RESUMO
Tomato Dicer-like2 (slDCL2) is a key component of resistance pathways against potato virus X (PVX) and tobacco mosaic virus (TMV). It is also required for production of endogenous small RNAs, including miR6026 and other noncanonical microRNAs (miRNAs). The slDCL2 mRNAs are targets of these slDCL2-dependent RNAs in a feedback loop that was disrupted by target mimic RNAs of miR6026. In lines expressing these RNAs, there was correspondingly enhanced resistance against PVX and TMV. These findings illustrate a novel miRNA pathway in plants and a crop protection strategy in which miRNA target mimicry elevates expression of defense-related mRNAs.
Assuntos
MicroRNAs/metabolismo , Vírus de RNA/fisiologia , Ribonuclease III/metabolismo , Solanum lycopersicum/genética , Solanum lycopersicum/virologia , Sistemas CRISPR-Cas , Suscetibilidade a Doenças , Solanum lycopersicum/enzimologia , Mutação , Doenças das Plantas/virologia , Pequeno RNA não Traduzido/metabolismo , Ribonuclease III/genéticaRESUMO
During meiosis, homologous chromosomes undergo crossover recombination, which is typically concentrated in narrow hot spots that are controlled by genetic and epigenetic information. Arabidopsis chromosomes are highly DNA methylated in the repetitive centromeres, which are also crossover-suppressed. Here we demonstrate that RNA-directed DNA methylation is sufficient to locally silence Arabidopsis euchromatic crossover hot spots and is associated with increased nucleosome density and H3K9me2. However, loss of CG DNA methylation maintenance in met1 triggers epigenetic crossover remodeling at the chromosome scale, with pericentromeric decreases and euchromatic increases in recombination. We used recombination mutants that alter interfering and noninterfering crossover repair pathways (fancm and zip4) to demonstrate that remodeling primarily involves redistribution of interfering crossovers. Using whole-genome bisulfite sequencing, we show that crossover remodeling is driven by loss of CG methylation within the centromeric regions. Using cytogenetics, we profiled meiotic DNA double-strand break (DSB) foci in met1 and found them unchanged relative to wild type. We propose that met1 chromosome structure is altered, causing centromere-proximal DSBs to be inhibited from maturation into interfering crossovers. These data demonstrate that DNA methylation is sufficient to silence crossover hot spots and plays a key role in establishing domains of meiotic recombination along chromosomes.
Assuntos
Arabidopsis/genética , Cromossomos de Plantas/genética , Troca Genética/genética , Metilação de DNA , Epigenômica , Recombinação Homóloga/genética , Meiose/genética , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Centrômero/genética , DNA (Citosina-5-)-Metiltransferases/genética , Quebras de DNA de Cadeia Dupla , Mutação , NucleossomosRESUMO
Meiotic recombination initiates from DNA double-strand breaks (DSBs) generated by SPO11 topoisomerase-like complexes. Meiotic DSB frequency varies extensively along eukaryotic chromosomes, with hotspots controlled by chromatin and DNA sequence. To map meiotic DSBs throughout a plant genome, we purified and sequenced Arabidopsis thaliana SPO11-1-oligonucleotides. SPO11-1-oligos are elevated in gene promoters, terminators, and introns, which is driven by AT-sequence richness that excludes nucleosomes and allows SPO11-1 access. A positive relationship was observed between SPO11-1-oligos and crossovers genome-wide, although fine-scale correlations were weaker. This may reflect the influence of interhomolog polymorphism on crossover formation, downstream from DSB formation. Although H3K4me3 is enriched in proximity to SPO11-1-oligo hotspots at gene 5' ends, H3K4me3 levels do not correlate with DSBs. Repetitive transposons are thought to be recombination silenced during meiosis, to prevent nonallelic interactions and genome instability. Unexpectedly, we found high SPO11-1-oligo levels in nucleosome-depleted Helitron/Pogo/Tc1/Mariner DNA transposons, whereas retrotransposons were coldspots. High SPO11-1-oligo transposons are enriched within gene regulatory regions and in proximity to immunity genes, suggesting a role as recombination enhancers. As transposon mobility in plant genomes is restricted by DNA methylation, we used the met1 DNA methyltransferase mutant to investigate the role of heterochromatin in SPO11-1-oligo distributions. Epigenetic activation of meiotic DSBs in proximity to centromeres and transposons occurred in met1 mutants, coincident with reduced nucleosome occupancy, gain of transcription, and H3K4me3. Together, our work reveals a complex relationship between chromatin and meiotic DSBs within A. thaliana genes and transposons, with significance for the diversity and evolution of plant genomes.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Metilação de DNA/genética , Nucleossomos/genética , Cromossomos Fúngicos , Quebras de DNA de Cadeia Dupla , Elementos de DNA Transponíveis/genética , Epigênese Genética/genética , Meiose/genética , Sequências Reguladoras de Ácido Nucleico/genéticaRESUMO
The ability for cut tissues to join and form a chimeric organism is a remarkable property of many plants; however, grafting is poorly characterized at the molecular level. To better understand this process, we monitored genome-wide gene expression changes in grafted Arabidopsis thaliana hypocotyls. We observed a sequential activation of genes associated with cambium, phloem, and xylem formation. Tissues above and below the graft rapidly developed an asymmetry such that many genes were more highly expressed on one side than on the other. This asymmetry correlated with sugar-responsive genes, and we observed an accumulation of starch above the graft junction. This accumulation decreased along with asymmetry once the sugar-transporting vascular tissues reconnected. Despite the initial starvation response below the graft, many genes associated with vascular formation were rapidly activated in grafted tissues but not in cut and separated tissues, indicating that a recognition mechanism was activated independently of functional vascular connections. Auxin, which is transported cell to cell, had a rapidly elevated response that was symmetric, suggesting that auxin was perceived by the root within hours of tissue attachment to activate the vascular regeneration process. A subset of genes was expressed only in grafted tissues, indicating that wound healing proceeded via different mechanisms depending on the presence or absence of adjoining tissues. Such a recognition process could have broader relevance for tissue regeneration, intertissue communication, and tissue fusion events.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Feixe Vascular de Plantas/fisiologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cruzamento , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos/metabolismo , Feixe Vascular de Plantas/genética , Regeneração , TranscriptomaRESUMO
RNA silencing at the transcriptional and posttranscriptional levels regulates endogenous gene expression, controls invading transposable elements (TEs), and protects the cell against viruses. Key components of the mechanism are small RNAs (sRNAs) of 21-24 nt that guide the silencing machinery to their nucleic acid targets in a nucleotide sequence-specific manner. Transcriptional gene silencing is associated with 24-nt sRNAs and RNA-directed DNA methylation (RdDM) at cytosine residues in three DNA sequence contexts (CG, CHG, and CHH). We previously demonstrated that 24-nt sRNAs are mobile from shoot to root in Arabidopsis thaliana and confirmed that they mediate DNA methylation at three sites in recipient cells. In this study, we extend this finding by demonstrating that RdDM of thousands of loci in root tissues is dependent upon mobile sRNAs from the shoot and that mobile sRNA-dependent DNA methylation occurs predominantly in non-CG contexts. Mobile sRNA-dependent non-CG methylation is largely dependent on the DOMAINS REARRANGED METHYLTRANSFERASES 1/2 (DRM1/DRM2) RdDM pathway but is independent of the CHROMOMETHYLASE (CMT)2/3 DNA methyltransferases. Specific superfamilies of TEs, including those typically found in gene-rich euchromatic regions, lose DNA methylation in a mutant lacking 22- to 24-nt sRNAs (dicer-like 2, 3, 4 triple mutant). Transcriptome analyses identified a small number of genes whose expression in roots is associated with mobile sRNAs and connected to DNA methylation directly or indirectly. Finally, we demonstrate that sRNAs from shoots of one accession move across a graft union and target DNA methylation de novo at normally unmethylated sites in the genomes of root cells from a different accession.
Assuntos
Arabidopsis/genética , Metilação de DNA/genética , Genoma de Planta , RNA de Plantas/genética , Alelos , Elementos de DNA Transponíveis/genética , Regulação da Expressão Gênica de Plantas , Loci Gênicos , Raízes de Plantas/genética , RNA de Plantas/metabolismoRESUMO
Meiotic crossover frequency varies extensively along chromosomes and is typically concentrated in hotspots. As recombination increases genetic diversity, hotspots are predicted to occur at immunity genes, where variation may be beneficial. A major component of plant immunity is recognition of pathogen Avirulence (Avr) effectors by resistance (R) genes that encode NBS-LRR domain proteins. Therefore, we sought to test whether NBS-LRR genes would overlap with meiotic crossover hotspots using experimental genetics in Arabidopsis thaliana. NBS-LRR genes tend to physically cluster in plant genomes; for example, in Arabidopsis most are located in large clusters on the south arms of chromosomes 1 and 5. We experimentally mapped 1,439 crossovers within these clusters and observed NBS-LRR gene associated hotspots, which were also detected as historical hotspots via analysis of linkage disequilibrium. However, we also observed NBS-LRR gene coldspots, which in some cases correlate with structural heterozygosity. To study recombination at the fine-scale we used high-throughput sequencing to analyze ~1,000 crossovers within the RESISTANCE TO ALBUGO CANDIDA1 (RAC1) R gene hotspot. This revealed elevated intragenic crossovers, overlapping nucleosome-occupied exons that encode the TIR, NBS and LRR domains. The highest RAC1 recombination frequency was promoter-proximal and overlapped CTT-repeat DNA sequence motifs, which have previously been associated with plant crossover hotspots. Additionally, we show a significant influence of natural genetic variation on NBS-LRR cluster recombination rates, using crosses between Arabidopsis ecotypes. In conclusion, we show that a subset of NBS-LRR genes are strong hotspots, whereas others are coldspots. This reveals a complex recombination landscape in Arabidopsis NBS-LRR genes, which we propose results from varying coevolutionary pressures exerted by host-pathogen relationships, and is influenced by structural heterozygosity.
Assuntos
Arabidopsis/genética , Resistência à Doença/genética , Recombinação Genética , Alelos , Proteínas de Arabidopsis/genética , Cruzamentos Genéticos , Genes de Plantas , Variação Genética , Heterozigoto , Desequilíbrio de Ligação , Meiose , Família Multigênica , Hibridização de Ácido Nucleico , Doenças das Plantas/genética , Pólen/metabolismoRESUMO
Contents Summary 540 I. Introduction 540 II. There are different types of sRNA mobility 541 III. Mechanisms of sRNA movement 541 IV. Long-distance, shoot-root, mobile siRNAs influence DNA methylation in recipient tissues 541 V. Classes of interactions between shoot-root mobile siRNAs and DNA methylation 542 VI. Loci targeted directly and indirectly by shoot-root mobile siRNAs are associated with different histone modifications 543 VII. Is mobile siRNA-regulated DNA methylation important in specific tissues or under specific conditions? 543 VIII. Mobile sRNAs can be used to modify plant traits 544 IX. Conclusions 544 Acknowledgements 544 References 544 SUMMARY: RNA-directed DNA methylation (RdDM) at cytosine residues regulates gene expression, silences transposable elements and influences genome stability. The mechanisms responsible for RdDM are guided to target loci by small RNAs (sRNAs) that can move within plants cell to cell and long distance. Here we discuss recent advances in the understanding of interactions between mobile sRNAs and DNA methylation. We describe the mechanisms of sRNA movement, the differences between known classes of mobile sRNA-DNA methylation interactions and the limits of current knowledge. Finally, we discuss potential applications of mobile sRNAs in modifying plant traits.
Assuntos
Metilação de DNA/genética , Genoma de Planta , Transporte de RNA , RNA de Plantas/metabolismo , Loci Gênicos , Histonas/metabolismo , Transporte de RNA/genéticaRESUMO
BACKGROUND: Cytosine methylation is widespread in most eukaryotic genomes and is known to play a substantial role in various regulatory pathways. Unmethylated cytosines may be converted to uracil through the addition of sodium bisulphite, allowing genome-wide quantification of cytosine methylation via high-throughput sequencing. The data thus acquired allows the discovery of methylation 'loci'; contiguous regions of methylation consistently methylated across biological replicates. The mapping of these loci allows for associations with other genomic factors to be identified, and for analyses of differential methylation to take place. RESULTS: The segmentSeq R package is extended to identify methylation loci from high-throughput sequencing data from multiple experimental conditions. A statistical model is then developed that accounts for biological replication and variable rates of non-conversion of cytosines in each sample to compute posterior likelihoods of methylation at each locus within an empirical Bayesian framework. The same model is used as a basis for analysis of differential methylation between multiple experimental conditions with the baySeq R package. We demonstrate the capability of this method to analyse complex data sets in an analysis of data derived from multiple Dicer-like mutants in Arabidopsis. This reveals several novel behaviours at distinct sets of loci in response to loss of one or more of the Dicer-like proteins that indicate an antagonistic relationship between the Dicer-like proteins at at least some methylation loci. Finally, we show in simulation studies that this approach can be significantly more powerful in the detection of differential methylation than many existing methods in data derived from both mammalian and plant systems. CONCLUSIONS: The methods developed here make it possible to analyse high-throughput sequencing of the methylome of any given organism under a diverse set of experimental conditions. The methods are able to identify methylation loci and evaluate the likelihood that a region is truly methylated under any given experimental condition, allowing for downstream analyses that characterise differences between methylated and non-methylated regions of the genome. Futhermore, diverse patterns of differential methylation may also be characterised from these data.
Assuntos
Citosina/metabolismo , Metilação de DNA , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNA , Arabidopsis/genética , Teorema de Bayes , Loci Gênicos , Genoma , Genômica/métodos , Modelos Estatísticos , Ribonuclease III/genética , SoftwareRESUMO
Ribosome profiling is a technique that permits genome-wide, quantitative analysis of translation and has found broad application in recent years. Here we describe a modified profiling protocol and software package designed to benefit more broadly the translation community in terms of simplicity and utility. The protocol, applicable to diverse organisms, including organelles, is based largely on previously published profiling methodologies, but uses duplex-specific nuclease (DSN) as a convenient, species-independent way to reduce rRNA contamination. We show that DSN-based depletion compares favorably with other commonly used rRNA depletion strategies and introduces little bias. The profiling protocol typically produces high levels of triplet periodicity, facilitating the detection of coding sequences, including upstream, downstream, and overlapping open reading frames (ORFs) and an alternative ribosome conformation evident during termination of protein synthesis. In addition, we provide a software package that presents a set of methods for parsing ribosomal profiling data from multiple samples, aligning reads to coding sequences, inferring alternative ORFs, and plotting average and transcript-specific aspects of the data. Methods are also provided for extracting the data in a form suitable for differential analysis of translation and translational efficiency.
Assuntos
Endonucleases/metabolismo , Ribossomos/metabolismo , Software , Chlamydomonas reinhardtii/genética , Biologia Computacional , Fases de Leitura AbertaRESUMO
MOTIVATION: High-throughput data are now commonplace in biological research. Rapidly changing technologies and application mean that novel methods for detecting differential behaviour that account for a 'large P, small n' setting are required at an increasing rate. The development of such methods is, in general, being done on an ad hoc basis, requiring further development cycles and a lack of standardization between analyses. RESULTS: We present here a generalized method for identifying differential behaviour within high-throughput biological data through empirical Bayesian methods. This approach is based on our baySeq algorithm for identification of differential expression in RNA-seq data based on a negative binomial distribution, and in paired data based on a beta-binomial distribution. Here we show how the same empirical Bayesian approach can be applied to any parametric distribution, removing the need for lengthy development of novel methods for differently distributed data. Comparisons with existing methods developed to address specific problems in high-throughput biological data show that these generic methods can achieve equivalent or better performance. A number of enhancements to the basic algorithm are also presented to increase flexibility and reduce computational costs. AVAILABILITY AND IMPLEMENTATION: The methods are implemented in the R baySeq (v2) package, available on Bioconductor http://www.bioconductor.org/packages/release/bioc/html/baySeq.html. CONTACT: tjh48@cam.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Algoritmos , Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA , Animais , Teorema de Bayes , Distribuição Binomial , Ratos , SoftwareRESUMO
Small (s)RNAs of 21 to 24 nucleotides are associated with RNA silencing and methylation of DNA cytosine residues. All sizes can move from cell-to-cell and long distance in plants, directing RNA silencing in destination cells. Twenty-four nucleotide sRNAs are the predominant long-distance mobile species. Thousands move from shoot to root, where they target methylation of transposable elements both directly and indirectly. We derive several classes of interaction between small RNAs and methylation and use these to explore the mechanisms of methylation and gene expression that associate with mobile sRNA signaling.
Assuntos
Citosina/química , Metilação de DNA , Plantas/genética , Pequeno RNA não Traduzido/genética , Elementos de DNA Transponíveis/genética , DNA de Plantas/química , DNA de Plantas/genética , Regulação da Expressão Gênica de Plantas , Raízes de Plantas/genética , Brotos de Planta/genética , RNA de Plantas/genéticaRESUMO
BACKGROUND: Pairing of samples arises naturally in many genomic experiments; for example, gene expression in tumour and normal tissue from the same patients. Methods for analysing high-throughput sequencing data from such experiments are required to identify differential expression, both within paired samples and between pairs under different experimental conditions. RESULTS: We develop an empirical Bayesian method based on the beta-binomial distribution to model paired data from high-throughput sequencing experiments. We examine the performance of this method on simulated and real data in a variety of scenarios. Our methods are implemented as part of the RbaySeq package (versions 1.11.6 and greater) available from Bioconductor (http://www.bioconductor.org). CONCLUSIONS: We compare our approach to alternatives based on generalised linear modelling approaches and show that our method offers significant gains in performance on simulated data. In testing on real data from oral squamous cell carcinoma patients, we discover greater enrichment of previously identified head and neck squamous cell carcinoma associated gene sets than has previously been achieved through a generalised linear modelling approach, suggesting that similar gains in performance may be found in real data. Our methods thus show real and substantial improvements in analyses of high-throughput sequencing data from paired samples.
Assuntos
Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Teorema de Bayes , Distribuição Binomial , Carcinoma de Células Escamosas/genética , Genômica , Neoplasias de Cabeça e Pescoço/genética , Humanos , Modelos Lineares , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
The formation of heterochromatin at the centromeres in fission yeast depends on transcription of the outer repeats. These transcripts are processed into siRNAs that target homologous loci for heterochromatin formation. Here, high throughput sequencing of small RNA provides a comprehensive analysis of centromere-derived small RNAs. We found that the centromeric small RNAs are Dcr1 dependent, carry 5'-monophosphates and are associated with Ago1. The majority of centromeric small RNAs originate from two remarkably well-conserved sequences that are present in all centromeres. The high degree of similarity suggests that this non-coding sequence in itself may be of importance. Consistent with this, secondary structure-probing experiments indicate that this centromeric RNA is partially double-stranded and is processed by Dicer in vitro. We further demonstrate the existence of small centromeric RNA in rdp1Delta cells. Our data suggest a pathway for siRNA generation that is distinct from the well-documented model involving RITS/RDRC. We propose that primary transcripts fold into hairpin-like structures that may be processed by Dcr1 into siRNAs, and that these siRNAs may initiate heterochromatin formation independent of RDRC activity.
Assuntos
Centrômero/ultraestrutura , Regulação Fúngica da Expressão Gênica , RNA Interferente Pequeno/metabolismo , Schizosaccharomyces/fisiologia , Sequência de Bases , Centrômero/metabolismo , Heterocromatina/química , Dados de Sequência Molecular , Família Multigênica , Mutação , Conformação de Ácido Nucleico , RNA/química , RNA/metabolismo , Interferência de RNA , RNA de Cadeia Dupla/química , RNA Interferente Pequeno/química , Schizosaccharomyces/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
MOTIVATION: Small interfering RNAs (siRNAs) are produced from much longer sequences of double-stranded RNA precursors through cleavage by Dicer or a Dicer-like protein. These small RNAs play a key role in genetic and epigenetic regulation; however, a full understanding of the mechanisms by which they operate depends on the characterization of the precursors from which they are derived. RESULTS: High-throughput sequencing of small RNA populations allows the locations of the double-stranded RNA precursors to be inferred. We have developed methods to analyse small RNA sequencing data from multiple biological sources, taking into account replicate information, to identify robust sets of siRNA precursors. Our methods show good performance on both a set of small RNA sequencing data in Arabidopsis thaliana and simulated datasets. AVAILABILITY: Our methods are available as the Bioconductor (www.bioconductor.org) package segmentSeq (version 1.5.6 and above).
Assuntos
Arabidopsis/genética , RNA de Plantas/genética , RNA Interferente Pequeno/genética , Simulação por Computador , Genoma de Planta , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de RNARESUMO
Argonaute (AGO) effectors of RNA silencing bind small RNA (sRNA) molecules and mediate mRNA cleavage, translational repression, or epigenetic DNA modification. In many organisms, these targeting mechanisms are devolved to different products of AGO multigene families. To investigate the basis of AGO functional diversification, we characterized three closely related Arabidopsis thaliana AGOs (AGO4, AGO6, and AGO9) implicated in RNA-directed DNA methylation. All three AGOs bound 5' adenosine 24-nucleotide sRNAs, but each exhibited different preferences for sRNAs from different heterochromatin-associated loci. This difference was reduced when AGO6 and AGO9 were expressed from the AGO4 promoter, indicating that the functional diversification was partially due to differential expression of the corresponding genes. However, the AGO4-directed pattern of sRNA accumulation and DNA methylation was not fully recapitulated with AGO6 or AGO9 expressed from the AGO4 promoter. Here, we show that sRNA length and 5' nucleotide do not account for the observed functional diversification of these AGOs. Instead, the selectivity of sRNA binding is determined by the coincident expression of the AGO and sRNA-generating loci, and epigenetic modification is influenced by interactions between the AGO protein and the different target loci. These findings highlight the importance of tissue specificity and AGO-associated proteins in influencing epigenetic modifications.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Metilação de DNA , Regulação da Expressão Gênica de Plantas , RNA de Plantas/genética , Proteínas de Arabidopsis/metabolismo , Genoma de PlantaRESUMO
BACKGROUND: High throughput sequencing has become an important technology for studying expression levels in many types of genomic, and particularly transcriptomic, data. One key way of analysing such data is to look for elements of the data which display particular patterns of differential expression in order to take these forward for further analysis and validation. RESULTS: We propose a framework for defining patterns of differential expression and develop a novel algorithm, baySeq, which uses an empirical Bayes approach to detect these patterns of differential expression within a set of sequencing samples. The method assumes a negative binomial distribution for the data and derives an empirically determined prior distribution from the entire dataset. We examine the performance of the method on real and simulated data. CONCLUSIONS: Our method performs at least as well, and often better, than existing methods for analyses of pairwise differential expression in both real and simulated data. When we compare methods for the analysis of data from experimental designs involving multiple sample groups, our method again shows substantial gains in performance. We believe that this approach thus represents an important step forward for the analysis of count data from sequencing experiments.
Assuntos
Algoritmos , Teorema de Bayes , Perfilação da Expressão Gênica/métodos , Arabidopsis/genética , Sequência de Bases , RNA de Plantas/genética , Projetos de PesquisaRESUMO
Based on 98 public and internal small RNA high throughput sequencing libraries, we mapped small RNAs to the genome of the model organism Arabidopsis thaliana and defined loci based on their expression using an empirical Bayesian approach. The resulting loci were subsequently classified based on their genetic and epigenetic context as well as their expression properties. We present the results of this classification, which broadly conforms to previously reported divisions between transcriptional and post-transcriptional gene silencing small RNAs, and to PolIV and PolV dependencies. However, we are able to demonstrate the existence of further subdivisions in the small RNA population of functional significance. Moreover, we present a framework for similar analyses of small RNA populations in all species.
Assuntos
Arabidopsis/genética , Epigênese Genética/genética , Epigenômica/métodos , Proteínas de Arabidopsis/genética , Teorema de Bayes , Regulação da Expressão Gênica de Plantas/genética , Genoma de Planta/genética , Sequenciamento de Nucleotídeos em Larga Escala , Interferência de RNA/fisiologia , RNA de Plantas/genética , RNA Interferente Pequeno/metabolismoRESUMO
The evolution of heterogametic sex chromosomes is often-but not always-accompanied by the evolution of dosage compensating mechanisms that mitigate the impact of sex-specific gene dosage on levels of gene expression. One emerging view of this process is that such mechanisms may only evolve in male-heterogametic (XY) species but not in female-heterogametic (ZW) species, which will consequently exhibit "incomplete" sex chromosome dosage compensation. However, recent results suggest that at least some Lepidoptera (moths and butterflies) may prove to be an exception to this prediction. Studies in bombycoid moths indicate the presence of a chromosome-wide epigenetic mechanism that effectively balances Z chromosome gene expression between the sexes by reducing Z-linked expression in males. In contrast, strong sex chromosome dosage effects without any reduction in male Z-linked expression were previously reported in a pyralid moth, suggesting a lack of any such dosage compensating mechanism. Here we report an analysis of sex chromosome dosage compensation in Heliconius butterflies, sampling multiple individuals for several different adult tissues (head, abdomen, leg, mouth, and antennae). Methodologically, we introduce a novel application of linear mixed-effects models to assess dosage compensation, offering a unified statistical framework that can estimate effects specific to chromosome, to sex, and their interactions (i.e., a dosage effect). Our results show substantially reduced Z-linked expression relative to autosomes in both sexes, as previously observed in bombycoid moths. This observation is consistent with an increasing body of evidence that some lepidopteran species possess an epigenetic dosage compensating mechanism that reduces Z chromosome expression in males to levels comparable with females. However, this mechanism appears to be imperfect in Heliconius, resulting in a modest dosage effect that produces an average 5-20% increase in male expression relative to females on the Z chromosome, depending on the tissue. Thus our results in Heliconius reflect a mixture of previous patterns reported for Lepidoptera. In Heliconius, a moderate pattern of incomplete dosage compensation persists apparently despite the presence of an epigenetic dosage compensating mechanism. The chromosomal distributions of sex-biased genes show an excess of male-biased and a dearth of female-biased genes on the Z chromosome relative to autosomes, consistent with predictions of sexually antagonistic evolution.
Assuntos
Borboletas/genética , Cromossomos de Insetos , Mecanismo Genético de Compensação de Dose , Cromossomos Sexuais , Animais , Feminino , Masculino , Análise de Sequência de DNARESUMO
: Cytosine methylation is a significant and widespread regulatory factor in plant systems. Methods for the high-throughput sequencing of methylation have allowed a greatly improved characterisation of the methylome. Here we discuss currently available methods for generation and analysis of high-throughput sequencing of methylation data. We also discuss the results previously acquired through sequencing plant methylomes, and highlight remaining challenges in this field.
RESUMO
PRDM9 directs human meiotic crossover hot spots to intergenic sequence motifs, whereas budding yeast hot spots overlap regions of low nucleosome density (LND) in gene promoters. To investigate hot spots in plants, which lack PRDM9, we used coalescent analysis of genetic variation in Arabidopsis thaliana. Crossovers increased toward gene promoters and terminators, and hot spots were associated with active chromatin modifications, including H2A.Z, histone H3 Lys4 trimethylation (H3K4me3), LND and low DNA methylation. Hot spot-enriched A-rich and CTT-repeat DNA motifs occurred upstream and downstream, respectively, of transcriptional start sites. Crossovers were asymmetric around promoters and were most frequent over CTT-repeat motifs and H2A.Z nucleosomes. Pollen typing, segregation and cytogenetic analysis showed decreased numbers of crossovers in the arp6 H2A.Z deposition mutant at multiple scales. During meiosis, H2A.Z forms overlapping chromosomal foci with the DMC1 and RAD51 recombinases. As arp6 reduced the number of DMC1 or RAD51 foci, H2A.Z may promote the formation or processing of meiotic DNA double-strand breaks. We propose that gene chromatin ancestrally designates hot spots within eukaryotes and PRDM9 is a derived state within vertebrates.