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1.
Plant Cell Physiol ; 2021 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-33638992

RESUMO

Humans are currently facing the problem of how to ensure that there is enough food to feed all of the world's population. Ensuring that the food supply is sufficient will likely require the modification of crop genomes to improve their agronomic traits. The development of engineered sequence-specific nucleases (SSNs) paved the way for targeted gene editing in organisms, including plants. SSNs generate a double-strand break (DSB) at the target DNA site in a sequence-specific manner. These DSBs are predominantly repaired via error-prone non-homologous end joining (NHEJ), and are only rarely repaired via error-free homology-directed repair (HDR) if an appropriate donor template is provided. Gene targeting (GT), i.e., the integration or replacement of a particular sequence, can be achieved with combinations of SSNs and repair donor templates. Although its efficiency is extremely low, GT has been achieved in some higher plants. Here, we provide an overview of SSN-facilitated GT in higher plants and discuss the potential of GT as a powerful tool for generating crop plants with desirable features.

2.
J Integr Plant Biol ; 2021 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-33438356

RESUMO

Heterochromatin is widespread in eukaryotic genomes and has diverse impacts depending on its genomic context. Previous studies have shown that a protein complex, the ASI1-AIPP1-EDM2 (AAE) complex, participates in polyadenylation regulation of several intronic heterochromatin-containing genes. However, the genome-wide functions of AAE are still unknown. Here, we show that the ASI1 and EDM2 mostly target the common genomic regions on a genome-wide level and preferentially interacts with genetic heterochromatin. Polyadenylation [(poly(A)] sequencing reveals that AAE complex has a substantial influence on poly(A) site usage of heterochromatin-containing genes, including not only intronic heterochromatin-containing genes but also the genes showing overlap with heterochromatin. Intriguingly, AAE is also involved in the alternative splicing regulation of a number of heterochromatin-overlapping genes, such as the disease resistance gene RPP4. We provided evidence that genic heterochromatin is indispensable for the recruitment of AAE in polyadenylation and splicing regulation. In addition to conferring RNA processing regulation at genic heterochromatin-containing genes, AAE also targets some transposable elements (TEs) outside of genes (including TEs sandwiched by genes and island TEs) for epigenetic silencing. Our results reveal new functions of AAE in RNA processing and epigenetic silencing, and thus represent important advances in epigenetic regulation. This article is protected by copyright. All rights reserved.

3.
Methods Mol Biol ; 2200: 121-146, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33175375

RESUMO

CRISPR/Cas9 system has emerged as a powerful genome engineering tool to study gene function and improve plant traits. Genome editing is achieved at a specific genome sequence by Cas9 endonuclease to generate double standard breaks (DSBs) directed by short guide RNAs (sgRNAs). The DSB is repaired by error-prone nonhomologous end joining (NHEJ) or error-free homology-directed repair (HDR) pathways, resulting in gene mutation or sequence replacement, respectively. These cellular DSB repair pathways can be exploited to knock out or replace genes. Also, cytidine or adenine base editors (CBEs or ABEs) fused to catalytically dead Cas9 (dCas9) or nickase Cas9 (nCas9) are used to perform precise base editing without generating DSBs. In this chapter, we describe a detailed procedure to carry out single/multiple gene mutations and precise base editing in the Arabidopsis genome by using CRISPR/Cas9-based system. Specifically, the steps of target gene selection, sgRNA design, vector construction, transformation, and analysis of transgenic lines are described. The protocol is potentially adaptable to perform genome editing in other plant species such as rice.

4.
Neurochem Int ; 142: 104902, 2020 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-33197527

RESUMO

The primary cilium is a solitary organelle that organizes a sensitive signaling hub in a highly ordered microenvironment. Cilia are plastic structures, changing their length in response to bioactive substances, and ciliary length may be regulated to ensure efficient signaling capacity. Mammalian brain neurons possess primary cilia that are enriched in a set of G protein-coupled receptors (GPCRs), including the feeding-related melanin-concentrating hormone (MCH) receptor 1 (MCHR1). We previously demonstrated a novel biological phenomenon, ciliary MCHR1-mediated cilia length shortening through Gi/o and Akt signaling, using a simple cell culture model of human retinal pigmented epithelial RPE1 cells exogenously expressing MCHR1. In the present study, we characterized the properties of endogenous MCHR1-expressing primary cilia in hippocampal neurons in rodents. Using cultured dissociated rat hippocampal neurons in vitro, we showed that MCH triggered cilia length reduction involved in MCHR1-Gi/o and -Akt signaling. In rat hippocampal slice cultures with preservation of the cytoarchitecture and cell populations, ciliary MCHR1 was abundantly located in the CA1 and CA3 regions, but not in the dentate gyrus. Notably, treatment of slice cultures with MCH induced Gi/o- and Akt-dependent cilia shortening in the CA1 region without influencing cilia length in the CA3 region. Regarding the in vivo mouse brain, we observed higher levels of ciliary MCHR1 in the CA1 and CA3 regions as well as in slice cultures. In the starved state mice, a marked increase in MCH mRNA expression was detected in the lateral hypothalamus. Furthermore, MCHR1-positive cilia length in the hippocampal CA1 region was significantly shortened in fasted mice compared with fed mice. The present findings focused on the hippocampus provide a potential approach to investigate how MCHR1-driven cilia shortening regulates neuronal activity and physiological function toward feeding and memory tasks.

5.
J Integr Plant Biol ; 2020 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-33236824

RESUMO

DNA methylation is an epigenetic mark important for genome stability and gene expression. In Arabidopsis thaliana, the 5-methylcytosine DNA glycosylase/demethylase DEMETER (DME) controls active DNA demethylation during the reproductive stage; however, the lethality of loss-of-function dme mutations has made it difficult to assess DME function in vegetative tissues. Here, we edited DME using CRISPR/Cas9 and created three weak dme mutants that produced a few viable seeds. We also performed central cell-specific complementation in a strong dme mutant and combined this line with mutations in the other three Arabidopsis demethylase genes to generate the dme ros1 dml2 dml3 (drdd) quadruple mutant. A DNA methylome analysis showed that DME is required for DNA demethylation at hundreds of genomic regions in vegetative tissues. A transcriptome analysis of the drdd mutant revealed that DME and the other three demethylases are important for plant responses to biotic and abiotic stresses in vegetative tissues. Despite the limited role of DME in regulating DNA methylation in vegetative tissues, the dme mutants showed increased susceptibility to bacterial and fungal pathogens. Our study highlights the important functions of DME in vegetative tissues and provides valuable genetic tools for future investigations of DNA demethylation in plants. This article is protected by copyright. All rights reserved.

6.
Nat Plants ; 6(8): 983-995, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32661278

RESUMO

Root-associated soil bacteria can strongly influence plant fitness. DNA methylation is an epigenetic mark important to many fundamental biological processes; however, its roles in plant interactions with beneficial microbes remain elusive. Here, we report that active DNA demethylation in Arabidopsis controls root secretion of myo-inositol and consequently plant growth promotion triggered by Bacillus megaterium strain YC4. Root-secreted myo-inositol is critical for YC4 colonization and preferentially attracts B. megaterium among the examined bacteria species. Active DNA demethylation antagonizes RNA-directed DNA methylation in controlling myo-inositol homeostasis. Importantly, we demonstrate that active DNA demethylation controls myo-inositol-mediated mutualism between YC4 and Solanum lycopersicum, thus suggesting a conserved nature of this epigenetic regulatory mechanism.

8.
Proc Natl Acad Sci U S A ; 116(33): 16641-16650, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31363048

RESUMO

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.


Assuntos
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/metabolismo
9.
Neurochem Res ; 44(7): 1736-1744, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31037609

RESUMO

Recent advances in human induced pluripotent stem cells (hiPSCs) offer new possibilities for biomedical research and clinical applications. Neurons differentiated from hiPSCs may be promising tools to develop novel treatment methods for various neurological diseases. However, the detailed process underlying functional maturation of hiPSC-derived neurons remains poorly understood. Here, we analyze the developmental architecture of hiPSC-derived cortical neurons, iCell GlutaNeurons, focusing on the primary cilium, a single sensory organelle that protrudes from the surface of most growth-arrested vertebrate cells. To characterize the neuronal cilia, cells were cultured for various periods and evaluated immunohistochemically by co-staining with antibodies against ciliary markers Arl13b and MAP2. Primary cilia were detected in neurons within days, and their prevalence and length increased with increasing days in culture. Treatment with the mood stabilizer lithium led to primary cilia length elongation, while treatment with the orexigenic neuropeptide melanin-concentrating hormone caused cilia length shortening in iCell GlutaNeurons. The present findings suggest that iCell GlutaNeurons develop neuronal primary cilia together with the signaling machinery for regulation of cilia length. Our approach to the primary cilium as a cellular antenna can be useful for both assessment of neuronal maturation and validation of pharmaceutical agents in hiPSC-derived neurons.


Assuntos
Cílios/metabolismo , Cílios/ultraestrutura , Células-Tronco Pluripotentes Induzidas/citologia , Neurônios/citologia , Fatores de Ribosilação do ADP/imunologia , Adenilil Ciclases/imunologia , Animais , Anticorpos/imunologia , Linhagem Celular , Cílios/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Humanos , Hormônios Hipotalâmicos/farmacologia , Imuno-Histoquímica , Lítio/farmacologia , Melaninas/farmacologia , Proteínas Associadas aos Microtúbulos/imunologia , Neurogênese/fisiologia , Neurônios/efeitos dos fármacos , Hormônios Hipofisários/farmacologia , Ratos Wistar , Receptores de Somatostatina/imunologia
10.
Sci Rep ; 9(1): 1979, 2019 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-30760833

RESUMO

Vigorous explosive eruptions that produce continuous high eruption plumes (Plinian eruptions) are generally assumed to tap a magma reservoir. The 1914 Plinian eruption at the Sakurajima volcano located on the Aira caldera rim is one such case, where the main magma reservoir was assumed to be located approximately 10 km beneath the caldera. However, we report that estimated magma storage depths immediately prior to the eruption were much shallower (0.9-3.2 km) on the basis of pressure at which volatiles within the phenocryst melt inclusions and plagioclase rims were finally equilibrated. The same is observed for two historic Plinian eruptions in 1471 and 1779. This depth is even shallower than the shallowest magma reservoir estimated from the pressure source for geodetic deformation during recent Vulcanian explosions (4 km beneath the crater). We propose that the magmas were fed from a thick conduit pre-charged from deeper reservoirs. The ground subsidence observed after 1914 within the Aira caldera may have been caused by conduit recharge following the eruption. Voluminous conduit recharge could be key to forecasting the next possible large eruption at the Sakurajima volcano.

11.
Nat Commun ; 10(1): 436, 2019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30683860

RESUMO

Broomcorn millet (Panicum miliaceum L.) is the most water-efficient cereal and one of the earliest domesticated plants. Here we report its high-quality, chromosome-scale genome assembly using a combination of short-read sequencing, single-molecule real-time sequencing, Hi-C, and a high-density genetic map. Phylogenetic analyses reveal two sets of homologous chromosomes that may have merged ~5.6 million years ago, both of which exhibit strong synteny with other grass species. Broomcorn millet contains 55,930 protein-coding genes and 339 microRNA genes. We find Paniceae-specific expansion in several subfamilies of the BTB (broad complex/tramtrack/bric-a-brac) subunit of ubiquitin E3 ligases, suggesting enhanced regulation of protein dynamics may have contributed to the evolution of broomcorn millet. In addition, we identify the coexistence of all three C4 subtypes of carbon fixation candidate genes. The genome sequence is a valuable resource for breeders and will provide the foundation for studying the exceptional stress tolerance as well as C4 biology.


Assuntos
Cromossomos de Plantas/química , Genoma de Planta , Panicum/genética , Filogenia , Proteínas de Plantas/genética , Adaptação Fisiológica/genética , Sequência de Bases , Evolução Biológica , Ciclo do Carbono , Mapeamento Cromossômico , Ontologia Genética , Sequenciamento de Nucleotídeos em Larga Escala , MicroRNAs/classificação , MicroRNAs/genética , Anotação de Sequência Molecular , Panicum/classificação , Melhoramento Vegetal , RNA de Plantas/genética , Estresse Fisiológico , Sintenia , Ubiquitina-Proteína Ligases/genética
12.
Cell Discov ; 4: 55, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30345072

RESUMO

DNA methylation is a conserved epigenetic mark that is critical for many biological processes in plants and mammals. In Arabidopsis, the antagonistic activities of RNA-directed DNA methylation (RdDM) and ROS1-dependent active DNA demethylation are key for the dynamic regulation of locus-specific DNA methylation. However, the molecular factors that coordinate RdDM and active demethylation are largely unknown. Here we report that CLSY4 and its three paralogous SWI2/SNF2-type chromatin-remodeling proteins function in both RdDM and DNA demethylation in Arabidopsis. We initially identified CLSY4 in a genetic screen for DNA demethylation factors and subsequently demonstrated that it also is important in RdDM. Comprehensive genetic analyses using single and high order mutants of CLSY family proteins revealed their roles as double agents in the balance between methylation and demethylation reactions. The four CLSY proteins collectively are necessary for the canonical RdDM pathway; at the same time, each CLSY likely mediates DNA demethylation at specific loci where DNA methylation depends on RdDM. These results indicate that the four chromatin-remodeling proteins have dual functions in regulating genomic DNA methylation, and thus provide new insights into the dynamic regulation of DNA methylation in a model multicellular eukaryotic organism.

13.
Proc Natl Acad Sci U S A ; 115(42): E9962-E9970, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30266793

RESUMO

Genomic imprinting is a form of epigenetic regulation resulting in differential gene expression that reflects the parent of origin. In plants, imprinted gene expression predominantly occurs in the seed endosperm. Maternal-specific DNA demethylation by the DNA demethylase DME frequently underlies genomic imprinting in endosperm. Whether other more ubiquitously expressed DNA demethylases regulate imprinting is unknown. Here, we found that the DNA demethylase ROS1 regulates the imprinting of DOGL4 DOGL4 is expressed from the maternal allele in endosperm and displays preferential methylation and suppression of the paternal allele. We found that ROS1 negatively regulates imprinting by demethylating the paternal allele, preventing its hypermethylation and complete silencing. Furthermore, we found that DOGL4 negatively affects seed dormancy and response to the phytohormone abscisic acid and that ROS1 controls these processes by regulating DOGL4 Our results reveal roles for ROS1 in mitigating imprinted gene expression and regulating seed dormancy.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Impressão Genômica , Proteínas Nucleares/metabolismo , Dormência de Plantas , Sementes/fisiologia , Fatores de Transcrição/metabolismo , Proteínas de Arabidopsis/genética , DNA de Plantas/genética , Proteínas de Ligação a DNA/genética , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Proteínas Nucleares/genética , Fatores de Transcrição/genética
14.
Nat Commun ; 9(1): 1967, 2018 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-29773790

RESUMO

Homologous recombination-based gene targeting is a powerful tool for precise genome modification and has been widely used in organisms ranging from yeast to higher organisms such as Drosophila and mouse. However, gene targeting in higher plants, including the most widely used model plant Arabidopsis thaliana, remains challenging. Here we report a sequential transformation method for gene targeting in Arabidopsis. We find that parental lines expressing the bacterial endonuclease Cas9 from the egg cell- and early embryo-specific DD45 gene promoter can improve the frequency of single-guide RNA-targeted gene knock-ins and sequence replacements via homologous recombination at several endogenous sites in the Arabidopsis genome. These heritable gene targeting can be identified by regular PCR. Our approach enables routine and fine manipulation of the Arabidopsis genome.


Assuntos
Arabidopsis/genética , Sistemas CRISPR-Cas , Técnicas de Introdução de Genes/métodos , Marcação de Genes/métodos , Substituição de Aminoácidos/genética , Proteínas de Arabidopsis/genética , Genoma de Planta/genética , Proteínas Nucleares/genética , Plantas Geneticamente Modificadas/genética , RNA Guia/genética , Transformação Genética/genética
15.
Mol Cell Endocrinol ; 474: 184-193, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-29574003

RESUMO

Beige adipocytes are thermogenic adipocytes with developmental and anatomical properties distinct from those of classical brown adipocytes. Recent studies have revealed several key molecular regulators of beige adipocyte development. CD105, also called endoglin, is a membrane protein composed of TGF-ß receptor complex. It regulates TGF-ß-family signal transduction and vascular formation in vivo. We report here that CD105 maintains the thermogenic gene program of beige adipocytes by regulating Smad2 signaling. Cd105-/- adipocyte precursors showed augmented Smad2 activation and decreased expression of thermogenic genes such as Ucp1 and Prdm16-which encodes a transcriptional regulatory protein for thermogenesis-after adipogenic differentiation. Smad2 signaling augmentation by the constitutively active form of Smad2 decreased the expression of thermogenic genes in beige adipocytes. Loss of thermogenic activity in Cd105-/- beige adipocytes was rescued by Prdm16 expression. These data reveal a novel function of CD105 in beige adipocytes: maintaining their thermogenic program by regulating Smad2 signaling.


Assuntos
Adipócitos Bege/metabolismo , Endoglina/metabolismo , Transdução de Sinais , Proteína Smad2/metabolismo , Termogênese , Adipócitos Bege/citologia , Animais , Diferenciação Celular/genética , Células Cultivadas , Citoproteção , Proteínas de Ligação a DNA/metabolismo , Endoglina/deficiência , Regulação da Expressão Gênica , Masculino , Camundongos Endogâmicos C57BL , Fosforilação , Termogênese/genética , Fatores de Transcrição/metabolismo , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismo
16.
Genome Biol ; 18(1): 103, 2017 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-28569170

RESUMO

BACKGROUND: The chromodomain helicase DNA-binding family of ATP-dependent chromatin remodeling factors play essential roles during eukaryote growth and development. They are recruited by specific transcription factors and regulate the expression of developmentally important genes. Here, we describe an unexpected role in non-coding RNA-directed DNA methylation in Arabidopsis thaliana. RESULTS: Through forward genetic screens we identified PKL, a gene required for developmental regulation in plants, as a factor promoting transcriptional silencing at the transgenic RD29A promoter. Mutation of PKL results in DNA methylation changes at more than half of the loci that are targeted by RNA-directed DNA methylation (RdDM). A small number of transposable elements and genes had reduced DNA methylation correlated with derepression in the pkl mutant, though for the majority, decreases in DNA methylation are not sufficient to cause release of silencing. The changes in DNA methylation in the pkl mutant are positively correlated with changes in 24-nt siRNA levels. In addition, PKL is required for the accumulation of Pol V-dependent transcripts and for the positioning of Pol V-stabilized nucleosomes at several tested loci, indicating that RNA polymerase V-related functions are impaired in the pkl mutant. CONCLUSIONS: PKL is required for transcriptional silencing and has significant effects on RdDM in plants. The changes in DNA methylation in the pkl mutant are correlated with changes in the non-coding RNAs produced by Pol IV and Pol V. We propose that at RdDM target regions, PKL may be required to create a chromatin environment that influences non-coding RNA production, DNA methylation, and transcriptional silencing.


Assuntos
Metilação de DNA/genética , RNA Polimerases Dirigidas por DNA/genética , RNA não Traduzido/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Mutação/genética , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , Transcrição Genética/genética
17.
Sci Rep ; 7(1): 301, 2017 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-28331197

RESUMO

It has been reported that double-stranded break (DSB)-induced small RNAs (diRNAs) are generated via the RNA-directed DNA methylation pathway and function in DSB repair in Arabidposis. However, important questions remain regarding the biogenesis and function of diRNAs. Here, we used CRISPR/Cas9- or TALEN-triggered DSBs to characterize diRNAs in Arabidopsis and rice. We found that 21-nt diRNAs were generated from a 35S promoter::GU-US reporter transgene targeted by CRISPR/Cas9. Unexpectedly, Pol II transcription of the transgene was required for efficient diRNA production and the level of diRNA accumulation correlated with the expression level of the transgene. diRNAs were not detected from CRISPR/Cas9- or TALEN-induced DSBs within the examined endogenous genes in Arabidopsis or rice. We also found that DCL4 and RDR6 that are known to be involved in posttranscriptional gene silencing were required to generate diRNAs. Our results suggest that DSBs are necessary but not sufficient for efficient diRNA generation and a high level of diRNAs is not necessary for DSB repair.


Assuntos
Arabidopsis/metabolismo , Oryza/metabolismo , Interferência de RNA , Pequeno RNA não Traduzido/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sistemas CRISPR-Cas , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Marcação de Genes , Oryza/genética , Ribonuclease III/metabolismo , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição
18.
Nucleic Acids Res ; 45(1): 181-197, 2017 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-27672037

RESUMO

Epigenetic regulation is important for organismal development and response to the environment. Alteration in epigenetic status has been known mostly from the perspective of enzymatic actions of DNA methylation and/or histone modifications. In a genetic screen for cellular factors involved in preventing epigenetic silencing, we isolated an Arabidopsis mutant defective in SAC3B, a component of the conserved TREX-2 complex that couples mRNA transcription with nuleo-cytoplasmic export. Arabidopsis SAC3B dysfunction causes gene silencing at transgenic and endogenous loci, accompanied by elevation in the repressive histone mark H3K9me2 and by reduction in RNA polymerase Pol II occupancy. SAC3B dysfunction does not alter promoter DNA methylation level of the transgene d35S::LUC, although the DNA demethylase ROS1 is also required for d35S::LUC anti-silencing. THP1 and NUA were identified as SAC3B-associated proteins whose mutations also caused d35S::LUC silencing. RNA-DNA hybrid exists at the repressed loci but is unrelated to gene suppression by the sac3b mutation. Genome-wide analyses demonstrated minor but clear involvement of SAC3B in regulating siRNAs and DNA methylation, particularly at a group of TAS and TAS-like loci. Together our results revealed not only a critical role of mRNA-export factors in transcriptional anti-silencing but also the contribution of SAC3B in shaping plant epigenetic landscapes.


Assuntos
Transporte Ativo do Núcleo Celular/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Transporte/genética , Regulação da Expressão Gênica de Plantas , Inativação Gênica , RNA Mensageiro/genética , Ribonucleoproteínas/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Metilação de DNA , Genes Reporter , Loci Gênicos , Luciferases/genética , Luciferases/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Plantas Geneticamente Modificadas , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ribonucleoproteínas/metabolismo , Transcrição Genética
19.
Cell Res ; 27(2): 226-240, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27934869

RESUMO

Transposons are generally kept silent by epigenetic mechanisms including DNA methylation. Here, we identified a pair of Harbinger transposon-derived proteins (HDPs), HDP1 and HDP2, as anti-silencing factors in Arabidopsis. hdp1 and hdp2 mutants displayed an enhanced silencing of transgenes and some transposons. Phylogenetic analyses revealed that HDP1 and HDP2 were co-domesticated from the Harbinger transposon-encoded transposase and DNA-binding protein, respectively. HDP1 interacts with HDP2 in the nucleus, analogous to their transposon counterparts. Moreover, HDP1 and HDP2 are associated with IDM1, IDM2, IDM3 and MBD7 that constitute a histone acetyltransferase complex functioning in DNA demethylation. HDP2 and the methyl-DNA-binding protein MBD7 share a large set of common genomic binding sites, indicating that they jointly determine the target specificity of the histone acetyltransferase complex. Thus, our data revealed that HDP1 and HDP2 constitute a functional module that has been recruited to a histone acetyltransferase complex to prevent DNA hypermethylation and epigenetic silencing.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Desmetilação do DNA , Elementos de DNA Transponíveis/genética , Proteínas de Ligação a DNA/metabolismo , Histona Acetiltransferases/metabolismo , Proteínas Nucleares/metabolismo , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Sequência de Bases , Núcleo Celular/metabolismo , Metilação de DNA , DNA de Plantas/metabolismo , Proteínas de Ligação a DNA/química , Inativação Gênica , Proteínas Nucleares/química , Filogenia , Ligação Proteica , Domínios Proteicos , Transgenes
20.
Anal Chem ; 87(20): 10470-4, 2015 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-26378678

RESUMO

This work describes a sensor to be incorporated into the on-site monitoring system of airborne house dust mite (HDM) allergens. A surface acoustic wave (SAW) device was combined with self-assembled monolayers of a highly stable antibody capture protein on the SAW surface that have high resistance to pH change. A sandwich assay was used to measure a HDM allergen, Der f 1 derived from Dermatophagoides farinae. Capture antibodies were cross-linked to a protein G based capture layer (ORLA85) on the sensor surface, thereby only Der f 1 and detection antibodies were regenerated by changing pH, resulting in fast repetition of the measurement. The sensor was characterized through 10 repetitive measurements of Der f 1, which demonstrated high reproducibility of the sensor with the coefficient of variation of 5.6%. The limit of detection (LOD) of the sensor was 6.1 ng·mL(-1), encompassing the standard (20 ng·mL(-1)) set by the World Health Organization. Negligible sensor outputs were observed for five different major allergens including other HDM allergens which tend to have cross-reactivity to Der f 1 and their mixtures with Der f 1. Finally, the sensor lifetime was evaluated by conducting three measurements per day, and the sensor output did not substantially change for 4 days. These characteristics make the SAW immunosensor a promising candidate for incorporation into on-site allergen monitoring systems.


Assuntos
Poluição do Ar em Ambientes Fechados/análise , Alérgenos/análise , Antígenos de Dermatophagoides/análise , Proteínas de Bactérias/química , Poeira/análise , Ensaio de Imunoadsorção Enzimática , Som , Alérgenos/imunologia , Animais , Antígenos de Dermatophagoides/imunologia , Proteínas de Bactérias/imunologia , Gatos , Poeira/imunologia , Ensaio de Imunoadsorção Enzimática/instrumentação , Propriedades de Superfície
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