RESUMO
Trichomes are specialized epidermal structures that protect plants from biotic and abiotic stresses by synthesizing, storing, and secreting defensive compounds. This study investigates the role of the Gossypium arboreum DNA topoisomerase VI subunit B gene (GaTOP6B) in trichome development and branching. Sequence alignment revealed a high similarity between GaTOP6B and AtTOP6B, suggesting a conserved function in trichome regulation. Although AtTOP6B acts as a positive regulator of trichome development, functional analyses showed contrasting effects: Virus-induced gene silencing (VIGS) of GaTOP6B in cotton increased trichome density, while its overexpression in Arabidopsis decreased trichome density but enhanced branching. This demonstrates that GaTOP6B negatively regulates trichome number, indicating species-specific roles in trichome initiation and branching between cotton and Arabidopsis. Overexpression of the GaTOP6B promotes jasmonic acid synthesis, which in turn inhibits the G1/S or G2/M transitions, stalling the cell cycle. On the other hand, it suppresses brassinolide synthesis and signaling while promoting cytokinin degradation, further inhibiting mitosis. These hormonal interactions facilitate the transition of cells from the mitotic cycle to the endoreduplication cycle. As the level of endoreduplication increases, trichomes develop an increased number of branches. These findings highlight GaTOP6B's critical role as a regulator of trichome development, providing new genetic targets for improving cotton varieties in terms of enhanced adaptability and resilience.
Assuntos
Arabidopsis , Ciclopentanos , Endorreduplicação , Regulação da Expressão Gênica de Plantas , Gossypium , Oxilipinas , Proteínas de Plantas , Tricomas , Tricomas/genética , Tricomas/crescimento & desenvolvimento , Tricomas/metabolismo , Gossypium/genética , Gossypium/crescimento & desenvolvimento , Gossypium/metabolismo , Ciclopentanos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Oxilipinas/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Endorreduplicação/genética , Brassinosteroides/metabolismo , Plantas Geneticamente Modificadas , Genes de Plantas , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Esteroides HeterocíclicosRESUMO
Endoreduplication, during which cells increase their DNA content through successive rounds of full genome replication without cell division, is the major source of endopolyploidy in higher plants. Endoreduplication plays pivotal roles in plant growth and development and is associated with the activation of specific transcriptional programmes that are characteristic of each cell type, thereby defining their identity. In plants, endoreduplication is found in numerous organs and cell types, especially in agronomically valuable ones, such as the fleshy fruit (pericarp) of tomato presenting high ploidy levels. We used the tomato pericarp tissue as a model system to explore the transcriptomes associated with endoreduplication progression during fruit growth. We confirmed that expression globally scales with ploidy level and identified sets of differentially expressed genes presenting only developmental-specific, only ploidy-specific expression patterns or profiles resulting from an additive effect of ploidy and development. When comparing ploidy levels at a specific developmental stage, we found that non-endoreduplicated cells are defined by cell division state and cuticle synthesis while endoreduplicated cells are mainly defined by their metabolic activity changing rapidly over time. By combining this dataset with publicly available spatiotemporal pericarp expression data, we proposed a map describing the distribution of ploidy levels within the pericarp. These transcriptome-based predictions were validated by quantifying ploidy levels within the pericarp tissue. This in situ ploidy quantification revealed the dynamic progression of endoreduplication and its cell layer specificity during early fruit development. In summary, the study sheds light on the complex relationship between endoreduplication, cell differentiation and gene expression patterns in the tomato pericarp.
Assuntos
Endorreduplicação , Frutas , Regulação da Expressão Gênica de Plantas , Ploidias , Solanum lycopersicum , Transcriptoma , Solanum lycopersicum/genética , Solanum lycopersicum/crescimento & desenvolvimento , Solanum lycopersicum/metabolismo , Frutas/genética , Frutas/crescimento & desenvolvimento , Frutas/metabolismo , Endorreduplicação/genética , Perfilação da Expressão Gênica , Divisão Celular/genéticaRESUMO
BACKGROUND AND AIMS: Endoreduplication, the duplication of the nuclear genome without mitosis, is a common process in plants, especially in angiosperms and mosses. Accumulating evidence supports the relationship between endoreduplication and plastic responses to stress factors. Here, we investigated the level of endoreduplication in Ceratodon (Bryophyta), which includes the model organism Ceratodon purpureus. METHODS: We used flow cytometry to estimate the DNA content of 294 samples from 67 localities and found three well-defined cytotypes, two haploids and one diploid, the haploids corresponding to C. purpureus and Ceratodon amazonum, and the diploid to Ceratodon conicus, recombination occurring between the former two. KEY RESULTS: The endoreduplication index (EI) was significantly different for each cytotype, being higher in the two haploids. In addition, the EI of the haploids was higher during the hot and dry periods typical of the Mediterranean summer than during spring, whereas the EI of the diploid cytotype did not differ between seasons. CONCLUSIONS: Endopolyploidy may be essential in haploid mosses to buffer periods of drought and to respond rapidly to desiccation events. Our results also suggest that the EI is closely related to the basic ploidy level, but less so to the nuclear DNA content as previously suggested.
Assuntos
Briófitas , Bryopsida , Diploide , Haploidia , Endorreduplicação/genética , Secas , DNARESUMO
PREMISE: Endoreduplication, nonheritable duplication of a nuclear genome, is widespread in plants and plays a role in developmental processes related to cell differentiation. However, neither ecological nor cytological factors influencing intraspecific variation in endoreduplication are fully understood. METHODS: We cultivated plants covering the range-wide natural diversity of diploid and tetraploid populations of Arabidopsis arenosa in common conditions to investigate the effect of original ploidy level on endoreduplication. We also raised plants from several foothill and alpine populations from different lineages and of both ploidies to test for the effect of elevation. We determined the endoreduplication level in leaves of young plants by flow cytometry. Using RNA-seq data available for our populations, we analyzed gene expression analysis in individuals that differed in endoreduplication level. RESULTS: We found intraspecific variation in endoreduplication that was mainly driven by the original ploidy level of populations, with significantly higher endoreduplication in diploids. An effect of elevation was also found within each ploidy, yet its direction exhibited rather regional-specific patterns. Transcriptomic analysis comparing individuals with high vs. low endopolyploidy revealed a majority of differentially expressed genes related to the stress and hormone response and to modifications especially in the cell wall and in chloroplasts. CONCLUSIONS: Our results support the general assumption of higher potential of low-ploidy organisms to undergo endoreduplication and suggest that endoreduplication is further integrated within the stress response pathways for a fine-tune adjustment of the endoreduplication process to their local environment.
Assuntos
Arabidopsis , Arabidopsis/genética , Diploide , Endorreduplicação/genética , Ploidias , TetraploidiaRESUMO
Endoreduplication, a process in which DNA replication occurs in the absence of mitosis, is found in all eukaryotic kingdoms, especially plants, where it is assumed to be important for cell growth and cell fate maintenance. However, a comprehensive understanding of the mechanism regulating endoreduplication is still lacking. We previously reported that UBIQUITIN-SPECIFIC PROTEASE14 (UBP14), encoded by DA3, acts upstream of CYCLIN-DEPENDENT KINASE B1;1 (CDKB1;1) to influence endoreduplication and cell growth in Arabidopsis thaliana. The da3-1 mutant possesses large cotyledons with enlarged cells due to high ploidy levels. Here, we identified a suppressor of da3-1 (SUPPRESSOR OF da3-1 6; SUD6), encoding CYCLIN-DEPENDENT KINASE G2 (CDKG2), which promotes endoreduplication and cell growth. CDKG2/SUD6 physically associates with CDKB1;1 in vivo and in vitro. CDKB1;1 directly phosphorylates SUD6 and modulates its stability. Genetic analysis indicated that SUD6 acts downstream of DA3 and CDKB1;1 to control ploidy level and cell growth. Thus, our study establishes a regulatory cascade for UBP14/DA3-CDKB1;1-CDKG2/SUD6-mediated control of endoreduplication and cell growth in Arabidopsis.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Quinases Ciclina-Dependentes/genética , Endorreduplicação/genética , Ubiquitina/genéticaRESUMO
Although the evolutionary drivers of genome size change are known, the general patterns and mechanisms of plant genome size evolution are yet to be established. Here we aim to assess the relative importance of proliferation of repetitive DNA, chromosomal variation (including polyploidy), and the type of endoreplication for genome size evolution of the Pleurothallidinae, the most species-rich orchid lineage. Phylogenetic relationships between 341 Pleurothallidinae representatives were refined using a target enrichment hybrid capture combined with high-throughput sequencing approach. Genome size and the type of endoreplication were assessed using flow cytometry supplemented with karyological analysis and low-coverage Illumina sequencing for repeatome analysis on a subset of samples. Data were analyzed using phylogeny-based models. Genome size diversity (0.2-5.1 Gbp) was mostly independent of profound chromosome count variation (2n = 12-90) but tightly linked with the overall content of repetitive DNA elements. Species with partial endoreplication (PE) had significantly greater genome sizes, and genomic repeat content was tightly correlated with the size of the non-endoreplicated part of the genome. In PE species, repetitive DNA is preferentially accumulated in the non-endoreplicated parts of their genomes. Our results demonstrate that proliferation of repetitive DNA elements and PE together shape the patterns of genome size diversity in orchids.
Assuntos
Endorreduplicação/genética , Evolução Molecular , Tamanho do Genoma/genética , Genoma de Planta/genética , Orchidaceae/genética , Sequências Repetitivas de Ácido Nucleico/genética , Cromossomos de Plantas/genética , DNA de Cloroplastos/genética , DNA de Plantas/genética , Citometria de Fluxo , Variação Genética , Cariotipagem , Filogenia , Análise de Sequência de DNARESUMO
DNA endoreplication has been implicated as a cell strategy for cell growth and in tissue injury. Here, we demonstrate that barrier-to-autointegration factor (BAF) represses endoreplication in Drosophila myofibers. We show that BAF localization at the nuclear envelope is eliminated in flies with mutations of the linker of nucleoskeleton and cytoskeleton (LINC) complex in which the LEM-domain protein Otefin is excluded, or after disruption of the nucleus-sarcomere connections. Furthermore, BAF localization at the nuclear envelope requires the activity of the BAF kinase VRK1/Ball, and, consistently, non-phosphorylatable BAF-GFP is excluded from the nuclear envelope. Importantly, removal of BAF from the nuclear envelope correlates with increased DNA content in the myonuclei. E2F1, a key regulator of endoreplication, overlaps BAF localization at the myonuclear envelope, and BAF removal from the nuclear envelope results in increased E2F1 levels in the nucleoplasm and subsequent elevated DNA content. We suggest that LINC-dependent and phosphosensitive attachment of BAF to the nuclear envelope, through its binding to Otefin, tethers E2F1 to the nuclear envelope thus inhibiting its accumulation in the nucleoplasm.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Endorreduplicação/genética , Proteínas de Membrana/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Animais , Citoesqueleto/genética , Replicação do DNA/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Mutação/genética , Miofibrilas/genética , Membrana Nuclear/genética , Matriz Nuclear/genética , Protamina Quinase/genéticaRESUMO
Adult progenitor cells activation is a key event in the formation of adult organs. In Drosophila, formation of abdominal adult trachea depends on the specific activation of tracheal adult progenitors (tracheoblasts) at the Tr4 and Tr5 spiracular branches. Proliferation of these tracheoblasts generates a pool of tracheal cells that migrate toward the posterior part of the trachea by the activation of the branchless/fibroblast growth factor (Bnl/FGF) signaling to form the abdominal adult trachea. Here, we show that, in addition to migration, Bnl/FGF signaling, mediated by the transcription factor Pointed, is also required for tracheoblast proliferation. This tracheoblast activation relies on the expression of the FGF ligand bnl in their nearby branches. Finally, we show that, in the absence of the transcription factor Cut (Ct), Bnl/FGF signaling induces endoreplication of tracheoblasts partially by promoting fizzy-related expression. Altogether, our results suggest a dual role of Bnl/FGF signaling in tracheoblasts, inducing both proliferation and endoreplication, depending on the presence or absence of the transcription factor Ct, respectively.
Assuntos
Proliferação de Células/genética , Proteínas de Drosophila/metabolismo , Drosophila/genética , Drosophila/metabolismo , Endorreduplicação/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas Nucleares/metabolismo , Células-Tronco/metabolismo , Traqueia/citologia , Fatores de Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Movimento Celular/genética , Drosophila/crescimento & desenvolvimento , Feminino , Masculino , Morfogênese/genética , Transdução de Sinais/genética , Traqueia/crescimento & desenvolvimento , Traqueia/metabolismo , TransgenesRESUMO
In Drosophila, the larval prothoracic gland integrates nutritional status with developmental signals to regulate growth and maturation through the secretion of the steroid hormone ecdysone. While the nutritional signals and cellular pathways that regulate prothoracic gland function are relatively well studied, the transcriptional regulators that orchestrate the activity of this tissue remain less characterized. Here, we show that lysine demethylase 5 (KDM5) is essential for prothoracic gland function. Indeed, restoring kdm5 expression only in the prothoracic gland in an otherwise kdm5 null mutant animal is sufficient to rescue both the larval developmental delay and the pupal lethality caused by loss of KDM5. Our studies show that KDM5 functions by promoting the endoreplication of prothoracic gland cells, a process that increases ploidy and is rate limiting for the expression of ecdysone biosynthetic genes. Molecularly, we show that KDM5 activates the expression of the receptor tyrosine kinase torso, which then promotes polyploidization and growth through activation of the MAPK signaling pathway. Taken together, our studies provide key insights into the biological processes regulated by KDM5 and expand our understanding of the transcriptional regulators that coordinate animal development.
Assuntos
Relógios Biológicos/genética , Proteínas de Drosophila/fisiologia , Drosophila melanogaster , Desenvolvimento Embrionário/genética , Glândulas Endócrinas/embriologia , Histona Desmetilases/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Ecdisona/metabolismo , Embrião não Mamífero , Glândulas Endócrinas/metabolismo , Endorreduplicação/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Larva , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Organogênese/genética , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Fatores de TempoRESUMO
The development of a new organ is the result of coordinated events of cell division and expansion, in strong interaction with each other. This study presents a dynamic model of tomato fruit development that includes cell division, endoreduplication, and expansion processes. The model is used to investigate the potential interactions among these developmental processes within the context of the neo-cellular theory. In particular, different control schemes (either cell-autonomous or organ-controlled) are tested and compared to experimental data from two contrasting genotypes. The model shows that a pure cell-autonomous control fails to reproduce the observed cell-size distribution, and that an organ-wide control is required in order to get realistic cell-size variations. The model also supports the role of endoreduplication as an important determinant of the final cell size and suggests that a direct effect of endoreduplication on cell expansion is needed in order to obtain a significant correlation between size and ploidy, as observed in real data.
Assuntos
Tamanho Celular , Simulação por Computador , Frutas/citologia , Modelos Biológicos , Ploidias , Solanum lycopersicum/citologia , Proliferação de Células , Endorreduplicação/genética , Genótipo , Solanum lycopersicum/genética , Especificidade de Órgãos , Análise de Componente PrincipalRESUMO
BACKGROUND: The floral transition is a complex developmental event, fine-tuned by various environmental and endogenous cues to ensure the success of offspring production. Leaves are key organs in sensing floral inductive signals, such as a change in light regime, and in the production of the mobile florigen. CONSTANS and FLOWERING LOCUS T are major players in leaves in response to photoperiod. Morphological and molecular events during the floral transition have been intensively studied in the shoot apical meristem. To better understand the concomitant processes in leaves, which are less described, we investigated the nuclear changes in fully developed leaves during the time course of the floral transition. RESULTS: We highlighted new putative regulatory candidates of flowering in leaves. We observed differential expression profiles of genes related to cellular, hormonal and metabolic actions, but also of genes encoding long non-coding RNAs and new natural antisense transcripts. In addition, we detected a significant increase in ploidy level during the floral transition, indicating endoreduplication. CONCLUSIONS: Our data indicate that differentiated mature leaves, possess physiological plasticity and undergo extensive nuclear reprogramming during the floral transition. The dynamic events point at functionally related networks of transcription factors and novel regulatory motifs, but also complex hormonal and metabolic changes.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Reprogramação Celular/genética , Endorreduplicação/genética , Florígeno/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Flores/genética , Flores/crescimento & desenvolvimento , Flores/fisiologia , Flores/efeitos da radiação , Regulação da Expressão Gênica de Plantas , Meristema/genética , Meristema/crescimento & desenvolvimento , Meristema/fisiologia , Meristema/efeitos da radiação , Fotoperíodo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/fisiologia , Folhas de Planta/efeitos da radiação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
MAIN CONCLUSION: Cotton GaTOP6B is involved in cellular endoreduplication and a positive response to drought stress via promoting plant leaf and root growth. Drought is deemed as one of adverse conditions that could cause substantial reductions in crop yields worldwide. Since cotton exhibits a moderate-tolerant phenotype under water-deficit conditions, the plant could therefore be used to characterize potential new genes regulating drought tolerance in crop plants. In this work, GaTOP6B, encoding DNA topoisomerase VI subunit B, was identified in Asian cotton (Gossypium arboreum). Virus-induced gene silencing (VIGS) and overexpression (OE) were used to investigate the biological function of GaTOP6B in G. arboreum and Arabidopsis thaliana under drought stress. The GaTOP6B-silencing plants showed a reduced ploidy level, and displayed a compromised tolerance phenotype including lowered relative water content (RWC), decreased proline content and antioxidative enzyme activity, and an increased malondialdehyde (MDA) content under drought stress. GaTOP6B-overexpressing Arabidopsis lines, however, had increased ploidy levels, and were more tolerant to drought treatment, associated with improved RWC maintenance, higher proline accumulation, and reduced stomatal aperture under drought stress. Transcriptome analysis showed that genes involved in the processes like cell cycle, transcription and signal transduction, were substantially up-regulated in GaTOP6B-overexpressing Arabidopsis, promoting plant growth and development. More specifically, under drought stress, the genes involved in the biosynthesis of secondary metabolites such as phenylpropanoid, starch and sucrose were selectively enhanced to improve tolerance in plants. Taken together, the results demonstrated that GaTOP6B could coordinately regulate plant leaf and root growth via cellular endoreduplication, and positively respond to drought stress. Thus, GaTOP6B could be a competent candidate gene for improvement of drought tolerance in crop species.
Assuntos
Endorreduplicação/genética , Genes de Plantas/fisiologia , Gossypium/genética , Arabidopsis , Proteínas Arqueais/genética , Proteínas Arqueais/fisiologia , Clorofila/metabolismo , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/fisiologia , Desidratação , Citometria de Fluxo , Genes de Plantas/genética , Gossypium/crescimento & desenvolvimento , Gossypium/metabolismo , Gossypium/fisiologia , Malondialdeído/metabolismo , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Raízes de Plantas/crescimento & desenvolvimento , Transpiração Vegetal , Plantas Geneticamente Modificadas , Prolina/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
TCP (TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR) proteins, a family of plant-specific transcription factors, play important roles in many developmental processes. However, genetic and functional redundancy among class I TCP limits the analysis of their biological roles. Here, we identified a dominant-negative mutant of Arabidopsis thaliana TCP7 named leaf curling-upward (lcu), which exhibits smaller leaf cells and shorter hypocotyls than the wild type, due to defective endoreplication. A septuple loss-of-function mutant of TCP7, TCP8, TCP14, TCP15, TCP21, TCP22, and TCP23 displayed similar developmental defects to those of lcu. Genome-wide RNA-sequencing showed that lcu and the septuple mutant share many misexpressed genes. Intriguingly, TCP7 directly targets the CYCLIN D1;1 (CYCD1;1) locus and activates its transcription. We determined that the C-terminus of TCP7 accounts for its transcriptional activation activity. Furthermore, the mutant protein LCU exhibited reduced transcriptional activation activity due to the introduction of an EAR-like repressive domain at its C-terminus. Together, these observations indicate that TCP7 plays important roles during leaf and hypocotyl development, redundantly, with at least six class I TCPs, and regulates the expression of CYCD1;1 to affect endoreplication in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis , Ciclina D3/metabolismo , Endorreduplicação/genética , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Hipocótilo/crescimento & desenvolvimento , Folhas de Planta/crescimento & desenvolvimentoRESUMO
Anaplastic thyroid cancer (ATC) is among the most lethal malignancies. The mitotic kinase PLK1 is overexpressed in the majority of ATCs and PLK1 inhibitors have shown preclinical efficacy. However, they also cause mitotic slippage and endoreduplication, leading to the generation of tetraploid, genetically unstable cell populations. We hypothesized that PI3K activity may facilitate mitotic slippage upon PLK1 inhibition, and thus tested the effect of combining PLK1 and PI3K inhibitors in ATC models, in vitro and in vivo. Treatment with BI6727 and BKM120 resulted in a significant synergistic effect in ATC cells, independent of the levels of AKT activity. Combination of the two drugs enhanced growth suppression at doses for which the single drugs showed no effect, and led to a massive reduction of the tetraploid cells population. Furthermore, combined treatment in PI3Khigh cell lines showed a significant induction of apoptosis. Finally, combined inhibition of PI3K and PLK1 was extremely effective in vivo, in an immunocompetent allograft model of ATC. Our results demonstrate a clear therapeutic potential of combining PLK1 and PI3K inhibitors in anaplastic thyroid tumors.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Endorreduplicação/efeitos dos fármacos , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Carcinoma Anaplásico da Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/tratamento farmacológico , Aminopiridinas/administração & dosagem , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Endorreduplicação/genética , Humanos , Camundongos , Morfolinas/administração & dosagem , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Pteridinas/administração & dosagem , Carcinoma Anaplásico da Tireoide/genética , Carcinoma Anaplásico da Tireoide/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Quinase 1 Polo-LikeRESUMO
KEY MESSAGE: DNA replication and continuous process of transcription during ongoing amitotic division accelerate the development of four-celled pea suspensor containing nuclei which create transient gradient of polyploidy necessary for correct embryo development. A suspensor, the link between embryo proper and surrounding tissues, differs significantly in size, morphology, and degree of polyploidy among the species. The suspensor of Pisum sativum consists of four polynuclear cells (two hemispherical and two elongated) formed in two layers. Their nuclei undergo endoreplication reaching, respectively, up to 256C and 128-256C DNA levels in its hemispherical and elongated parts. Our study shows that endoreplication first appears in the spherical part of the suspensor, and, subsequently, in the elongated one. At the next stages of suspensor development, the increase in DNA content takes place also in a similar order. Thus, despite simple construction of the suspensor, its development, supported by endoreplication, creates a certain gradient of polyploidy, which occurs in more extensive suspensors. Moreover, the rapid development of suspensor is supported both by the initiation of DNA replication prior to the completion of amitotic division of its polyploidal nuclei and by a continuous process of transcription, which is silenced by chromatin condensation throughout mitosis. Furthermore, the increase in DNA content correlates with the greater amount of transcripts; however, the multiplication of DNA copies does not entail an increase (but fluctuation) in the mean transcriptional activity of a particular nucleus during the next stages of suspensor development.
Assuntos
Endorreduplicação/genética , Pisum sativum/anatomia & histologia , Pisum sativum/genética , Sementes/anatomia & histologia , Sementes/genética , Núcleo Celular/metabolismo , DNA de Plantas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
The largest subunit of the origin recognition complex (ORC1) is essential for assembly of the prereplicative complex, firing of DNA replication origins, and faithful duplication of the genome. Here, we generated knock-in mice with LoxP sites flanking exons encoding the critical ATPase domain of ORC1. Global or tissue-specific ablation of ORC1 function in mouse embryo fibroblasts and fetal and adult diploid tissues blocked DNA replication, cell lineage expansion, and organ development. Remarkably, ORC1 ablation in extraembryonic trophoblasts and hepatocytes, two polyploid cell types in mice, failed to impede genome endoreduplication and organ development and function. Thus, ORC1 in mice is essential for mitotic cell divisions but dispensable for endoreduplication. We propose that DNA replication of mammalian polyploid genomes uses a distinct ORC1-independent mechanism.
Assuntos
Endorreduplicação/genética , Genoma/genética , Complexo de Reconhecimento de Origem/genética , Complexo de Reconhecimento de Origem/metabolismo , Adenosina Trifosfatases/genética , Animais , Divisão Celular/genética , Proliferação de Células/genética , Desenvolvimento Embrionário/genética , Ativação Enzimática , Feminino , Deleção de Genes , Hepatócitos/citologia , Regeneração Hepática/genética , Camundongos , Mitose/genética , Placenta/fisiologia , GravidezRESUMO
Universal minicircle sequence binding proteins (UMSBPs) are CCHC-type zinc-finger proteins that bind a single-stranded G-rich sequence, UMS, conserved at the replication origins of the mitochondrial (kinetoplast) DNA of trypanosomatids. Here, we report that Trypanosoma brucei TbUMSBP2, which has been previously proposed to function in the replication and segregation of the mitochondrial DNA, colocalizes with telomeres at the nucleus and is essential for their structure, protection and function. Knockdown of TbUMSBP2 resulted in telomere clustering in one or few foci, phosphorylation of histone H2A at the vicinity of the telomeres, impaired nuclear division, endoreduplication and cell growth arrest. Furthermore, TbUMSBP2 depletion caused rapid reduction in the G-rich telomeric overhang, and an increase in C-rich single-stranded telomeric DNA and in extrachromosomal telomeric circles. These results indicate that TbUMSBP2 is essential for the integrity and function of telomeres. The sequence similarity between the mitochondrial UMS and the telomeric overhang and the finding that UMSBPs bind both sequences suggest a common origin and/or function of these interactions in the replication and maintenance of the genomes in the two organelles. This feature could have converged or preserved during the evolution of the nuclear and mitochondrial genomes from their ancestral (likely circular) genome in early diverged protists.
Assuntos
Cromossomos/genética , Proteínas de Ligação a DNA/genética , Proteínas de Protozoários/genética , Telômero/genética , Trypanosoma brucei brucei/genética , Divisão do Núcleo Celular/genética , DNA Mitocondrial/genética , Endorreduplicação/genética , Genoma de Protozoário/genética , Histonas/metabolismo , Fosforilação , Ligação Proteica/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Trypanosoma brucei brucei/crescimento & desenvolvimentoRESUMO
In the development of cell-based medicinal products, it is crucial to guarantee that the application of such an advanced therapy medicinal product (ATMP) is safe for the patients. The consensus of the European regulatory authorities is: "In conclusion, on the basis of the state of art, conventional karyotyping can be considered a valuable and useful technique to analyse chromosomal stability during preclinical studies". 408 chondrocyte samples (84 monolayers and 324 spheroids) from six patients were analysed using trypsin-Giemsa staining, spectral karyotyping and fluorescence in situ hybridisation, to evaluate the genetic stability of chondrocyte samples from non-clinical studies. Single nucleotide polymorphism (SNP) array analysis was performed on chondrocyte spheroids from five of the six donors. Applying this combination of techniques, the genetic analyses performed revealed no significant genetic instability until passage 3 in monolayer cells and interphase cells from spheroid cultures at different time points. Clonal occurrence of polyploid metaphases and endoreduplications were identified associated with prolonged cultivation time. Also, gonosomal losses were observed in chondrocyte spheroids, with increasing passage and duration of the differentiation phase. Interestingly, in one of the donors, chromosomal aberrations that are also described in extraskeletal myxoid chondrosarcoma were identified. The SNP array analysis exhibited chromosomal aberrations in two donors and copy neutral losses of heterozygosity regions in four donors. This study showed the necessity of combined genetic analyses at defined cultivation time points in quality studies within the field of cell therapy.
Assuntos
Corantes Azur/metabolismo , Condrócitos/metabolismo , Bandeamento Cromossômico , Loci Gênicos , Genômica/métodos , Hibridização in Situ Fluorescente , Polimorfismo de Nucleotídeo Único/genética , Cariotipagem Espectral , Idoso , Biópsia , Células Cultivadas , Aberrações Cromossômicas , Cromossomos Humanos/genética , Variações do Número de Cópias de DNA/genética , Endorreduplicação/genética , Feminino , Humanos , Perda de Heterozigosidade/genética , Masculino , Pessoa de Meia-Idade , Poliploidia , Esferoides Celulares/citologiaRESUMO
KEY MESSAGE: DSBs differently affect endoreduplication and organ size in radish cotyledons and hypocotyls in different light conditions, suggesting that DSBs-mediated endoreduplication varies based on different developmental and environmental cues. Endoreduplication induced by DNA double strand breaks (DSBs) in Arabidopsis thaliana roots and cultured cells has been reported in recent years. In this study, we investigated whether DSBs-mediated endoreduplication also occurs in other tissues, such as cotyledons and hypocotyls of radish (Raphanus sativus var. longipinnatus) plants. To induce DSBs, UV irradiation and Zeocin treatment were applied to in vitro-cultured radish seedlings, and ploidy distribution of the treated tissues was analyzed by flow cytometry. Consequently, frequencies of the higher ploidy (8C) cells and cycle values in the cotyledon tissues increased with increasing doses of UV irradiation and concentrations of Zeocin, irrespective of light conditions. UV-stimulated endoreduplication was also observed in four Brassica species. In hypocotyls, UV treatments decreased the frequencies of higher ploidy (32C) cells and cycle values in dark-grown seedlings, whereas Zeocin treatments increased the frequencies of higher ploidy (16C and 32C) cells and cycle values in light- and dark-grown seedlings. Among the treatments, organ sizes did not simply correlate with cycle values. The effects of treatments on endoreduplication and organ size differed based on organ and light conditions, indicating that DSBs-mediated endoreduplication may involve a multifaceted response to different developmental and environmental cues.