RESUMEN
The histone variant H2A.Z plays a critical role in chromatin-based processes such as transcription, replication, and repair in eukaryotes. Although many H2A.Z-associated processes and features are conserved in plants and animals, a distinguishing feature of plant chromatin is the enrichment of H2A.Z in the bodies of genes that exhibit dynamic expression, particularly in response to differentiation and the environment. Recent work sheds new light on the plant machinery that enables dynamic changes in H2A.Z enrichment and identifies additional chromatin-based pathways that contribute to transcriptional properties of H2A.Z-enriched chromatin. In particular, analysis of a variety of responsive loci reveals a repressive role for H2A.Z in expression of responsive genes and identifies roles for SWR1 and INO80 chromatin remodelers in enabling dynamic regulation of H2A.Z levels and transcription. These studies lay the groundwork for understanding how this ancient histone variant is harnessed by plants to enable responsive and dynamic gene expression (Graphical Abstract).
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Histonas , Nucleosomas , Animales , Histonas/genética , Histonas/metabolismo , Cromatina/genética , Ensamble y Desensamble de Cromatina/genética , Plantas/genética , Plantas/metabolismoRESUMEN
Selective, tissue-specific gene expression is facilitated by the epigenetic modification H3K27me3 (trimethylation of lysine 27 on histone H3) in plants and animals. Much remains to be learned about how H3K27me3-enriched chromatin states are constructed and maintained. Here, we identify a genetic interaction in Arabidopsis thaliana between the chromodomain helicase DNA binding chromatin remodeler PICKLE (PKL), which promotes H3K27me3 enrichment, and the SWR1-family remodeler PHOTOPERIOD INDEPENDENT EARLY FLOWERING1 (PIE1), which incorporates the histone variant H2A.Z. Chromatin immunoprecipitation-sequencing and RNA-sequencing reveal that PKL, PIE1, and the H3K27 methyltransferase CURLY LEAF act in a common gene expression pathway and are required for H3K27me3 levels genome-wide. Additionally, H3K27me3-enriched genes are largely a subset of H2A.Z-enriched genes, further supporting the functional linkage between these marks. We also found that recombinant PKL acts as a prenucleosome maturation factor, indicating that it promotes retention of H3K27me3. These data support the existence of an epigenetic pathway in which PIE1 promotes H2A.Z, which in turn promotes H3K27me3 deposition. After deposition, PKL promotes retention of H3K27me3 after DNA replication and/or transcription. Our analyses thus reveal roles for H2A.Z and ATP-dependent remodelers in construction and maintenance of H3K27me3-enriched chromatin in plants.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Histonas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Ensamble y Desensamble de Cromatina/genética , Ensamble y Desensamble de Cromatina/fisiología , Epigénesis Genética/genética , Epigénesis Genética/fisiología , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Histonas/genética , FotoperiodoRESUMEN
PICKLE (PKL) is an ATP-dependent chromodomain-helicase-DNA-binding domain (CHD3) chromatin remodeling enzyme in Arabidopsis (Arabidopsis thaliana). Previous studies showed that PKL promotes embryonic-to-vegetative transition by inhibiting expression of seed-specific genes during seed germination. The pkl mutants display a low penetrance of the "pickle root" phenotype, with a thick and green primary root that retains embryonic characteristics. The penetrance of this pickle root phenotype in pkl is dramatically increased in gibberellin (GA)-deficient conditions. At adult stages, the pkl mutants are semidwarfs with delayed flowering time, which resemble reduced GA-signaling mutants. These findings suggest that PKL may play a positive role in regulating GA signaling. A recent biochemical analysis further showed that PKL and GA signaling repressors DELLAs antagonistically regulate hypocotyl cell elongation genes by direct protein-protein interaction. To elucidate further the role of PKL in GA signaling and plant development, we studied the genetic interaction between PKL and DELLAs using the hextuple mutant containing pkl and della pentuple (dP) mutations. Here, we show that PKL is required for most of GA-promoted developmental processes, including vegetative growth such as hypocotyl, leaf, and inflorescence stem elongation, and phase transitions such as juvenile-to-adult leaf and vegetative-to-reproductive phase. The removal of all DELLA functions (in the dP background) cannot rescue these phenotypes in pkl RNA-sequencing analysis using the ga1 (a GA-deficient mutant), pkl, and the ga1 pkl double mutant further shows that expression of 80% of GA-responsive genes in seedlings is PKL dependent, including genes that function in cell elongation, cell division, and phase transitions. These results indicate that the CHD3 chromatin remodeler PKL is required for regulating gene expression during most of GA-regulated developmental processes.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , ADN Helicasas/metabolismo , Giberelinas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , ADN Helicasas/genética , Regulación de la Expresión Génica de las Plantas , Germinación , Familia de Multigenes , Mutación , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Semillas/genética , Semillas/crecimiento & desarrollo , Transducción de SeñalRESUMEN
Agrobacterium-mediated transformation is a core technology for basic plant science and agricultural biotechnology. Improving transformation frequency is a major goal for plant transgenesis. We previously showed that T-DNA insertions in some histone genes decreased transformation susceptibility, whereas overexpression of several Arabidopsis H2A and H4 isoforms increased transformation. Overexpression of several histone H2B and H3 isoforms had little effect on transformation frequency. However, overexpression of histone H3-11 (HTR11) enhanced transformation. HTR11 is a unique H3 variant that lacks lysine at positions 9 and 27. The modification status of these lysine residues in canonical H3 proteins plays a critical role in epigenetic determination of gene expression. We mutated histone H3-4 (HTR4), a canonical H3.3 protein that does not increase transformation when overexpressed, by replacing either or both K9 and K27 with the amino acids in HTR11 (either K9I, K27Q, or both). Overexpression of HTR4 with the K27Q but not the K9I substitution enhanced transformation. HTR4K27Q was incorporated into chromatin, and HTR4K27Q overexpression lines exhibited deregulated expression of H3K27me3-enriched genes. These results demonstrate that mutation of K27 in H3.3 is sufficient to perturb H3K27me3-dependent expression in plants as in animals and suggest a distinct epigenetic role for histone HTR11. Further, these observations implicate manipulation of H3K27me3-dependent gene expression as a novel strategy to increase transformation susceptibility.
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Agrobacterium/metabolismo , Epigénesis Genética , Histonas/metabolismo , Lisina/metabolismo , Transformación Genética , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/microbiología , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Cromatina/metabolismo , Evolución Molecular , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Histonas/química , Metilación , Raíces de Plantas/genética , Plantas Modificadas GenéticamenteRESUMEN
Genome-wide chromatin immunoprecipitation (ChIP) studies have brought significant insight into the genomic localization of chromatin-associated proteins and histone modifications. The large amount of data generated by these analyses, however, require approaches that enable rapid validation and analysis of biological relevance. Furthermore, there are still protein and modification targets that are difficult to detect using standard ChIP methods. To address these issues, we developed an immediate chromatin immunoprecipitation procedure which we call ZipChip. ZipChip significantly reduces the time and increases sensitivity allowing for rapid screening of multiple loci. Here we describe how ZipChIP enables detection of histone modifications (H3K4 mono- and trimethylation) and two yeast histone demethylases, Jhd2 and Rph1, which were previously difficult to detect using standard methods. Furthermore, we demonstrate the versatility of ZipChIP by analyzing the enrichment of the histone deacetylase Sir2 at heterochromatin in yeast and enrichment of the chromatin remodeler, PICKLE, at euchromatin in Arabidopsis thaliana.
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Inmunoprecipitación de Cromatina/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Actinas/genética , Actinas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cromatina/genética , Cromatina/metabolismo , Inmunoprecipitación de Cromatina/estadística & datos numéricos , ADN Helicasas/genética , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Genes Fúngicos , Genes de Plantas , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Histonas/genética , Histonas/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Sistemas de Lectura Abierta , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Sirtuina 2/genética , Sirtuina 2/metabolismoRESUMEN
The chromatin remodeler CHD5 plays a critical role in tumor suppression and neurogenesis in mammals. CHD5 contributes to gene expression during neurogenesis, but there is still much to learn regarding how this class of remodelers contributes to differentiation and development. CHD5 remodelers are vertebrate-specific, raising the prospect that CHD5 plays one or more conserved roles in this phylum. Expression of chd5 in adult fish closely mirrors expression of CHD5 in adult mammals. Knockdown of Chd5 during embryogenesis suggests new roles for CHD5 remodelers based on resulting defects in craniofacial development including reduced head and eye size as well as reduced cartilage formation in the head. In addition, knockdown of Chd5 results in altered expression of neural markers in the developing brain and eye as well as a profound defect in differentiation of dopaminergic amacrine cells. Recombinant zebrafish Chd5 protein exhibits nucleosome remodeling activity in vitro, suggesting that it is the loss of this activity that contributes to the observed phenotypes. Our studies indicate that zebrafish is an appropriate model for functional characterization of CHD5 remodelers in vertebrates and highlight the potential of this model for generating novel insights into the role of this vital class of remodelers.
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ADN Helicasas/fisiología , Desarrollo Embrionario/genética , Cabeza/embriología , Proteínas de Pez Cebra/fisiología , Pez Cebra/embriología , Adenosina Trifosfatasas/genética , Animales , Animales Modificados Genéticamente , Encéfalo/embriología , Encéfalo/metabolismo , Cartílago/embriología , Cartílago/metabolismo , Diferenciación Celular/genética , Ensamble y Desensamble de Cromatina/genética , ADN Helicasas/genética , Neuronas Dopaminérgicas/fisiología , Embrión no Mamífero , Ojo/embriología , Ojo/metabolismo , Neurogénesis/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
PICKLE plays a critical role in repression of genes that regulate development identity in Arabidopsis thaliana. PICKLE codes for a putative ATP-dependent chromatin remodeler that exhibits sequence similarity to members of subfamily II of animal CHD remodelers, which includes remodelers such as CHD3/Mi-2 that also restrict expression of developmental regulators. Whereas animal CHD3 remodelers are a component of the Mi-2/NuRD complex that promotes histone deacetylation, PICKLE promotes trimethylation of histone H3 lysine 27 suggesting that it acts via a distinct epigenetic pathway. Here, we examine whether PICKLE is also a member of a multisubunit complex and characterize the biochemical properties of recombinant PICKLE protein. Phylogenetic analysis indicates that PICKLE-related proteins in plants share a common ancestor with members of subfamily II of animal CHD remodelers. Biochemical characterization of PICKLE in planta, however, reveals that PICKLE primarily exists as a monomer. Recombinant PICKLE protein is an ATPase that is stimulated by ssDNA and mononucleosomes and binds to both naked DNA and mononucleosomes. Furthermore, recombinant PICKLE exhibits ATP-dependent chromatin remodeling activity. These studies demonstrate that subfamily II CHD proteins in plants, such as PICKLE, retain ATP-dependent chromatin remodeling activity but act through a mechanism that does not involve the ubiquitous Mi-2/NuRD complex.
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Proteínas de Arabidopsis , Arabidopsis/genética , Ensamble y Desensamble de Cromatina/genética , Cromatina/genética , ADN Helicasas , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/genética , Secuencia de Aminoácidos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Regulación de la Expresión Génica de las Plantas , Histonas/genética , Lisina/genética , Lisina/metabolismo , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Datos de Secuencia Molecular , FilogeniaRESUMEN
In Arabidopsis (Arabidopsis thaliana), the ATP-dependent chromatin remodeler PICKLE (PKL) determines expression of genes associated with developmental identity. PKL promotes the epigenetic mark trimethylation of histone H3 lysine 27 (H3K27me3) that facilitates repression of tissue-specific genes in plants. It has previously been proposed that PKL acts indirectly to promote H3K27me3 by promoting expression of the POLYCOMB REPRESSIVE COMPLEX2 complex that generates H3K27me3. We undertook expression and chromatin immunoprecipitation analyses to further characterize the contribution of PKL to gene expression and developmental identity. Our expression data support a critical and specific role for PKL in expression of H3K27me3-enriched loci but do not support a role for PKL in expression of POLYCOMB REPRESSIVE COMPLEX2. Moreover, our chromatin immunoprecipitation data reveal that PKL protein is present at the promoter region of multiple H3K27me3-enriched loci, indicating that PKL directly acts on these loci. In particular, we find that PKL is present at LEAFY COTYLEDON1 and LEAFY COTYLEDON2 during germination, which is when PKL acts to repress these master regulators of embryonic identity. Surprisingly, we also find that PKL is present at the promoters of actively transcribed genes that are ubiquitously expressed such as ACTIN7 and POLYUBIQUITIN10 that do not exhibit PKL-dependent expression. Taken together, our data contravene the previous model of PKL action and instead support a direct role for PKL in determining levels of H3K27me3 at repressed loci. Our data also raise the possibility that PKL facilitates a common chromatin remodeling process that is not restricted to H3K27me3-enriched regions.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Histonas/metabolismo , Actinas/genética , Actinas/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Ensamble y Desensamble de Cromatina , Inmunoprecipitación de Cromatina , ADN Helicasas , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Sitios Genéticos , Germinación , Lisina/genética , Lisina/metabolismo , Metilación , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Brotes de la Planta/genética , Brotes de la Planta/crecimiento & desarrollo , Brotes de la Planta/metabolismo , Complejo Represivo Polycomb 2 , Poliubiquitina/genética , Poliubiquitina/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Semillas/genética , Semillas/metabolismoRESUMEN
Introduction: The domestic dog, Canis familiaris, is quickly gaining traction as an advantageous model for use in the study of cancer, one of the leading causes of death worldwide. Naturally occurring canine cancers share clinical, histological, and molecular characteristics with the corresponding human diseases. Methods: In this study, we take a deep-learning approach to test how similar the gene expression profile of canine glioma and bladder cancer (BLCA) tumors are to the corresponding human tumors. We likewise develop a tool for identifying misclassified or outlier samples in large canine oncological datasets, analogous to that which was developed for human datasets. Results: We test a number of machine learning algorithms and found that a convolutional neural network outperformed logistic regression and random forest approaches. We use a recently developed RNA-seq-based convolutional neural network, TULIP, to test the robustness of a human-data-trained primary tumor classification tool on cross-species primary tumor prediction. Our study ultimately highlights the molecular similarities between canine and human BLCA and glioma tumors, showing that protein-coding one-to-one homologs shared between humans and canines, are sufficient to distinguish between BLCA and gliomas. Discussion: The results of this study indicate that using protein-coding one-to-one homologs as the features in the input layer of TULIP performs good primary tumor prediction in both humans and canines. Furthermore, our analysis shows that our selected features also contain the majority of features with known clinical relevance in BLCA and gliomas. Our success in using a human-data-trained model for cross-species primary tumor prediction also sheds light on the conservation of oncological pathways in humans and canines, further underscoring the importance of the canine model system in the study of human disease.
RESUMEN
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.
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Metilación de ADN/genética , ARN Polimerasas Dirigidas por ADN/genética , ARN no Traducido/genética , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Mutación/genética , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genética , Transcripción Genética/genéticaRESUMEN
Angiosperm reproduction requires the integrated development of multiple tissues with different genotypes. To achieve successful fertilization, the haploid female gametophytes and diploid ovary must coordinate their development, after which the male gametes must navigate through the maternal sporophytic tissues to reach the female gametes. After fertilization, seed development requires coordinated development of the maternal diploid integuments, the triploid endosperm, and the diploid zygote. Transcription and signaling factors contribute to communication between these tissues, and roles for epigenetic regulation have been described for some of these processes. Here we identify a broad role for CHD3 chromatin remodelers in Arabidopsis thaliana reproductive development. Plants lacking the CHD3 remodeler, PICKLE, exhibit various reproductive defects including abnormal development of the integuments, female gametophyte, and pollen tube, as well as delayed progression of ovule and embryo development. Genetic analyses demonstrate that these phenotypes result from loss of PICKLE in the maternal sporophyte. The paralogous gene PICKLE RELATED 2 is preferentially expressed in the endosperm and acts antagonistically with respect to PICKLE in the seed: loss of PICKLE RELATED 2 suppresses the large seed phenotype of pickle seeds. Surprisingly, the alteration of seed size in pickle plants is sufficient to determine the expression of embryonic traits in the seedling primary root. These findings establish an important role for CHD3 remodelers in plant reproduction and highlight how the epigenetic status of one tissue can impact the development of genetically distinct tissues.
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Proteínas de Arabidopsis/genética , Arabidopsis/genética , ADN Helicasas/genética , Células Germinativas de las Plantas/metabolismo , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/metabolismo , ADN Helicasas/metabolismo , Endospermo/crecimiento & desarrollo , Endospermo/metabolismo , Epigénesis Genética , Células Germinativas de las Plantas/crecimiento & desarrolloRESUMEN
The developmental program of seeds is promoted by master regulators that are expressed in a seed-specific manner. Ectopic expression studies reveal that expression of these master regulators and other transcriptional regulators is sufficient to promote seed-associated traits, including generation of somatic embryos. Recent work highlights the importance of chromatin-associated factors in restricting expression of seed-specific genes, in particular PcG proteins and ATP-dependent remodelers. This review summarizes what is known regarding factors that promote zygotic and/or somatic embryogenesis and the chromatin machinery that represses their expression. Characterization of the regulation of seed-specific genes reveals that plant chromatin-based repression systems exhibit broad conservation with and surprising differences from animal repression systems.
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Epigénesis Genética/genética , Regulación de la Expresión Génica de las Plantas , Semillas/genética , Cromatina/genética , Cromatina/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Modelos BiológicosRESUMEN
CHD3 proteins are ATP-dependent chromatin remodelers that contribute to repression of developmentally regulated genes in both animal and plant systems. In animals, this repression has been linked to a multiple subunit complex, Mi-2/NuRD, whose constituents include a CHD3 protein, a histone deacetylase, and a methyl-CpG-binding domain protein. In Arabidopsis, PICKLE (PKL) codes for a CHD3 protein that acts during germination to repress expression of seed-associated genes. Repression of seed-associated traits is promoted in pkl seedlings by the plant growth regulator gibberellin (GA). We undertook a microarray analysis to determine how PKL and GA act to promote the transition from seed to seedling. We found that PKL and GA act in separate pathways to repress expression of seed-specific genes. Comparison of genomic datasets revealed that PKL-dependent genes are enriched for trimethylation of histone H3 lysine 27 (H3K27me3), a repressive epigenetic mark. Chromatin immunoprecipitation studies demonstrate that PKL promotes H3K27me3 in both germinating seedlings and in adult plants but do not identify a connection between PKL-dependent expression and acetylation levels. Taken together, our analyses illuminate a new pathway by which CHD3 remodelers contribute to repression in eukaryotes.
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Proteínas de Arabidopsis/metabolismo , Histona Desacetilasas/metabolismo , Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/efectos de los fármacos , ADN Helicasas , Regulación de la Expresión Génica de las Plantas , Germinación , Metilación , Miconazol/farmacología , Plantones/efectos de los fármacos , Plantones/genética , Plantones/metabolismo , Semillas/genética , Semillas/fisiologíaRESUMEN
PICKLE (PKL) codes for a CHD3 chromatin remodeling factor that plays multiple roles in Arabidopsis growth and development. Previous analysis of the expression of genes that exhibit PKL-dependent regulation suggested that PKL acts during germination to repress expression of embryonic traits. In this study, we examined the expression of PKL protein to investigate when and where PKL acts to regulate development. A PKL:eGFP translational fusion is preferentially localized in the nucleus of cells, consistent with the proposed role for PKL as a chromatin remodeling factor. A steroid-inducible version of PKL [a fusion of PKL to the glucocorticoid receptor (PKL:GR)] was used to examine when PKL acts to repress expression of embryonic traits. We found that activation of PKL:GR during germination was sufficient to repress expression of embryonic traits in the primary roots of pkl seedlings, whereas activation of PKL:GR after germination had little effect. In contrast, we observed that PKL is required continuously after germination to repress expression of PHERES1, a type I MADS box gene that is normally expressed during early embryogenesis in wild-type plants. Thus, PKL acts at multiple points during development to regulate patterns of gene expression in Arabidopsis.
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Proteínas de Arabidopsis/fisiología , Arabidopsis/embriología , Arabidopsis/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Germinación/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Diferenciación Celular/genética , Núcleo Celular/metabolismo , ADN Helicasas , Proteínas de Dominio MADS/metabolismo , Fenotipo , Raíces de Plantas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Semillas/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Embryos express several unique differentiation characteristics, including the accumulation of a number of metabolites that are generally considered to be unique to seeds. PICKLE (PKL) codes for a CHD3-chromatin remodeling factor that is necessary for repression of embryonic traits in seedlings of Arabidopsis thaliana (L.) Heynh. In pkl mutants, primary roots are capable of expressing many embryonic traits after germination and are referred to as "pickle roots". In an attempt to examine the breadth of PKL-dependent repression of embryo-specific differentiation pathways, we determined the extent to which a variety of embryo-specific compounds accumulate in pickle roots. We found that pickle roots accumulate triacylglycerol with a fatty acid composition that is similar to that found in seeds. The major seed storage proteins are also present in pickle roots. In addition to these two well-characterized seed storage compounds, we observed that pickle roots accumulate phytate, a form of stored phosphate that is preferentially accumulated in seeds. Seeds of members of the Brassicaceae also accumulate a variety of unique secondary metabolites, including sinapate esters and glucosinolates. Surprisingly, the levels of secondary metabolites in pickle roots were not suggestive of an embryonic differentiation state, but did reveal that a mutation in PKL results in substantial changes in root secondary metabolism. Taken together, these data suggest that PKL is responsible for regulating some but not all aspects of the embryonic program as it relates to the accumulation of embryo-specific metabolites.
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Proteínas de Arabidopsis/genética , Arabidopsis/embriología , Arabidopsis/genética , Genes de Plantas , Arabidopsis/metabolismo , Secuencia de Bases , ADN Helicasas , ADN de Plantas/genética , Metabolismo de los Lípidos , Mutación , Ácido Fítico/metabolismo , Proteínas de Plantas/metabolismoRESUMEN
In angiosperms, germination represents an important developmental transition during which embryonic identity is repressed and vegetative identity emerges. PICKLE (PKL) encodes a CHD3-chromatin-remodeling factor necessary for the repression of expression of LEAFY COTYLEDON1 (LEC1), a central regulator of embryogenesis. A candidate gene approach and microarray analysis identified nine additional genes that exhibit PKL-dependent repression of expression during germination. Transcripts for all three LEAFY COTYLEDON genes, LEC1, LEC2, and FUS3, exhibit PKL-dependent repression, and all three transcripts are elevated more than 100-fold in pkl primary roots that inappropriately express embryonic traits (pickle roots). Three other genes that exhibit PKL-dependent regulation have expression patterns correlated with zygotic or somatic embryogenesis, and one gene encodes a putative Lin-11, Isl-1, MEC-3 (LIM) domain transcriptional regulator that is preferentially expressed in siliques. Genes that exhibit PKL-dependent repression during germination are not necessarily regulated by PKL at other points in development. Our data suggest that PKL selectively regulates a suite of genes during germination to repress embryonic identity. In particular, we propose that PKL acts as a master regulator of the LEAFY COTYLEDON genes, and that joint derepression of these genes is likely to contribute substantially to expression of embryonic identity in pkl seedlings.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/embriología , Arabidopsis/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Germinación , Reguladores del Crecimiento de las Plantas/metabolismo , Semillas/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , ADN Helicasas , Regulación del Desarrollo de la Expresión Génica , Genes de Plantas/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Hojas de la Planta/genética , Raíces de Plantas/genética , Semillas/embriología , Semillas/metabolismoRESUMEN
A seed marks the transition between two developmental states; a plant is an embryo during seed formation, whereas it is a seedling after emergence from the seed. Two factors have been identified in Arabidopsis that play a role in establishment of repression of the embryonic state: PKL (PICKLE), which codes for a putative CHD3 chromatin remodeling factor, and gibberellin (GA), a plant growth regulator. Previous observations have also suggested that PKL mediates some aspects of GA responsiveness in the adult plant. To investigate possible mechanisms by which PKL and GA might act to repress the embryonic state, we further characterized the ability of PKL and GA to repress embryonic traits and reexamined the role of PKL in mediating GA-dependent responses. We found that PKL acts throughout the seedling to repress expression of embryonic traits. Although the ability of pkl seedlings to express embryonic traits is strongly induced by inhibiting GA biosynthesis, it is only marginally responsive to abscisic acid and SPY (SPINDLY), factors that have previously been demonstrated to inhibit GA-dependent responses during germination. We also observed that pkl plants exhibit the phenotypic hallmarks of a mutation in a positive regulator of a GA response pathway including reduced GA responsiveness and increased synthesis of bioactive GAs. These observations indicate that PKL may mediate a subset of GA-dependent responses during shoot development.