RESUMEN
BACKGROUND: Polycomb repressive complex 2 (PRC2) is an epigenetic transcriptional repression system, whose catalytic subunit (ENHANCER OF ZESTE HOMOLOG 2, EZH2 in animals) is responsible for trimethylating histone H3 at lysine 27 (H3K27me3). In mammals, gain-of-function mutations as well as overexpression of EZH2 have been associated with several tumors, therefore making this subunit a suitable target for the development of selective inhibitors. Indeed, highly specific small-molecule inhibitors of EZH2 have been reported. In plants, mutations in some PRC2 components lead to embryonic lethality, but no trial with any inhibitor has ever been reported. RESULTS: We show here that the 1,5-bis (3-bromo-4-methoxyphenyl)penta-1,4-dien-3-one compound (RDS 3434), previously reported as an EZH2 inhibitor in human leukemia cells, is active on the Arabidopsis catalytic subunit of PRC2, since treatment with the drug reduces the total amount of H3K27me3 in a dose-dependent fashion. Consistently, we show that the expression level of two PRC2 targets is significantly increased following treatment with the RDS 3434 compound. Finally, we show that impairment of H3K27 trimethylation in Arabidopsis seeds and seedlings affects both seed germination and root growth. CONCLUSIONS: Our results provide a useful tool for the plant community in investigating how PRC2 affects transcriptional control in plant development.
Asunto(s)
Proteínas de Arabidopsis/antagonistas & inhibidores , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Histonas/metabolismo , Proteínas Represoras/antagonistas & inhibidores , Arabidopsis/efectos de los fármacos , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteína Potenciadora del Homólogo Zeste 2 , Inhibidores Enzimáticos/farmacología , Regulación del Desarrollo de la Expresión Génica , Lisina/metabolismo , Metilación , Complejo Represivo Polycomb 2 , Proteínas Represoras/genética , Rutina/análogos & derivados , Rutina/farmacología , Plantones/efectos de los fármacos , Plantones/genética , Plantones/crecimiento & desarrollo , Plantones/metabolismo , Semillas/efectos de los fármacos , Semillas/genética , Semillas/crecimiento & desarrollo , Semillas/metabolismoRESUMEN
BACKGROUND: In seeds, the transition from dormancy to germination is regulated by abscisic acid (ABA) and gibberellins (GAs), and involves chromatin remodelling. Particularly, the repressive mark H3K27 trimethylation (H3K27me3) has been shown to target many master regulators of this transition. DAG1 (DOF AFFECTING GERMINATION1), is a negative regulator of seed germination in Arabidopsis, and directly represses the GA biosynthetic gene GA3ox1 (gibberellin 3-ß-dioxygenase 1). We set to investigate the role of DAG1 in seed dormancy and maturation with respect to epigenetic and hormonal control. RESULTS: We show that DAG1 expression is controlled at the epigenetic level through the H3K27me3 mark during the seed-to-seedling transition, and that DAG1 directly represses also the ABA catabolic gene CYP707A2; consistently, the ABA level is lower while the GA level is higher in dag1 mutant seeds. Furthermore, both DAG1 expression and protein stability are controlled by GAs. CONCLUSIONS: Our results point to DAG1 as a key player in the control of the developmental switch between seed dormancy and germination.
Asunto(s)
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Unión al ADN/metabolismo , Giberelinas/metabolismo , Plantones/metabolismo , Semillas/metabolismo , Factores de Transcripción/metabolismo , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Unión al ADN/genética , Plantones/genética , Plantones/crecimiento & desarrollo , Semillas/genética , Semillas/crecimiento & desarrollo , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: The transcription factor DOF AFFECTING GERMINATION1 (DAG1) is a repressor of the light-mediated seed germination process. DAG1 acts downstream PHYTOCHROME INTERACTING FACTOR3-LIKE 5 (PIL5), the master repressor, and negatively regulates gibberellin biosynthesis by directly repressing the biosynthetic gene AtGA3ox1. The Dof protein DOF AFFECTING GERMINATION (DAG2) shares a high degree of aminoacidic identity with DAG1. While DAG1 inactivation considerably increases the germination capability of seeds, the dag2 mutant has seeds with a germination potential substantially lower than the wild-type, indicating that these factors may play opposite roles in seed germination. RESULTS: We show here that DAG2 expression is positively regulated by environmental factors triggering germination, whereas its expression is repressed by PIL5 and DAG1; by Chromatin Immuno Precipitation (ChIP) analysis we prove that DAG1 directly regulates DAG2. In addition, we show that Red light significantly reduces germination of dag2 mutant seeds. CONCLUSIONS: In agreement with the seed germination phenotype of the dag2 mutant previously published, the present data prove that DAG2 is a positive regulator of the light-mediated seed germination process, and particularly reveal that this protein plays its main role downstream of PIL5 and DAG1 in the phytochrome B (phyB)-mediated pathway.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/embriología , Proteínas de Unión al ADN/metabolismo , Germinación/efectos de la radiación , Luz , Proteínas Represoras/metabolismo , Semillas/embriología , Factores de Transcripción/metabolismo , Ácido Abscísico/farmacología , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Unión al ADN/genética , Oscuridad , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Genes de Plantas , Germinación/genética , Giberelinas/metabolismo , Giberelinas/farmacología , Mutación/genética , Fitocromo B/metabolismo , Semillas/efectos de la radiación , Factores de Transcripción/genéticaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is characterized by extremely poor prognosis. PDAC presents with molecularly distinct subtypes, with the basal-like one being associated with enhanced chemoresistance. Splicing dysregulation contributes to PDAC; however, its involvement in subtype specification remains elusive. Herein, we uncover a subtype-specific splicing signature associated with prognosis in PDAC and the splicing factor Quaking (QKI) as a determinant of the basal-like signature. Single-cell sequencing analyses highlight QKI as a marker of the basal-like phenotype. QKI represses splicing events associated with the classical subtype while promoting basal-like events associated with shorter survival. QKI favors a plastic, quasi-mesenchymal phenotype that supports migration and chemoresistance in PDAC organoids and cell lines, and its expression is elevated in high-grade primary tumors and metastatic lesions. These studies identify a splicing signature that defines PDAC subtypes and indicate that QKI promotes an undifferentiated, plastic phenotype, which renders PDAC cells chemoresistant and adaptable to environmental changes.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Empalme Alternativo/genética , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Línea Celular , FenotipoRESUMEN
Signal transduction pathways transmit the information received from external and internal cues and generate a response that allows the cell to adapt to changes in the surrounding environment. Signaling pathways trigger rapid responses by changing the activity or localization of existing molecules, as well as long-term responses that require the activation of gene expression programs. All steps involved in the regulation of gene expression, from transcription to processing and utilization of new transcripts, are modulated by multiple signal transduction pathways. This review provides a broad overview of the post-translational regulation of factors involved in RNA processing events by signal transduction pathways, with particular focus on the regulation of pre-mRNA splicing, cleavage and polyadenylation. The effects of several post-translational modifications (i.e., sumoylation, ubiquitination, methylation, acetylation and phosphorylation) on the expression, subcellular localization, stability and affinity for RNA and protein partners of many RNA-binding proteins are highlighted. Moreover, examples of how some of the most common signal transduction pathways can modulate biological processes through changes in RNA processing regulation are illustrated. Lastly, we discuss challenges and opportunities of therapeutic approaches that correct RNA processing defects and target signaling molecules.
Asunto(s)
Poliadenilación/genética , Procesamiento Postranscripcional del ARN/genética , ARN/genética , Transducción de Señal/genética , Empalme Alternativo/genética , Humanos , Metilación , Fosforilación/genética , Proteínas de Unión al ARN/genética , Sumoilación/genética , Ubiquitinación/genéticaRESUMEN
In both animals and plants, development involves anatomical modifications. In the root of Arabidopsis thaliana, maturation of the ground tissue (GT)-a tissue comprising all cells between epidermal and vascular ones-is a paradigmatic example of these modifications, as it generates an additional tissue layer, the middle cortex (MC).1-4 In early post-embryonic phases, the Arabidopsis root GT is composed of one layer of endodermis and one of cortex. A second cortex layer, the MC, is generated by asymmetric cell divisions in about 80% of Arabidopsis primary roots, in a time window spanning from 7 to 14 days post-germination (dpg). The cell cycle regulator CYCLIN D6;1 (CYCD6;1) plays a central role in this process, as its accumulation in the endodermis triggers the formation of MC.5 The phytohormone gibberellin (GA) is a key regulator of the timing of MC formation, as alterations in its signaling and homeostasis result in precocious endodermal asymmetric cell divisions.3,6,7 However, little is known on how GAs are regulated during GT maturation. Here, we show that the HOMEODOMAIN LEUCINE ZIPPER III (HD-ZIPIII) transcription factor PHABULOSA (PHB) is a master regulator of MC formation, controlling the accumulation of CYCD6;1 in the endodermis in a cell non-autonomous manner. We show that PHB activates the GA catabolic gene GIBBERELLIN 2 OXIDASE 2 (GA2ox2) in the vascular tissue, thus regulating the stability of the DELLA protein GIBBERELLIN INSENSITIVE (GAI)-a GA signaling repressor-in the root and, hence, CYCD6;1 expression in the endodermis.
Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Ciclinas/genética , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Proteínas de Homeodominio/metabolismo , Arabidopsis/genética , División Celular Asimétrica/genética , Giberelinas/metabolismo , Proteínas de Homeodominio/genética , MicroARNs/metabolismo , Oxigenasas de Función Mixta/genética , Raíces de Plantas/crecimiento & desarrollo , Plantas Modificadas GenéticamenteRESUMEN
The DOF (DNA binding with one finger) family of plant-specific transcription factors (TF) was first identified in maize in 1995. Since then, DOF proteins have been shown to be present in the whole plant kingdom, including the unicellular alga Chlamydomonas reinhardtii. The DOF TF family is characterised by a highly conserved DNA binding domain (DOF domain), consisting of a CX2C-X21-CX2C motif, which is able to form a zinc finger structure. Early in the study of DOF proteins, their relevance for seed biology became clear. Indeed, the PROLAMIN BINDING FACTOR (PBF), one of the first DOF proteins characterised, controls the endosperm-specific expression of the zein genes in maize. Subsequently, several DOF proteins from both monocots and dicots have been shown to be primarily involved in seed development, dormancy and germination, as well as in seedling development and other light-mediated processes. In the last two decades, the molecular network underlying these processes have been outlined, and the main molecular players and their interactions have been identified. In this review, we will focus on the DOF TFs involved in these molecular networks, and on their interaction with other proteins.
RESUMEN
The transition from a dormant to a germinating seed represents a crucial developmental switch in the life cycle of a plant. Subsequent transition from a germinating seed to an autotrophic organism also requires a robust and multi-layered control. Seed germination and seedling growth are multistep processes, involving both internal and external signals, which lead to a fine-tuning control network. In recent years, numerous studies have contributed to elucidate the molecular mechanisms underlying these processes: from light signaling and light-hormone crosstalk to the effects of abiotic stresses, from epigenetic regulation to translational control. However, there are still many open questions and molecular elements to be identified. This review will focus on the different aspects of the molecular control of seed dormancy and germination, pointing out new molecular elements and how these integrate in the signaling pathways already known.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Germinación/genética , Latencia en las Plantas/genética , Semillas/genética , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacología , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/metabolismo , Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Luz , Fototransducción , Fotosíntesis/genética , Reguladores del Crecimiento de las Plantas/metabolismo , Reguladores del Crecimiento de las Plantas/farmacología , Plantones/genética , Plantones/crecimiento & desarrollo , Plantones/metabolismo , Plantones/efectos de la radiación , Semillas/crecimiento & desarrollo , Semillas/metabolismo , Semillas/efectos de la radiación , Estrés FisiológicoRESUMEN
Transcriptome analyses allow the distinguishing of pancreatic ductal adenocarcinoma (PDAC) subtypes, exhibiting different prognoses and chemotherapy responses. However, RNA extraction from pancreatic tissue is cumbersome and has been performed mainly from surgical samples, which are representative of < 20% of cases. The majority of PDAC patients undergo endoscopic ultrasound (EUS)-guided tissue acquisition (EUS-TA), but RNA has been rarely extracted from EUS-TA with scanty results. Herein, we aimed to determine the best conditions for RNA extraction and analysis from PDAC EUS-TA samples in order to carry out molecular analyses. PDAC cases underwent diagnostic EUS-TA, with needles being a 25G fine needle aspiration (FNA) in all patients and then either a 20G lateral core-trap fine needle biopsy (FNB) or a 25G Franseen FNB; the conservation methods were either snap freezing, RNALater or Trizol. RNA concentration and quality (RNA integrity index; RIN) were analyzed and a panel of genes was investigated for tissue contamination and markers of molecular subtype and aggressivity through qRT-PCR. Seventy-four samples from 37 patients were collected. The median RNA concentration was significantly higher in Trizol samples (10.33 ng/uL) compared with snap frozen (0.64 ng/uL; p < 0.0001) and RNALater (0.19 ng/uL; p < 0.0001). The RIN was similar between Trizol (5.15) and snap frozen samples (5.85), while for both methods it was higher compared with RNALater (2.7). Among the needles, no substantial difference was seen in terms of RNA concentration and quality. qRT-PCR analyses revealed that samples from all needles were suitable for the detection of PDAC subtype markers (GATA6 and ZEB1) and splice variants associated with mutational status (GAP17) as well as for the detection of contaminating tissue around PDAC cells. This is the first study that specifically investigates the best methodology for RNA extraction from EUS-TA. A higher amount of good quality RNA is obtainable with conservation in Trizol with a clear superiority of neither FNA nor FNB needles. RNA samples from EUS-TA are suitable for transcriptome analysis including the investigation of molecular subtype and splice variants expression.
Asunto(s)
Neoplasias Pancreáticas/genética , ARN/genética , Anciano , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Femenino , Humanos , Masculino , Agujas , Páncreas/patología , Neoplasias Pancreáticas/patología , Estudios Prospectivos , Neoplasias PancreáticasRESUMEN
Hypocotyl elongation of Arabidopsis seedlings is influenced by light and numerous growth factors. Light induces inhibition of hypocotyl elongation (photomorphogenesis), whereas in the dark hypocotyl elongation is promoted (skotomorphogenesis). Abscisic acid (ABA) plays a major role in inhibition of hypocotyl elongation, but the molecular mechanism remains unclear. We investigated the effect of ABA during photo- and skotomorphogenesis, making use of appropriate mutants, and we show that ABA negatively controls hypocotyl elongation acting on gibberellin (GA) metabolic genes, increasing the amount of the DELLA proteins GAI and RGA, thus affecting GA signalling, and (ultimately) repressing auxin biosynthetic genes.
RESUMEN
Hypocotyl elongation is influenced by light and hormones, but the molecular mechanisms underlying this process are not yet fully elucidated. We had previously suggested that the Arabidopsis DOF transcription factor DAG1 may be a negative component of the mechanism of light-mediated inhibition of hypocotyl elongation, as light-grown dag1 knock-out mutant seedlings show significant shorter hypocotyls than the wild type. By using high-throughput RNA-seq, we compared the transcriptome profile of dag1 and wild type hypocotyls and seedlings. We identified more than 250 genes differentially expressed in dag1 hypocotyls, and their analysis suggests that DAG1 is involved in the promotion of hypocotyl elongation through the control of ABA, ethylene and auxin signaling. Consistently, ChIP-qPCR results show that DAG1 directly binds to the promoters of WRKY18 encoding a transcription factor involved in ABA signaling, of the ethylene- induced gene ETHYLENE RESPONSE FACTOR (ERF2), and of the SMALL AUXIN UP RNA 67 (SAUR67), an auxin-responding gene encoding a protein promoting hypocotyl cell expansion.