNA replication by the H3.1K27 methyltransferases ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) and ATXR6, which catalyze the deposition of K27me1 on replication-dependent H3.1 variants. The loss of H3.1K27me1 in atxr5 atxr6 double mutants leads to heterochromatin defects, including transcriptional de-repression and genomic instability, but the molecular mechanisms involved remain largely unknown. In this study, we identified the transcriptional co-activator and conserved histone acetyltransferase GCN5 as a mediator of transcriptional de-repression and genomic instability in the absence of H3.1K27me1. GCN5 is part of a SAGA-like complex in plants that requires the GCN5-interacting protein ADA2b and the chromatin remodeler CHR6 to mediate the heterochromatic defects in atxr5 atxr6 mutants. Our results also indicate that Arabidopsis GCN5 acetylates multiple lysine residues on H3.1 variants, but H3.1K27 and H3.1K36 play essential functions in inducing genomic instability in the absence of H3.1K27me1. Finally, we show that H3.1K36 acetylation by GCN5 is negatively regulated by H3.1K27me1 in vitro. Overall, this work reveals a key molecular role for H3.1K27me1 in maintaining transcriptional silencing and genome stability in heterochromatin by restricting GCN5-mediated histone acetylation in plants.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Inestabilidad Genómica , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Acetilación , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Silenciador del Gen , Genoma de Planta , Heterocromatina/genética , Heterocromatina/metabolismo , Histona Acetiltransferasas/genética , Histonas/genética , Lisina/genética , Metilación , Metiltransferasas/genética , Metiltransferasas/metabolismo , Mutación , Plantas Modificadas Genéticamente , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Flowering of the reference legume Medicago truncatula is promoted by winter cold (vernalization) followed by long-day photoperiods (VLD) similar to winter annual Arabidopsis. However, Medicago lacks FLC and CO, key regulators of Arabidopsis VLD flowering. Most plants have two INHIBITOR OF GROWTH (ING) genes (ING1 and ING2), encoding proteins with an ING domain with two anti-parallel alpha-helices and a plant homeodomain (PHD) finger, but their genetic role has not been previously described. In Medicago, Mting1 gene-edited mutants developed and flowered normally, but an Mting2-1 Tnt1 insertion mutant and gene-edited Mting2 mutants had developmental abnormalities including delayed flowering particularly in VLD, compact architecture, abnormal leaves with extra leaflets but no trichomes, and smaller seeds and barrels. Mting2 mutants had reduced expression of activators of flowering, including the FT-like gene MtFTa1, and increased expression of the candidate repressor MtTFL1c, consistent with the delayed flowering of the mutant. MtING2 overexpression complemented Mting2-1, but did not accelerate flowering in wild type. The MtING2 PHD finger bound H3K4me2/3 peptides weakly in vitro, but analysis of gene-edited mutants indicated that it was dispensable to MtING2 function in wild-type plants. RNA sequencing experiments indicated that >7000 genes are mis-expressed in the Mting2-1 mutant, consistent with its strong mutant phenotypes. Interestingly, ChIP-seq analysis identified >5000 novel H3K4me3 locations in the genome of Mting2-1 mutants compared to wild type R108. Overall, our mutant study has uncovered an important physiological role of a plant ING2 gene in development, flowering, and gene expression, which likely involves an epigenetic mechanism.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Medicago truncatula , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Plantas/metabolismo , Dedos de Zinc PHD , Flores , Medicago truncatula/genética , Medicago truncatula/metabolismo , Expresión Génica , Regulación de la Expresión Génica de las Plantas/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Dominio MADS/genéticaRESUMEN
The nucleosome is composed of histones and DNA. Prior to their deposition on chromatin, histones are shielded by specialized and diverse proteins known as histone chaperones. They escort histones during their entire cellular life and ensure their proper incorporation in chromatin. Physarum polycephalum is a Mycetozoan, a clade located at the crown of the eukaryotic tree. We previously found that histones, which are highly conserved between plants and animals, are also highly conserved in Physarum. However, histone chaperones differ significantly between animal and plant kingdoms, and this thus probed us to further study the conservation of histone chaperones in Physarum and their evolution relative to animal and plants. Most of the known histone chaperones and their functional domains are conserved as well as key residues required for histone and chaperone interactions. Physarum is divergent from yeast, plants and animals, but PpHIRA, PpCABIN1 and PpSPT6 are similar in structure to plant orthologues. PpFACT is closely related to the yeast complex, and the Physarum genome encodes the animal-specific APFL chaperone. Furthermore, we performed RNA sequencing to monitor chaperone expression during the cell cycle and uncovered two distinct patterns during S-phase. In summary, our study demonstrates the conserved role of histone chaperones in handling histones in an early-branching eukaryote.
Asunto(s)
Histonas , Physarum polycephalum , Animales , Histonas/metabolismo , Physarum polycephalum/genética , Physarum polycephalum/metabolismo , Chaperonas de Histonas/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromatina/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismoRESUMEN
BACKGROUND: The three-dimensional nuclear arrangement of chromatin impacts many cellular processes operating at the DNA level in animal and plant systems. Chromatin organization is a dynamic process that can be affected by biotic and abiotic stresses. Three-dimensional imaging technology allows to follow these dynamic changes, but only a few semi-automated processing methods currently exist for quantitative analysis of the 3D chromatin organization. RESULTS: We present an automated method, Nuclear Object DetectionJ (NODeJ), developed as an imageJ plugin. This program segments and analyzes high intensity domains in nuclei from 3D images. NODeJ performs a Laplacian convolution on the mask of a nucleus to enhance the contrast of intra-nuclear objects and allow their detection. We reanalyzed public datasets and determined that NODeJ is able to accurately identify heterochromatin domains from a diverse set of Arabidopsis thaliana nuclei stained with DAPI or Hoechst. NODeJ is also able to detect signals in nuclei from DNA FISH experiments, allowing for the analysis of specific targets of interest. CONCLUSION AND AVAILABILITY: NODeJ allows for efficient automated analysis of subnuclear structures by avoiding the semi-automated steps, resulting in reduced processing time and analytical bias. NODeJ is written in Java and provided as an ImageJ plugin with a command line option to perform more high-throughput analyses. NODeJ can be downloaded from https://gitlab.com/axpoulet/image2danalysis/-/releases with source code, documentation and further information avaliable at https://gitlab.com/axpoulet/image2danalysis . The images used in this study are publicly available at https://www.brookes.ac.uk/indepth/images/ and https://doi.org/10.15454/1HSOIE .
Asunto(s)
Arabidopsis , Procesamiento de Imagen Asistido por Computador , Animales , Arabidopsis/genética , Núcleo Celular/genética , Cromatina , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Programas InformáticosRESUMEN
Replication-dependent histone H3.1 and replication-independent histone H3.3 are nearly identical proteins in most multicellular eukaryotes. The N-terminal tails of these H3 variants, where the majority of histone post-translational modifications are made, typically differ by only one amino acid. Despite extensive sequence similarity with H3.3, the H3.1 variant has been hypothesized to play unique roles in cells, as it is specifically expressed and inserted into chromatin during DNA replication. However, identifying a function that is unique to H3.1 during replication has remained elusive. In this review, we discuss recent findings regarding the involvement of the H3.1 variant in regulating the TSK/TONSL-mediated resolution of stalled or broken replication forks. Uncovering this new function for the H3.1 variant has been made possible by the identification of the first proteins containing domains that can selectively bind or modify the H3.1 variant. The functional characterization of H3-variant-specific readers and writers reveals another layer of chromatin-based information regulating transcription, DNA replication, and DNA repair.
Asunto(s)
Eucariontes , Histonas , Cromatina/genética , Reparación del ADN , Replicación del ADN , Eucariontes/genética , Eucariontes/metabolismo , Inestabilidad Genómica , Histonas/metabolismo , Humanos , FN-kappa B/metabolismoRESUMEN
Eukaryotic centromeres contain the kinetochore, which connects chromosomes to the spindle allowing segregation. During meiosis, centromeres are suppressed for inter-homolog crossover, as recombination in these regions can cause chromosome missegregation and aneuploidy. Plant centromeres are surrounded by transposon-dense pericentromeric heterochromatin that is epigenetically silenced by histone 3 lysine 9 dimethylation (H3K9me2), and DNA methylation in CG and non-CG sequence contexts. However, the role of these chromatin modifications in control of meiotic recombination in the pericentromeres is not fully understood. Here, we show that disruption of Arabidopsis thaliana H3K9me2 and non-CG DNA methylation pathways, for example, via mutation of the H3K9 methyltransferase genes KYP/SUVH4 SUVH5 SUVH6, or the CHG DNA methyltransferase gene CMT3, increases meiotic recombination in proximity to the centromeres. Using immunocytological detection of MLH1 foci and genotyping by sequencing of recombinant plants, we observe that H3K9me2 and non-CG DNA methylation pathway mutants show increased pericentromeric crossovers. Increased pericentromeric recombination in H3K9me2/non-CG mutants occurs in hybrid and inbred backgrounds and likely involves contributions from both the interfering and noninterfering crossover repair pathways. We also show that meiotic DNA double-strand breaks (DSBs) increase in H3K9me2/non-CG mutants within the pericentromeres, via purification and sequencing of SPO11-1-oligonucleotides. Therefore, H3K9me2 and non-CG DNA methylation exert a repressive effect on both meiotic DSB and crossover formation in plant pericentromeric heterochromatin. Our results may account for selection of enhancer trap Dissociation (Ds) transposons into the CMT3 gene by recombination with proximal transposon launch-pads.
Asunto(s)
Arabidopsis/genética , Centrómero/genética , ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN/genética , Proteínas de Arabidopsis/genética , Roturas del ADN de Doble Cadena , Epigénesis Genética/genética , Genoma de Planta/genética , Heterocromatina/genética , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Recombinación Homóloga/genética , Meiosis/genética , Metiltransferasas/genéticaRESUMEN
The CRISPR/Cas9 system has greatly improved our ability to engineer targeted mutations in eukaryotic genomes. While CRISPR/Cas9 appears to work universally, the efficiency of targeted mutagenesis and the adverse generation of off-target mutations vary greatly between different organisms. In this study, we report that Arabidopsis plants subjected to heat stress at 37°C show much higher frequencies of CRISPR-induced mutations compared to plants grown continuously at the standard temperature (22°C). Using quantitative assays relying on green fluorescent protein (GFP) reporter genes, we found that targeted mutagenesis by CRISPR/Cas9 in Arabidopsis is increased by approximately 5-fold in somatic tissues and up to 100-fold in the germline upon heat treatment. This effect of temperature on the mutation rate is not limited to Arabidopsis, as we observed a similar increase in targeted mutations by CRISPR/Cas9 in Citrus plants exposed to heat stress at 37°C. In vitro assays demonstrate that Cas9 from Streptococcus pyogenes (SpCas9) is more active in creating double-stranded DNA breaks at 37°C than at 22°C, thus indicating a potential contributing mechanism for the in vivo effect of temperature on CRISPR/Cas9. This study reveals the importance of temperature in modulating SpCas9 activity in eukaryotes, and provides a simple method to increase on-target mutagenesis in plants using CRISPR/Cas9.
Asunto(s)
Arabidopsis/genética , Sistemas CRISPR-Cas/genética , Genoma de Planta/genética , Streptococcus pyogenes/enzimología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Calor , Mutagénesis Sitio-Dirigida , Mutación , Plantas Modificadas Genéticamente , Estrés FisiológicoRESUMEN
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
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
RNA molecules such as small-interfering RNAs (siRNAs) and antisense RNAs (asRNAs) trigger chromatin silencing of target loci. In the model plant Arabidopsis, RNA-triggered chromatin silencing involves repressive histone modifications such as histone deacetylation, histone H3 lysine-9 methylation, and H3 lysine-27 monomethylation. Here, we report that two Arabidopsis homologs of the human histone-binding proteins Retinoblastoma-Associated Protein 46/48 (RbAp46/48), known as MSI4 (or FVE) and MSI5, function in partial redundancy in chromatin silencing of various loci targeted by siRNAs or asRNAs. We show that MSI5 acts in partial redundancy with FVE to silence FLOWERING LOCUS C (FLC), which is a crucial floral repressor subject to asRNA-mediated silencing, FLC homologs, and other loci including transposable and repetitive elements which are targets of siRNA-directed DNA Methylation (RdDM). Both FVE and MSI5 associate with HISTONE DEACETYLASE 6 (HDA6) to form complexes and directly interact with the target loci, leading to histone deacetylation and transcriptional silencing. In addition, these two genes function in de novo CHH (Hâ=âA, T, or C) methylation and maintenance of symmetric cytosine methylation (mainly CHG methylation) at endogenous RdDM target loci, and they are also required for establishment of cytosine methylation in the previously unmethylated sequences directed by the RdDM pathway. This reveals an important functional divergence of the plant RbAp46/48 relatives from animal counterparts.
Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas Portadoras/metabolismo , Cromatina/genética , Silenciador del Gen , Proteínas de Dominio MADS/genética , Proteína 4 de Unión a Retinoblastoma/metabolismo , Arabidopsis/metabolismo , Proteínas Portadoras/genética , Cromatina/metabolismo , Metilación de ADN/genética , Elementos Transponibles de ADN/genética , Flores/genética , Flores/crecimiento & desarrollo , Histona Desacetilasas/metabolismo , Proteínas de Dominio MADS/metabolismo , ARN Interferente Pequeño/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Proteína 4 de Unión a Retinoblastoma/genética , Homología de Secuencia de Aminoácido , Factores de TranscripciónRESUMEN
The replication-dependent histone H3.1 variant, ubiquitous in multicellular eukaryotes, has been proposed to play key roles during chromatin replication due to its unique expression pattern restricted to the S phase of the cell cycle. Here, we describe recent discoveries in plants regarding molecular mechanisms and cellular pathways involving H3.1 that contribute to the maintenance of genomic and epigenomic information. First, we highlight new advances concerning the contribution of the histone chaperone CAF-1 and the TSK-H3.1 DNA repair pathway in preventing genomic instability during replication. We then summarize the evidence connecting H3.1 to specific roles required for the mitotic inheritance of epigenetic states. Finally, we discuss the recent identification of a specific interaction between H3.1 and DNA polymerase epsilon and its functional implications.
Asunto(s)
Epigenómica , Histonas , Histonas/genética , Histonas/metabolismo , Cromatina/genética , Ciclo Celular , Epigénesis Genética , Replicación del ADN/genéticaRESUMEN
Transformation via Agrobacterium tumefaciens (Agrobacterium) is the predominant method used to introduce exogenous DNA into plants. Transfer DNA (T-DNA) originating from Agrobacterium can be integrated as a single copy or in concatenated forms in plant genomes, but the mechanisms affecting final T-DNA structure remain unknown. In this study, we demonstrate that the inclusion of retrotransposon (RT)-derived sequences in T-DNA can increase transgene copy number by more than 50-fold in Arabidopsis thaliana (Arabidopsis). RT-mediated amplification of T-DNA results in large concatemers in the Arabidopsis genome, which are primarily induced by the long terminal repeats (LTRs) of RTs. T-DNA amplification is dependent on the activity of DNA repair proteins associated with theta-mediated end joining (TMEJ). Finally, we show that T-DNA amplification can increase the frequency of targeted mutagenesis and gene targeting. Overall, this work uncovers molecular determinants that modulate T-DNA copy number in Arabidopsis and demonstrates the utility of inducing T-DNA amplification for plant gene editing.
RESUMEN
Transformation via Agrobacterium tumefaciens is the predominant method used to introduce exogenous DNA into plant genomes1,2. Transfer DNA (T-DNA) originating from Agrobacterium can be integrated as a single copy or in complex concatenated forms3,4, but the mechanisms affecting final T-DNA structure remain unknown. Here we demonstrate that inclusion of retrotransposon (RT)-derived sequences in T-DNA can increase T-DNA copy number by more than 50-fold in Arabidopsis thaliana. These additional T-DNA copies are organized into large concatemers, an effect primarily induced by the long terminal repeats (LTRs) of RTs that can be replicated using non-LTR DNA repeats. We found that T-DNA concatenation is dependent on the activity of the DNA repair proteins MRE11, RAD17 and ATR. Finally, we show that T-DNA concatenation can be used to increase the frequency of targeted mutagenesis and gene targeting. Overall, this work uncovers molecular determinants that modulate T-DNA copy number in Arabidopsis and demonstrates the utility of inducing T-DNA concatenation for plant gene editing.
Asunto(s)
Arabidopsis , Edición Génica , Genoma de Planta , Retroelementos/genética , Genes de Plantas , Arabidopsis/genéticaRESUMEN
OBJECTIVE: To determine the overall tracking errors inherent to the co-calibration procedure of AlignRT InBore™'s (Vision RT Ltd., London, UK) ceiling-mounted and ring-mounted cameras. METHODS: Extrinsic calibration errors related to the mismatch between ceiling and InBore cameras' isocentres and treatment isocentre were determined using MV images and the SRS package and compared to traditional plate-based error. Next, using a realistic anthropomorphic female phantom, intrinsic calibration errors were determined while varying source-skin distance (80 to 100 cm), breast board inclination (0° to 12.5°), room lighting conditions (0 to 258 lx), skin colour (dark, white and natural skin colour), and pod occlusion. RESULTS: MV images of the cube proved plate-based calibration to suffer from large errors especially in the vertical direction (up to 2 mm). Intrinsic calibration errors were considerably lower. Indeed, RTD values of ceiling and InBore cameras showed little variability with isocentre depth (within 1.0 mm/0.4°), surface orientation and breast board inclination (within 0.7 mm/0.3°), changing lighting conditions (within 0.1 mm/0.2°), skin colour/tone (within 0.3 mm/0.3°) and camera pod occlusion (within 0.3 mm/0.2°). CONCLUSION: The use of MV-images proved critical to maintain co-calibrating errors of ceiling and InBore cameras to Halcyon's treatment isocentre below 1 mm.
Asunto(s)
Fantasmas de Imagen , Calibración , Humanos , Femenino , Piel/diagnóstico por imagenRESUMEN
The H3 methyltransferases ATXR5 and ATXR6 deposit H3.1K27me1 to heterochromatin to prevent genomic instability and transposon re-activation. Here, we report that atxr5 atxr6 mutants display robust resistance to Geminivirus. The viral resistance is correlated with activation of DNA repair pathways, but not with transposon re-activation or heterochromatin amplification. We identify RAD51 and RPA1A as partners of virus-encoded Rep protein. The two DNA repair proteins show increased binding to heterochromatic regions and defense-related genes in atxr5 atxr6 vs wild-type plants. Consequently, the proteins have reduced binding to viral DNA in the mutant, thus hampering viral amplification. Additionally, RAD51 recruitment to the host genome arise via BRCA1, HOP2, and CYCB1;1, and this recruitment is essential for viral resistance in atxr5 atxr6. Thus, Geminiviruses adapt to healthy plants by hijacking DNA repair pathways, whereas the unstable genome, triggered by reduced H3.1K27me1, could retain DNA repairing proteins to suppress viral amplification in atxr5 atxr6.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Geminiviridae , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Heterocromatina/metabolismo , Geminiviridae/genética , Histonas/metabolismo , Replicación del ADN , Reparación del ADN/genética , Metiltransferasas/metabolismoRESUMEN
Epigenetic inheritance refers to the faithful replication of DNA methylation and histone modification independent of DNA sequence. Nucleosomes block access to DNA methyltransferases, unless they are remodeled by DECREASE IN DNA METHYLATION1 (DDM1 Lsh/HELLS ), a Snf2-like master regulator of epigenetic inheritance. We show that DDM1 activity results in replacement of the transcriptional histone variant H3.3 for the replicative variant H3.1 during the cell cycle. In ddm1 mutants, DNA methylation can be restored by loss of the H3.3 chaperone HIRA, while the H3.1 chaperone CAF-1 becomes essential. The single-particle cryo-EM structure at 3.2 Å of DDM1 with a variant nucleosome reveals direct engagement at SHL2 with histone H3.3 at or near variant residues required for assembly, as well as with the deacetylated H4 tail. An N-terminal autoinhibitory domain binds H2A variants to allow remodeling, while a disulfide bond in the helicase domain is essential for activity in vivo and in vitro . We show that differential remodeling of H3 and H2A variants in vitro reflects preferential deposition in vivo . DDM1 co-localizes with H3.1 and H3.3 during the cell cycle, and with the DNA methyltransferase MET1 Dnmt1 . DDM1 localization to the chromosome is blocked by H4K16 acetylation, which accumulates at DDM1 targets in ddm1 mutants, as does the sperm cell specific H3.3 variant MGH3 in pollen, which acts as a placeholder nucleosome in the germline and contributes to epigenetic inheritance.