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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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Montagem e Desmontagem da Cromatina , Heterocromatina/metabolismo , Histonas/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Metilação de DNA , Elementos de DNA Transponíveis , Estudo de Associação Genômica Ampla , Histonas/química , Histonas/genética , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
The systematic determination of protein function is a key goal of modern biology, but remains challenging with current approaches. Here we present ORFtag, a versatile, cost-effective and highly efficient method for the massively parallel tagging and functional interrogation of proteins at the proteome scale. ORFtag uses retroviral vectors bearing a promoter, peptide tag and splice donor to generate fusions between the tag and endogenous open reading frames (ORFs). We demonstrate the utility of ORFtag through functional screens for transcriptional activators, repressors and posttranscriptional regulators in mouse embryonic stem cells. Each screen recovers known and identifies new regulators, including long ORFs inaccessible by other methods. Among other hits, we find that Zfp574 is a highly selective transcriptional activator and that oncogenic fusions often function as transactivators.
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BackgroundThe COVID-19 pandemic was largely driven by genetic mutations of SARS-CoV-2, leading in some instances to enhanced infectiousness of the virus or its capacity to evade the host immune system. To closely monitor SARS-CoV-2 evolution and resulting variants at genomic-level, an innovative pipeline termed SARSeq was developed in Austria.AimWe discuss technical aspects of the SARSeq pipeline, describe its performance and present noteworthy results it enabled during the pandemic in Austria.MethodsThe SARSeq pipeline was set up as a collaboration between private and public clinical diagnostic laboratories, a public health agency, and an academic institution. Representative SARS-CoV-2 positive specimens from each of the nine Austrian provinces were obtained from SARS-CoV-2 testing laboratories and processed centrally in an academic setting for S-gene sequencing and analysis.ResultsSARS-CoV-2 sequences from up to 2,880 cases weekly resulted in 222,784 characterised case samples in January 2021-March 2023. Consequently, Austria delivered the fourth densest genomic surveillance worldwide in a very resource-efficient manner. While most SARS-CoV-2 variants during the study showed comparable kinetic behaviour in all of Austria, some, like Beta, had a more focused spread. This highlighted multifaceted aspects of local population-level acquired immunity. The nationwide surveillance system enabled reliable nowcasting. Measured early growth kinetics of variants were predictive of later incidence peaks.ConclusionWith low automation, labour, and cost requirements, SARSeq is adaptable to monitor other pathogens and advantageous even for resource-limited countries. This multiplexed genomic surveillance system has potential as a rapid response tool for future emerging threats.
Assuntos
COVID-19 , Genoma Viral , SARS-CoV-2 , Humanos , Áustria/epidemiologia , SARS-CoV-2/genética , COVID-19/epidemiologia , COVID-19/virologia , COVID-19/diagnóstico , Mutação , Genômica/métodos , Pandemias , Evolução Molecular , Sequenciamento Completo do Genoma/métodosRESUMO
Chromatin regulation of eukaryotic genomes depends on the formation of nucleosome complexes between histone proteins and DNA. Histone variants, which are diversified by sequence or expression pattern, can profoundly alter chromatin properties. While variants in histone H2A and H3 families are well characterized, the extent of diversification of histone H2B proteins is less understood. Here, we report a systematic analysis of the histone H2B family in plants, which have undergone substantial divergence during the evolution of each major group in the plant kingdom. By characterising Arabidopsis H2Bs, we substantiate this diversification and reveal potential functional specialization that parallels the phylogenetic structure of emergent clades in eudicots. In addition, we identify a new class of highly divergent H2B variants, H2B.S, that specifically accumulate during chromatin compaction of dry seed embryos in multiple species of flowering plants. Our findings thus identify unsuspected diverse properties among histone H2B proteins in plants that has manifested into potentially novel groups of histone variants.
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Arabidopsis/genética , Cromatina/genética , Evolução Molecular , Histonas/genética , Arabidopsis/classificação , Eucariotos , Genoma de Planta/genética , Histonas/classificação , Família Multigênica/genéticaRESUMO
Active DNA demethylation is critical for controlling the DNA methylomes in plants and mammals. However, little is known about how DNA demethylases are recruited to target loci, and the involvement of chromatin marks in this process. Here, we identify 2 components of the SWR1 chromatin-remodeling complex, PIE1 and ARP6, as required for ROS1-mediated DNA demethylation, and discover 2 SWR1-associated bromodomain-containing proteins, AtMBD9 and nuclear protein X1 (NPX1). AtMBD9 and NPX1 recognize histone acetylation marks established by increased DNA methylation 1 (IDM1), a known regulator of DNA demethylation, redundantly facilitating H2A.Z deposition at IDM1 target loci. We show that at some genomic regions, H2A.Z and DNA methylation marks coexist, and H2A.Z physically interacts with ROS1 to regulate DNA demethylation and antisilencing. Our results unveil a mechanism through which DNA demethylases can be recruited to specific target loci exhibiting particular histone marks, providing a conceptual framework to understand how chromatin marks regulate DNA demethylation.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Desmetilação do DNA , Histonas/metabolismo , Complexos Multiproteicos/metabolismo , Acetilação , Cromatina/metabolismo , Inativação Gênica , Modelos Biológicos , Mutação/genética , Ligação Proteica , Subunidades Proteicas/metabolismoRESUMO
In eukaryotes, variants of core histone H2A are selectively incorporated in distinct functional domains of chromatin and are distinguished by conserved sequences of their C-terminal tail, the L1 loop and the docking domain, suggesting that each variant confers specific properties to the nucleosome. Chromatin of flowering plants contains four types of H2A variants, which biochemical properties have not been characterized. We report that in contrast with animals, in Arabidopsis thaliana H2A variants define only four major types of homotypic nucleosomes containing exclusively H2A, H2A.Z, H2A.X or H2A.W. In vitro assays show that the L1 loop and the docking domain confer distinct stability of the nucleosome. In vivo and in vitro assays suggest that the L1 loop and the docking domain cooperate with the C-terminal tail to regulate chromatin accessibility. Based on these findings we conclude that the type of H2A variant in the nucleosome impacts on its interaction with DNA and propose that H2A variants regulate the dynamics of chromatin accessibility. In plants, the predominance of homotypic nucleosomes with specific physical properties and their specific localization to distinct domains suggest that H2A variants play a dominant role in chromatin dynamics and function.
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Proteínas de Arabidopsis/metabolismo , Cromatina/metabolismo , Histonas/metabolismo , Nucleossomos/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Arabidopsis/genética , Sítios de Ligação/genética , Cromatina/genética , DNA/genética , DNA/metabolismo , Variação Genética , Histonas/genética , Humanos , Nucleossomos/genética , Plantas Geneticamente Modificadas , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Comprehensive proteomic analysis is essential to elucidate molecular pathways and protein functions. Despite tremendous progress in proteomics, current studies still suffer from limited proteomic coverage and dynamic range. Here, we utilize micropillar array columns (µPACs) together with wide-window acquisition and the AI-based CHIMERYS search engine to achieve excellent proteomic comprehensiveness for bulk proteomics, affinity purification mass spectrometry and single cell proteomics. Our data show that µPACs identify ≤50% more peptides and ≤24% more proteins, while offering improved throughput, which is critical for large (clinical) proteomics studies. Combining wide precursor isolation widths of m/z 4-12 with the CHIMERYS search engine identified +51-74% and +59-150% more proteins and peptides, respectively, for single cell, co-immunoprecipitation, and multi-species samples over a conventional workflow at well-controlled false discovery rates. The workflow further offers excellent precision, with CVs <7% for low input bulk samples, and accuracy, with deviations <10% from expected fold changes for regular abundance two-proteome mixes. Compared to a conventional workflow, our entire optimized platform discovered 92% more potential interactors in a protein-protein interaction study on the chromatin remodeler Smarca5/Snf2h. These include previously described Smarca5 binding partners and undescribed ones including Arid1a, another chromatin remodeler with key roles in neurodevelopmental and malignant disorders.
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Peptídeos , Proteômica , Proteômica/métodos , Proteoma/metabolismo , Cromatina , Inteligência ArtificialRESUMO
Polycomb Repressive Complexes 1 and 2 (PRC1, PRC2) are conserved epigenetic regulators that promote transcriptional gene silencing. PRC1 and PRC2 converge on shared targets, catalyzing repressive histone modifications. Additionally, a subset of PRC1/PRC2 targets engage in long-range interactions whose functions in gene silencing are poorly understood. Using a CRISPR screen in mouse embryonic stem cells, we found that the cohesin regulator PDS5A links transcriptional silencing by Polycomb and 3D genome organization. PDS5A deletion impairs cohesin unloading and results in derepression of a subset of endogenous PRC1/PRC2 target genes. Importantly, derepression is not linked to loss of Polycomb chromatin domains. Instead, PDS5A removal causes aberrant cohesin activity leading to ectopic insulation sites, which disrupt the formation of ultra-long Polycomb loops. We show that these loops are important for robust silencing at a subset of PRC1/PRC2 target genes and that maintenance of cohesin-dependent genome architecture is critical for Polycomb regulation.
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Coesinas , Proteínas Nucleares , Proteínas do Grupo Polycomb , Animais , Camundongos , Cromatina/genética , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 2/genética , Proteínas do Grupo Polycomb/genética , Proteínas Nucleares/genéticaRESUMO
How different intrinsic sequence variations and regulatory modifications of histones combine in nucleosomes remain unclear. To test the importance of histone variants in the organization of chromatin we investigated how histone variants and histone modifications assemble in the Arabidopsis thaliana genome. We showed that a limited number of chromatin states divide euchromatin and heterochromatin into several subdomains. We found that histone variants are as significant as histone modifications in determining the composition of chromatin states. Particularly strong associations were observed between H2A variants and specific combinations of histone modifications. To study the role of H2A variants in organizing chromatin states we determined the role of the chromatin remodeler DECREASED IN DNA METHYLATION (DDM1) in the organization of chromatin states. We showed that the loss of DDM1 prevented the exchange of the histone variant H2A.Z to H2A.W in constitutive heterochromatin, resulting in significant effects on the definition and distribution of chromatin states in and outside of constitutive heterochromatin. We thus propose that dynamic exchanges of histone variants control the organization of histone modifications into chromatin states, acting as molecular landmarks.
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Proteínas de Arabidopsis , Arabidopsis , Cromatina/genética , Histonas/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Heterocromatina/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Nucleossomos/genéticaRESUMO
Ascl1 and Ngn2, closely related proneural transcription factors, are able to convert mouse embryonic stem cells into induced neurons. Despite their similarities, these factors elicit only partially overlapping transcriptional programs, and it remains unknown whether cells are converted via distinct mechanisms. Here we show that Ascl1 and Ngn2 induce mutually exclusive side populations by binding and activating distinct lineage drivers. Furthermore, Ascl1 rapidly dismantles the pluripotency network and installs neuronal and trophoblast cell fates, while Ngn2 generates a neural stem cell-like intermediate supported by incomplete shutdown of the pluripotency network. Using CRISPR-Cas9 knockout screening, we find that Ascl1 relies more on factors regulating pluripotency and the cell cycle, such as Tcf7l1. In the absence of Tcf7l1, Ascl1 still represses core pluripotency genes but fails to exit the cell cycle. However, overexpression of Cdkn1c induces cell cycle exit and restores the generation of neurons. These findings highlight that cell type conversion can occur through two distinct mechanistic paths, even when induced by closely related transcription factors.
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Células-Tronco Embrionárias Murinas , Células-Tronco Neurais , Animais , Camundongos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Ciclo Celular/genética , Neurônios , Fatores de TranscriçãoRESUMO
ZNF462 haploinsufficiency is linked to Weiss-Kruszka syndrome, a genetic disorder characterized by neurodevelopmental defects, including autism. Though conserved in vertebrates and essential for embryonic development, the molecular functions of ZNF462 remain unclear. We identified its murine homologue ZFP462 in a screen for mediators of epigenetic gene silencing. Here we show that ZFP462 safeguards neural lineage specification of mouse embryonic stem cells (ESCs) by targeting the H3K9-specific histone methyltransferase complex G9A/GLP to silence meso-endodermal genes. ZFP462 binds to transposable elements that are potential enhancers harbouring pluripotency and meso-endoderm transcription factor binding sites. Recruiting G9A/GLP, ZFP462 seeds heterochromatin, restricting transcription factor binding. Loss of ZFP462 in ESCs results in increased chromatin accessibility at target sites and ectopic expression of meso-endodermal genes. Taken together, ZFP462 confers lineage and locus specificity to the broadly expressed epigenetic regulator G9A/GLP. Our results suggest that aberrant activation of lineage non-specific genes in the neuronal lineage underlies ZNF462-associated neurodevelopmental pathology.
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Heterocromatina , Histona-Lisina N-Metiltransferase , Animais , Camundongos , Heterocromatina/genética , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Cromatina , Células-Tronco Embrionárias , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas do Tecido Nervoso/genéticaRESUMO
Mobile transposable elements (TEs) not only participate in genome evolution but also threaten genome integrity. In healthy cells, TEs that encode all of the components that are necessary for their mobility are specifically silenced, yet the precise mechanism remains unknown. Here, we characterize the mechanism used by a conserved class of chromatin remodelers that prevent TE mobility. In the Arabidopsis chromatin remodeler DECREASE IN DNA METHYLATION 1 (DDM1), we identify two conserved binding domains for the histone variant H2A.W, which marks plant heterochromatin. DDM1 is necessary and sufficient for the deposition of H2A.W onto potentially mobile TEs, yet does not act on TE fragments or host protein-coding genes. DDM1-mediated H2A.W deposition changes the properties of chromatin, resulting in the silencing of TEs and, therefore, prevents their mobility. This distinct mechanism provides insights into the interplay between TEs and their host in the contexts of evolution and disease, and potentiates innovative strategies for targeted gene silencing.
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Proteínas de Arabidopsis/genética , Elementos de DNA Transponíveis/genética , Proteínas de Ligação a DNA/genética , Evolução Molecular , Histonas/genética , Fatores de Transcrição/genética , Arabidopsis/genética , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Metilação de DNA/genética , Regulação da Expressão Gênica de Plantas/genética , Inativação Gênica , Genoma de Planta/genética , Heterocromatina/genéticaRESUMO
In flowering plants, heterochromatin is demarcated by the histone variant H2A.W, elevated levels of the linker histone H1, and specific epigenetic modifications, such as high levels of DNA methylation at both CG and non-CG sites. How H2A.W regulates heterochromatin organization and interacts with other heterochromatic features is unclear. Here, we create a h2a.w null mutant via CRISPR-Cas9, h2a.w-2, to analyze the in vivo function of H2A.W. We find that H2A.W antagonizes deposition of H1 at heterochromatin and that non-CG methylation and accessibility are moderately decreased in h2a.w-2 heterochromatin. Compared to H1 loss alone, combined loss of H1 and H2A.W greatly increases accessibility and facilitates non-CG DNA methylation in heterochromatin, suggesting co-regulation of heterochromatic features by H2A.W and H1. Our results suggest that H2A.W helps maintain optimal heterochromatin accessibility and DNA methylation by promoting chromatin compaction together with H1, while also inhibiting excessive H1 incorporation.
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Proteínas de Arabidopsis/genética , Arabidopsis/genética , Metilação de DNA , Regulação da Expressão Gênica de Plantas , Heterocromatina/genética , Histonas/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cromatina/genética , Cromatina/metabolismo , DNA de Plantas/química , DNA de Plantas/genética , Variação Genética , Heterocromatina/metabolismo , Histonas/metabolismo , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Sequenciamento Completo do Genoma/métodosRESUMO
The COVID-19 pandemic has demonstrated the need for massively-parallel, cost-effective tests monitoring viral spread. Here we present SARSeq, saliva analysis by RNA sequencing, a method to detect SARS-CoV-2 and other respiratory viruses on tens of thousands of samples in parallel. SARSeq relies on next generation sequencing of multiple amplicons generated in a multiplexed RT-PCR reaction. Two-dimensional, unique dual indexing, using four indices per sample, enables unambiguous and scalable assignment of reads to individual samples. We calibrate SARSeq on SARS-CoV-2 synthetic RNA, virions, and hundreds of human samples of various types. Robustness and sensitivity were virtually identical to quantitative RT-PCR. Double-blinded benchmarking to gold standard quantitative-RT-PCR performed by human diagnostics laboratories confirms this high sensitivity. SARSeq can be used to detect Influenza A and B viruses and human rhinovirus in parallel, and can be expanded for detection of other pathogens. Thus, SARSeq is ideally suited for differential diagnostic of infections during a pandemic.
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Técnicas de Laboratório Clínico , Ensaios de Triagem em Larga Escala , Infecções Respiratórias/diagnóstico , Vírus/isolamento & purificação , COVID-19/diagnóstico , Diagnóstico Diferencial , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Reação em Cadeia da Polimerase , RNA Viral/genética , Infecções Respiratórias/virologia , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Saliva/virologia , Sensibilidade e Especificidade , Proteínas Virais/genética , Vírus/classificação , Vírus/genéticaRESUMO
Heterochromatin protein 1 (HP1) facilitates the formation of repressive heterochromatin domains by recruiting histone lysine methyltransferase enzymes to chromatin, resulting in increased levels of histone H3K9me3. To identify chemical inhibitors of the HP1-heterochromatin gene repression pathway, we combined a cell-based assay that utilized chemical-mediated recruitment of HP1 to an endogenous active gene with high-throughput flow cytometry. Here we characterized small molecule inhibitors that block HP1-mediated heterochromatin formation. Our lead compounds demonstrated dose-dependent inhibition of HP1-stimulated gene repression and were validated in an orthogonal cell-based system. One lead inhibitor was improved by a change in stereochemistry, resulting in compound 2, which was further used to decouple the inverse relationship between H3K9 and H3K4 methylation states. We identified molecular components that bound compound 2, either directly or indirectly, by chemical affinity purification with a biotin-tagged derivative, followed by quantitative proteomic techniques. In summary, our pathway-based chemical screening approach resulted in the discovery of new inhibitors of HP1-mediated heterochromatin formation while identifying exciting new molecular interactions in the pathway to explore in the future. This modular platform can be expanded to test a wide range of chromatin modification pathways yielding inhibitors that are cell permeable and function in a physiologically relevant setting.
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Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Proteínas Cromossômicas não Histona/metabolismo , Descoberta de Drogas , Heterocromatina/efeitos dos fármacos , Heterocromatina/metabolismo , Ensaios de Triagem em Larga Escala , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular , Cromatografia Líquida , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Proteínas Cromossômicas não Histona/química , Descoberta de Drogas/métodos , Citometria de Fluxo , Heterocromatina/genética , Histonas/metabolismo , Camundongos , Microscopia de Fluorescência , Modelos Biológicos , Estrutura Molecular , Fosfoproteínas/metabolismo , Ligação Proteica , Proteômica/métodos , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade , Espectrometria de Massas em TandemRESUMO
Polycomb group (PcG) proteins play critical roles in the epigenetic inheritance of cell fate. The Polycomb Repressive Complexes PRC1 and PRC2 catalyse distinct chromatin modifications to enforce gene silencing, but how transcriptional repression is propagated through mitotic cell divisions remains a key unresolved question. Using reversible tethering of PcG proteins to ectopic sites in mouse embryonic stem cells, here we show that PRC1 can trigger transcriptional repression and Polycomb-dependent chromatin modifications. We find that canonical PRC1 (cPRC1), but not variant PRC1, maintains gene silencing through cell division upon reversal of tethering. Propagation of gene repression is sustained by cis-acting histone modifications, PRC2-mediated H3K27me3 and cPRC1-mediated H2AK119ub1, promoting a sequence-independent feedback mechanism for PcG protein recruitment. Thus, the distinct PRC1 complexes present in vertebrates can differentially regulate epigenetic maintenance of gene silencing, potentially enabling dynamic heritable responses to complex stimuli. Our findings reveal how PcG repression is potentially inherited in vertebrates.
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Cromatina/metabolismo , Epigênese Genética , Inativação Gênica , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 2/genética , Processamento de Proteína Pós-Traducional , Animais , Linhagem Celular , Cromatina/química , Retroalimentação Fisiológica , Histonas/genética , Histonas/metabolismo , Padrões de Herança , Camundongos , Mitose , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Transcrição GênicaRESUMO
Fertilization comprises a complex series of cellular processes leading to the fusion of a male and female gamete. Many studies have been carried out to investigate each step of fertilization in plants; however, our comprehensive understanding of all the sequential events during fertilization is still limited. This is largely due to difficulty in investigating events in the female gametophyte, which is deeply embedded in the maternal tissue. Recent advances in confocal microcopy assisted by fluorescent marker lines have contributed to visualizing subcellular dynamics in real time during fertilization in vivo. In this chapter, we describe a method focusing on the investigation of F-actin dynamics in the central cell during male gamete nuclear migration. This method also allows the study of a wide range of early sexual reproduction events, from pollen tube guidance to the early stage of seed development.
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Actinas/metabolismo , Arabidopsis/metabolismo , Fertilização/fisiologia , Actinas/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fertilização/genética , Células Germinativas Vegetais/metabolismo , Óvulo Vegetal/genética , Óvulo Vegetal/metabolismoRESUMO
Histone modifications and histone variants barcode the genome and play major roles in epigenetic regulations. Chromatin immunoprecipitation (ChIP) coupled with next-generation sequencing (NGS) is a well-established method to investigate the landscape of epigenetic marks at a genomic level. Here, we describe procedures for conducting ChIP, subsequent NGS library construction, and data analysis on histone modifications and histone variants in Arabidopsis thaliana. We also describe an optimized nuclear isolation procedure to prepare chromatin for ChIP in the liverwort, Marchantia polymorpha, which is the emerging model plant ideal for evolutionary studies.
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Arabidopsis/genética , Histonas/metabolismo , Marchantia/genética , Imunoprecipitação da Cromatina , Processamento de Proteína Pós-Traducional/genéticaRESUMO
BACKGROUND: Gene bodies of vertebrates and flowering plants are occupied by the histone variant H3.3 and DNA methylation. The origin and significance of these profiles remain largely unknown. DNA methylation and H3.3 enrichment profiles over gene bodies are correlated and both have a similar dependence on gene transcription levels. This suggests a mechanistic link between H3.3 and gene body methylation. RESULTS: We engineered an H3.3 knockdown in Arabidopsis thaliana and observed transcription reduction that predominantly affects genes responsive to environmental cues. When H3.3 levels are reduced, gene bodies show a loss of DNA methylation correlated with transcription levels. To study the origin of changes in DNA methylation profiles when H3.3 levels are reduced, we examined genome-wide distributions of several histone H3 marks, H2A.Z, and linker histone H1. We report that in the absence of H3.3, H1 distribution increases in gene bodies in a transcription-dependent manner. CONCLUSIONS: We propose that H3.3 prevents recruitment of H1, inhibiting H1's promotion of chromatin folding that restricts access to DNA methyltransferases responsible for gene body methylation. Thus, gene body methylation is likely shaped by H3.3 dynamics in conjunction with transcriptional activity.
Assuntos
Arabidopsis/genética , DNA de Plantas/genética , Epigênese Genética , Genoma de Planta , Histonas/genética , Proteínas de Plantas/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cromatina/química , Cromatina/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , DNA de Plantas/metabolismo , Histonas/metabolismo , Proteínas de Plantas/metabolismo , Transcrição GênicaRESUMO
Among eukaryotes, the four core histones show an extremely high conservation of their structure and form nucleosomes that compact, protect, and regulate access to genetic information. Nevertheless, in multicellular eukaryotes the two families, histone H2A and histone H3, have diversified significantly in key residues. We present a phylogenetic analysis across the green plant lineage that reveals an early diversification of the H2A family in unicellular green algae and remarkable expansions of H2A variants in flowering plants. We define motifs and domains that differentiate plant H2A proteins into distinct variant classes. In non-flowering land plants, we identify a new class of H2A variants and propose their possible role in the emergence of the H2A.W variant class in flowering plants.