RESUMEN
bZIP transcription factors can function as homodimers or heterodimers through interactions with their disordered coiled-coil domain. Such dimer assemblies are known to influence DNA-binding specificity and/or the recruitment of binding partners, which can cause a functional switch of a transcription factor from being an activator to a repressor. We recently identified the genomic targets of a bZIP transcription factor called CREB3L1 in rat hypothalamic supraoptic nucleus by ChIP-seq. The objective of this study was to investigate the CREB3L1 protein-to-protein interactome of which little is known. For this approach, we created and screened a rat supraoptic nucleus yeast two-hybrid prey library with the bZIP region of rat CREB3L1 as the bait. Our yeast two-hybrid approach captured five putative CREB3L1 interacting prey proteins in the supraoptic nucleus. One interactor was selected by bioinformatic analyses for more detailed investigation by co-immunoprecipitation, immunofluorescent cellular localisation, and reporter assays in vitro. Here we identify dimerisation hub protein Dynein Light Chain LC8-Type 1 as a CREB3L1 interacting protein that in vitro enhances CREB3L1 activation of target genes.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Dineínas Citoplasmáticas , Proteínas del Tejido Nervioso , Activación Transcripcional , Animales , Humanos , Ratas , Arginina Vasopresina/metabolismo , Arginina Vasopresina/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Dineínas Citoplasmáticas/metabolismo , Dineínas Citoplasmáticas/genética , Multimerización de Proteína , Núcleo Supraóptico/metabolismo , Activación Transcripcional/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
The bZIP transcription factors play crucial roles in various aspects of plant biology, including development, defence mechanisms, senescence, and responses to both biotic and abiotic environmental stresses. Myristica fragrans Houtt. transcriptome analysis has identified 15 bZIP transcription factors, each exhibiting major conserved domains and motifs such as BRLZ, MFMR, and DOG1. Functional characterisation of these identified MfbZIP factors indicates their predominant localisation within the nucleus. Phylogenetic analysis reveals that MfbZIP factors cluster into three subgroups alongside annotated bZIP sequences from Magnolia sinica and Arabidopsis thaliana. Moreover, gene ontology (GO) analysis highlights several key functions of MfbZIP, including involvement in defence responses, abscisic acid-induced signalling pathways, and DNA-binding transcription factor activity. Further investigation through KEGG pathway analysis reveals that the amino acid sequences of MfbZIP contain binding motifs for proteins such as TGA, implicated in plant hormone signal transduction pathways associated with disease resistance. To confirm the disease-defence-related activity of the TGA binding protein within MfbZIP, we employed amino acid sequences for 3-D ab initio modelling. Subsequently, we analysed TGA-NPR1 interactions using docking and molecular dynamics simulation analysis. These analyses shed light on the functional and structural aspects of TGA, demonstrating its stable association with NPR1 protein and its significance in the expression of PR1 protein, thus playing a pivotal role in defence responses against pathogens.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Resistencia a la Enfermedad , Regulación de la Expresión Génica de las Plantas , Filogenia , Enfermedades de las Plantas , Proteínas de Plantas , Transcriptoma , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Resistencia a la Enfermedad/genética , Enfermedades de las Plantas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Perfilación de la Expresión Génica , Arabidopsis/genéticaRESUMEN
The basic leucine zipper (bZIP) is a widely found transcription factor family that plays regulatory roles in a variety of cellular processes including cell growth and development and various stress responses. However, the bZIP gene family has not been well studied at a genome-wide scale in Fusarium graminearum (Fg), a potent pathogen of cereal grains. In the present study, we conducted a genome-wide identification, characterization, and expression profiling of 22 F. graminearum bZIP (FgbZIP) genes at different developmental stages and under various abiotic stresses. All identified FgbZIPs were categorized into nine groups based on their sequence similarity and phylogenetic tree analysis. Furthermore, the gene structure analysis, conserved motif analysis, chromosomal localization, protein network studies, and synteny analysis were performed. The symmetry of the exon and intron varied with the phylogenetic groups. The post-translational modifications (PTMs) analysis also predicted several phosphorylation sites in FgbZIPs, indicating their functional diversity in cellular processes. The evolutionary study identified many orthogroups among eight species and also predicted several gene duplication events in F. graminearum. The protein modeling indicated the presence of a higher number of α-helices and random coils in their structures. The expression patterns of FgbZIP genes showed that 5 FgbZIP genes, including FgbZIP_1.1, FgbZIP_1.3, FgbZIP_2.6 FgbZIP_3.1 and FgbZIP_4.3, had high expression at different growth and conidiogenesis stages. Similarly, eight genes including FgbZIP_1.1, FgbZIP_1.6, FgbZIP_2.3, FgbZIP_2.4, FgbZIP_4.1, FgbZIP_4.2, FgbZIP_4.3 and FgbZIP_4.6 demonstrated their putative role in response to various abiotic stresses. In summary, these results provided basic information regarding FgbZIPs which are helpful for further functional analysis.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Regulación de la Expresión Génica de las Plantas , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Cromosomas de las Plantas/metabolismo , Fusarium , Perfilación de la Expresión Génica , Leucina Zippers/genética , Familia de Multigenes , FilogeniaRESUMEN
AFP1 interacts with ABI5 and negatively regulates the abscisic acid (ABA) signaling by accelerating ABI5's degradation during the seed germination phase in Arabidopsis, but the underlying mechanism remains unclear. Moreover, the molecular basis of the interaction between AFP1 homologs and ABI5 has yet to be elucidated. In this study, the patterns of their interactions with ABI5 were investigated in detail. We found that AFP2/3/4 can bind two regions of ABI5, one is ABI51aa to 135aa and another is ABI5202aa to 213aa. However, AFP1 only interacts with the second region of ABI5, i.e. ABI5202aa to 213aa. Prior research has shown that ABI51aa to 135aa is related to the transcriptional activity of ABI5. Thus, our results suggest that AFPs may also modulate ABI5, by directly binding to its transcriptional activation domain, thereby influencing its transcriptional activity. Further, interactions between AFPs and ABI5 were not affected if the Ser42th in the ABI5-SnRK2 motif were mutated respectively to Glu or Ala. Nevertheless, interactions between AFPs and ABI5 were eliminated if the Thr47th and Thr206th of ABI5 were mutated respectively to Glu or Ala. Since the two residues of Thr47th and Thr206th were located in the phosphorylation motifs of CKII, AFPs might regulate the activities of ABI5 transcription factor through a CKII-dependent pathway.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Sustitución de Aminoácidos , Proteínas de Arabidopsis/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Quinasa de la Caseína II/metabolismo , Mutagénesis Sitio-Dirigida , Fosforilación , Unión Proteica , Técnicas del Sistema de Dos HíbridosRESUMEN
The occurrence of frequent, extreme high temperatures affects agriculture and causes irreversible damage during the ripening period of grapes. Breeding high-temperature-tolerant varieties of grapes is the main way to deal with this challenge, thus necessitating research on the regulatory mechanism of high-temperature tolerance. Extreme high temperature causes the mismatch of proteins in the endoplasmic reticulum in plant cells and initiates the unfolded protein response (UPR). The transcription factor bZIP60 participates in the UPR process. In the present study, VvbZIP60 and VvbZIP60s (unconventional splicing of VvbZIP60) were cloned and expressed in a transgenic system to verify heat tolerance. VvbZIP60s was found to be a key gene in adapting to heat stress. VvbZIP60s/60u interacted with VvHSP83 as observed in two yeast hybrids, with bimolecular fluorescence complementation and pull-down assays. VvHSP83 is also a key gene for plants to adapt to heat stress by participating in the renaturation and degradation of denatured proteins under adversity, causing plants to resist high temperatures. This study provides a basis for analyzing the mechanism of high-temperature tolerance in grapes.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Plantas/metabolismo , Termotolerancia , Vitis/fisiología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Retículo Endoplásmico , Eliminación de Gen , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Isoformas de Proteínas , Empalme del ARN , Técnicas del Sistema de Dos Híbridos , Respuesta de Proteína Desplegada , Vitis/clasificación , Vitis/genéticaRESUMEN
Transcription factors are regulatory proteins known to modulate gene expression. These are the critical component of signaling pathways and help in mitigating various developmental and stress responses. Among them, bZIP, BBR, and BZR transcription factor families are well known to play a crucial role in regulating growth, development, and defense responses. However, limited data is available on these transcription factors in Triticum aestivum. In this study, bZIP, BBR, and BZR sequences from Brachypodium distachyon, Oryza sativa, Oryza barthii, Oryza brachyantha, T. aestivum, Triticum urartu, Sorghum bicolor, Zea mays were retrieved, and dendrograms were constructed to analyze the evolutionary relatedness among them. The sequences clustered into one group indicated a degree of evolutionary correlation highlighting the common lineage of cereal grains. This analysis also exhibited that these genes were highly conserved among studied monocots emphasizing their common ancestry. Furthermore, these transcription factor genes were evaluated for envisaging conserved motifs, gene structure, and subcellular localization in T. aestivum. This comprehensive computational analysis has provided an insight into transcription factor evolution that can also be useful in developing approaches for future functional characterization of these genes in T. aestivum. Furthermore, the data generated can be beneficial in future for genetic manipulation of economically important plants.
Asunto(s)
Genoma de Planta , Proteínas de Plantas/genética , Factores de Transcripción/genética , Triticum/genética , Secuencia de Aminoácidos , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/clasificación , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Brachypodium/genética , Brachypodium/metabolismo , Oryza/genética , Oryza/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/clasificación , Alineación de Secuencia , Sorghum/genética , Sorghum/metabolismo , Factores de Transcripción/química , Factores de Transcripción/clasificación , Triticum/metabolismo , Zea mays/genética , Zea mays/metabolismoRESUMEN
Aspergillus fumigatus is a human opportunistic pathogen showing emerging resistance against a limited repertoire of antifungal agents available. The GTPase Rho1 has been identified as an important regulator of the cell wall integrity signaling pathway that regulates the composition of the cell wall, a structure that is unique to fungi and serves as a target for antifungal compounds. Rom2, the guanine nucleotide exchange factor to Rho1, contains a C-terminal citron homology (CNH) domain of unknown function that is found in many other eukaryotic genes. Here, we show that the Rom2 CNH domain interacts directly with Rho1 to modulate ß-glucan and chitin synthesis. We report the structure of the Rom2 CNH domain, revealing that it adopts a seven-bladed ß-propeller fold containing three unusual loops. A model of the Rho1-Rom2 CNH complex suggests that the Rom2 CNH domain interacts with the Rho1 Switch II motif. This work uncovers the role of the Rom2 CNH domain as a scaffold for Rho1 signaling in fungal cell wall biosynthesis.
Asunto(s)
Aspergillus fumigatus/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Pared Celular/fisiología , Proteínas Fúngicas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Aspergillus fumigatus/genética , Aspergillus fumigatus/crecimiento & desarrollo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Conformación Proteica , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal , Proteínas de Unión al GTP rho/química , Proteínas de Unión al GTP rho/genéticaRESUMEN
TGA transcription factor is a member of the D subfamily of the basic region-leucine zippers (bZIP) family. It is a type of transcription factor that was first identified in plants and is the main regulator in plant development and physiological processes, including morphogenesis and seed formation in response to abiotic and biotic stress and maintaining plant growth. The present study examined the sequence of the MaTGA8 transcription factor, the sequence of which belonged to subfamily D of the bZIP and had multiple cis-acting elements such as the G-box, TCA-element, TGACG-element, and P-box. Quantitative real time polymerase chain reaction (qRT-PCR) analyses showed that MaTGA8 was significantly down-regulated by the soil-borne fungus Fusarium oxysporum f. sp. cubense race 4 (Foc TR4). Under the induction of salicylic acid (SA), MaTGA8 was down-regulated, while different members of the MaNPR1 family responded significantly differently. Among them, MaNPR11 and MaNPR3 showed an overall upward trend, and the expression level of MaNPR4, MaNPR8, and MaNPR13 was higher than other members. MaTGA8 is a nuclear-localized transcription factor through strong interaction with MaNPR11 or weaker interaction with MaNPR4, and it is implied that the MaPR gene can be activated. In addition, the MaTGA8 transgenic Arabidopsis has obvious disease resistance and higher chlorophyll content than the wild-type Arabidopsis with the infection of Foc TR4. These results indicate that MaTGA8 may enhance the resistance of bananas to Foc TR4 by interacting with MaNPR11 or MaNPR4. This study provides a basis for further research on the application of banana TGA transcription factors in Foc TR4 stress and disease resistance and molecular breeding programs.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Resistencia a la Enfermedad/genética , Musa/genética , Musa/microbiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/microbiología , Proteínas de Arabidopsis/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Fusariosis/genética , Fusarium/patogenicidad , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/química , Raíces de Plantas/genética , Raíces de Plantas/microbiología , Ácido Salicílico/farmacología , Alineación de Secuencia , Transducción de Señal/efectos de los fármacosRESUMEN
Proteins are folded to avoid exposure of the nonpolar groups to water because water-mediated interactions between nonpolar groups are a promising factor in the thermodynamic stabilities of proteins-which is a well-accepted view as one of the unique effects of hydrophobic interactions. This article poses a critical question for this classical view by conducting an accurate solvation free-energy calculation for a thermodynamic cycle of a protein folding using a liquid-state density functional theory. Here, the solvation-free energy for a leucine zipper formation was examined in the coiled-coil protein GCN4-p1, a typical model for hydrophobic interactions, which demonstrated that water-mediated interactions were unfavorable for the association of nonpolar groups in the native state, while the dispersion forces between them were, instead, responsible for the association. Furthermore, the present analysis well predicted the isolated helical state stabilized by pressure, which was previously observed in an experiment. We reviewed the problems in the classical concept and semiempirical presumption that the energetic cost of the hydration of nonpolar groups is a driving force of folding.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Modelos Moleculares , Pliegue de Proteína , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Agua , Estabilidad ProteicaRESUMEN
The progression of cancer involves not only the gradual evolution of cells by mutations in DNA but also alterations in the gene expression induced by those mutations and input from the surrounding microenvironment. Such alterations contribute to cancer cells' abilities to reprogram metabolic pathways and undergo epithelial-to-mesenchymal transition (EMT), which facilitate the survival of cancer cells and their metastasis to other organs. Recently, BTB and CNC homology 1 (BACH1), a heme-regulated transcription factor that represses genes involved in iron and heme metabolism in normal cells, was shown to shape the metabolism and metastatic potential of cancer cells. The growing list of BACH1 target genes in cancer cells reveals that BACH1 promotes metastasis by regulating various sets of genes beyond iron metabolism. BACH1 represses the expression of genes that mediate cell-cell adhesion and oxidative phosphorylation but activates the expression of genes required for glycolysis, cell motility, and matrix protein degradation. Furthermore, BACH1 represses FOXA1 gene encoding an activator of epithelial genes and activates SNAI2 encoding a repressor of epithelial genes, forming a feedforward loop of EMT. By synthesizing these observations, we propose a "two-faced BACH1 model", which accounts for the dynamic switching between metastasis and stress resistance along with cancer progression. We discuss here the possibility that BACH1-mediated promotion of cancer also brings increased sensitivity to iron-dependent cell death (ferroptosis) through crosstalk of BACH1 target genes, imposing programmed vulnerability upon cancer cells. We also discuss the future directions of this field, including the dynamics and plasticity of EMT.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/fisiología , Transición Epitelial-Mesenquimal/fisiología , Ferroptosis , Neoplasias/patología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Progresión de la Enfermedad , Hemo/metabolismo , Humanos , Metástasis de la Neoplasia , Estrés Oxidativo , Relación Estructura-ActividadRESUMEN
The acidic activation domain (AD) of yeast transcription factor Gal4 plays a dual role in transcription repression and activation through binding to Gal80 repressor and Mediator subunit Med15. The activation function of Gal4 arises from two hydrophobic regions within the 40-residue AD. We show by NMR that each AD region binds the Mediator subunit Med15 using a "fuzzy" protein interface. Remarkably, comparison of chemical shift perturbations shows that Gal4 and Gcn4, two intrinsically disordered ADs of different sequence, interact nearly identically with Med15. The finding that two ADs of different sequence use an identical fuzzy binding mechanism shows a common sequence-independent mechanism for AD-Mediator binding, similar to interactions within a hydrophobic cloud. In contrast, the same region of Gal4 AD interacts strongly with Gal80 via a distinct structured complex, implying that the structured binding partner of an intrinsically disordered protein dictates the type of protein-protein interaction.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteínas de Unión al ADN/metabolismo , Complejo Mediador/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Complejo Mediador/química , Complejo Mediador/genética , Metiltransferasas/química , Metiltransferasas/metabolismo , Modelos Moleculares , Dominios y Motivos de Interacción de Proteínas , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: The bZIP gene family, which is widely present in plants, participates in varied biological processes including growth and development and stress responses. How do the genes regulate such biological processes? Systems biology is powerful for mechanistic understanding of gene functions. However, such studies have not yet been reported in poplar. RESULTS: In this study, we identified 86 poplar bZIP transcription factors and described their conserved domains. According to the results of phylogenetic tree, we divided these members into 12 groups with specific gene structures and motif compositions. The corresponding genes that harbor a large number of segmental duplication events are unevenly distributed on the 17 poplar chromosomes. In addition, we further examined collinearity between these genes and the related genes from six other species. Evidence from transcriptomic data indicated that the bZIP genes in poplar displayed different expression patterns in roots, stems, and leaves. Furthermore, we identified 45 bZIP genes that respond to salt stress in the three tissues. We performed co-expression analysis on the representative genes, followed by gene set enrichment analysis. The results demonstrated that tissue differentially expressed genes, especially the co-expressing genes, are mainly involved in secondary metabolic and secondary metabolite biosynthetic processes. However, salt stress responsive genes and their co-expressing genes mainly participate in the regulation of metal ion transport, and methionine biosynthetic. CONCLUSIONS: Using comparative genomics and systems biology approaches, we, for the first time, systematically explore the structures and functions of the bZIP gene family in poplar. It appears that the bZIP gene family plays significant roles in regulation of poplar development and growth and salt stress responses through differential gene networks or biological processes. These findings provide the foundation for genetic breeding by engineering target regulators and corresponding gene networks into poplar lines.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Genoma de Planta , Familia de Multigenes , Populus/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Secuencia Conservada , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Estrés Salino/genéticaRESUMEN
Maintenance of stem-cell identity requires proper regulation of enhancer activity. Both transcription factors OCT4/SOX2/NANOG and histone methyltransferase complexes MLL/SET1 were shown to regulate enhancer activity, but how they are regulated in embryonic stem cells (ESCs) remains further studies. Here, we report a transcription factor BACH1, which directly interacts with OCT4/SOX2/NANOG (OSN) and MLL/SET1 methyltransferase complexes and maintains pluripotency in mouse ESCs (mESCs). BTB domain and bZIP domain of BACH1 are required for these interactions and pluripotency maintenance. Loss of BACH1 reduced the interaction between NANOG and MLL1/SET1 complexes, and decreased their occupancy on chromatin, and further decreased H3 lysine 4 trimethylation (H3K4me3) level on gene promoters and (super-) enhancers, leading to decreased enhancer activity and transcription activity, especially on stemness-related genes. Moreover, BACH1 recruited NANOG through chromatin looping and regulated remote NANOG binding, fine-tuning enhancer-promoter activity and gene expression. Collectively, these observations suggest that BACH1 maintains pluripotency in ESCs by recruiting NANOG and MLL/SET1 complexes to chromatin and maintaining the trimethylated state of H3K4 and enhancer-promoter activity, especially on stemness-related genes.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Células Madre Embrionarias/metabolismo , Elementos de Facilitación Genéticos , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteína Homeótica Nanog/metabolismo , Regiones Promotoras Genéticas , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/fisiología , Línea Celular , Células Cultivadas , Cromatina/metabolismo , Histonas/metabolismo , Ratones , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Dominios Proteicos , Factores de Transcripción SOXB1/metabolismoRESUMEN
Anthocyanins, a major class of compounds derived from the flavonoid pathway, are important pigments of apple fruit. They can also prevent certain diseases and are beneficial to human health. Fruit pigmentation is a key quality trait that influences consumer preference; therefore, it is of great importance to investigate its regulatory mechanism. Here, we identified a MYB transcription factor (TF), MdMYB114, whose transcript level increased in the skin of the deep red apple fruit. It was determined to belong to the R2R3-MYB TF family and was localized in the nucleus. MdMYB114 overexpression led to anthocyanin accumulation in apple calli. MdMYB114 was not able to form an MBW complex but could enhance anthocyanin biosynthesis and transport by directly binding to the promoters of MdANS, MdUFGT, and MdGST to promote their expression. In addition, multiple assays revealed that MdbZIP4-like, a basic leucine-zipper TF, could directly bind to the MdMYB114 promoter to enhance its expression. Taken collectively, our results provide evidence that MdMYB114 is a positive regulator of anthocyanin biosynthesis and transport and it functions downstream of MdbZIP4-like in apple fruit.
Asunto(s)
Antocianinas/biosíntesis , Proteínas de Arabidopsis/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Malus/genética , Proteínas de Plantas/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Frutas/metabolismo , Malus/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Alineación de Secuencia , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Torsional stress on DNA, introduced by molecular motors, constitutes an important regulatory mechanism of transcriptional control. Torsional stress can modulate specific binding of transcription factors to DNA and introduce local conformational changes that facilitate the opening of promoters and nucleosome remodelling. Using all-atom microsecond scale molecular dynamics simulations together with a torsional restraint that controls the total twist of a DNA fragment, we address the impact of torsional stress on DNA complexation with a human BZIP transcription factor, MafB. We gradually over- and underwind DNA alone and in complex with MafB by 0.5° per dinucleotide step, starting from the relaxed state to a maximum of 5° per dinucleotide step, monitoring the evolution of the protein-DNA contacts at different degrees of torsional strain. Our computations show that MafB changes the DNA sequence-specific response to torsional stress. The dinucleotide steps that are susceptible to absorbing most of the torsional stress become more torsionally rigid, as they are involved in protein-DNA contacts. Also, the protein undergoes substantial conformational changes to follow the stress-induced DNA deformation, but mostly maintains the specific contacts with DNA. This results in a significant asymmetric increase of free energy of DNA twisting transitions, relative to free DNA, where overtwisting is more energetically unfavourable. Our data suggest that specifically bound BZIP factors could act as torsional stress insulators, modulating the propagation of torsional stress along the chromatin fibre, which might promote cooperative binding of collaborative DNA-binding factors.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , ADN Superhelicoidal/química , ADN/química , Secuencia de Bases , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/fisiología , Fenómenos Biomecánicos , Cromatina , ADN/genética , Fragmentación del ADN , ADN Superhelicoidal/genética , Humanos , Simulación de Dinámica Molecular , Conformación de Ácido NucleicoRESUMEN
In mammalian cells, 5-methylcytosine (5mC) occurs in genomic double-stranded DNA (dsDNA) and is enzymatically oxidized to 5-hydroxymethylcytosine (5hmC), then to 5-formylcytosine (5fC), and finally to 5-carboxylcytosine (5caC). These cytosine modifications are enriched in regulatory regions of the genome. The effect of these oxidative products on five bZIP dimers (CREB1, ATF2, Zta, ATF3|cJun, and cFos|cJun) binding to five types of dsDNA was measured using protein binding microarrays. The five dsDNAs contain either cytosine in both DNA strands or cytosine in one strand and either 5mC, 5hmC, 5fC, or 5caC in the second strand. Some sequences containing the CEBP half-site GCAA are bound more strongly by all five bZIP domains when dsDNA contains 5mC, 5hmC, or 5fC. dsDNA containing 5caC in some TRE (AP-1)-like sequences, e.g., TGACTAA, is better bound by Zta, ATF3|cJun, and cFos|cJun.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Citosina/análogos & derivados , ADN/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/química , Secuencia de Aminoácidos , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Citosina/química , ADN/química , Ratones , Análisis por Matrices de Proteínas , Unión ProteicaRESUMEN
Radiotherapy, the most common therapy for the treatment of solid tumors, exerts its effects by inducing DNA damage. To fully understand the extent and nature of this damage, DNA models that mimic the in vivo situation should be utilized. In a cellular context, genomic DNA constantly interacts with proteins and these interactions could influence both the primary radical processes (triggered by ionizing radiation) and secondary reactions, ultimately leading to DNA damage. However, this is seldom addressed in the literature. In this work, we propose a general approach to tackle these shortcomings. We synthesized a protein-DNA complex that more closely represents DNA in the physiological environment than oligonucleotides solution itself, while being sufficiently simple to permit further chemical analyses. Using click chemistry, we obtained an oligonucleotide-peptide conjugate, which, if annealed with the complementary oligonucleotide strand, forms a complex that mimics the specific interactions between the GCN4 protein and DNA. The covalent bond connecting the oligonucleotide and peptide constitutes a part of substituted triazole, which forms due to the click reaction between the short peptide corresponding to the specific amino acid sequence of GCN4 protein (yeast transcription factor) and a DNA fragment that is recognized by the protein. DNAse footprinting demonstrated that the part of the DNA fragment that specifically interacts with the peptide in the complex is protected from DNAse activity. Moreover, the thermodynamic characteristics obtained using differential scanning calorimetry (DSC) are consistent with the interaction energies calculated at the level of metadynamics. Thus, we present an efficient approach to generate a well-defined DNA-peptide conjugate that mimics a real DNA-peptide complex. These complexes can be used to investigate DNA damage under conditions very similar to those present in the cell.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , ADN de Cadena Simple/química , ADN/química , Péptidos/química , Proteínas de Saccharomyces cerevisiae/química , Secuencia de Aminoácidos , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Sitios de Unión , Rastreo Diferencial de Calorimetría , Catálisis , Cromatografía Líquida de Alta Presión , Química Clic , Cobre/química , ADN/metabolismo , Daño del ADN , ADN de Cadena Simple/metabolismo , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Péptidos/metabolismo , Dominios Proteicos , Proteínas de Saccharomyces cerevisiae/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Temperatura de TransiciónRESUMEN
The unique structural flexibility of intrinsically disordered proteins (IDPs) is central to their diverse functions in cellular processes. Protein-protein interactions involving IDPs are frequently transient and dynamic in nature. Nuclear magnetic resonance (NMR) spectroscopy is an especially powerful tool for characterizing the structural propensities, dynamics, and interactions of IDPs. Here we describe applications of the Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiment in combination with NMR titrations to characterize the kinetics and mechanisms of interactions between intrinsically disordered proteins and their targets. We illustrate the method with reference to interactions between the activation domain of the human T-cell leukemia virus type-I (HTLV-1) basic leucine zipper protein (HBZ) and its cellular binding partner, the KIX domain of the transcriptional coactivator CBP.
Asunto(s)
Proteínas Intrínsecamente Desordenadas/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Mapeo de Interacción de Proteínas/métodos , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteína de Unión a CREB/metabolismo , Humanos , Proteínas Intrínsecamente Desordenadas/química , Cinética , Resonancia Magnética Nuclear Biomolecular/instrumentación , Unión Proteica , Dominios Proteicos , Proteínas de los Retroviridae/química , Proteínas de los Retroviridae/metabolismo , Programas InformáticosRESUMEN
Activator protein 1 (AP-1) is one of the largest families of basic leucine zipper (bZIP) transcription factors in eukaryotic cells. How AP-1 proteins achieve target DNA binding specificity remains elusive. In Saccharomyces cerevisiae, the AP-1-like protein (Yap) family comprises eight members (Yap1 to Yap8) that display distinct genomic target sites despite high sequence homology of their DNA binding bZIP domains. In contrast to the other members of the Yap family, which preferentially bind to short (7-8 bp) DNA motifs, Yap8 binds to an unusually long DNA motif (13 bp). It has been unclear what determines this unique specificity of Yap8. In this work, we use molecular and biochemical analyses combined with computer-based structural design and molecular dynamics simulations of Yap8-DNA interactions to better understand the structural basis of DNA binding specificity determinants. We identify specific residues in the N-terminal tail preceding the basic region, which define stable association of Yap8 with its target promoter. We propose that the N-terminal tail directly interacts with DNA and stabilizes Yap8 binding to the 13 bp motif. Thus, beside the core basic region, the adjacent N-terminal region contributes to alternative DNA binding selectivity within the AP-1 family.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , ADN de Hongos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , ADN de Hongos/química , Proteínas de Transporte de Membrana/genética , Simulación de Dinámica Molecular , Mutación , Regiones Promotoras Genéticas , Unión Proteica , Elementos de Respuesta , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Olive (Olea europaea.L) is an economically important oleaginous crop and its fruit cold-pressed oil is used for edible oil all over the world. The basic region-leucine zipper (bZIP) family is one of the largest transcription factors families among eukaryotic organisms; its members play vital roles in environmental signaling, stress response, plant growth, seed maturation, and fruit development. However, a comprehensive report on the bZIP gene family in olive is lacking. In this study, 103 OebZIP genes from the olive genome were identified and divided into 12 subfamilies according to their genetic relationship with 78 bZIPs of A. thaliana. Most OebZIP genes are clustered in the subgroup that has a similar gene structure and conserved motif distribution. According to the characteristics of the leucine zipper region, the dimerization characteristics of 103 OebZIP proteins were predicted. Gene duplication analyses revealed that 22 OebZIP genes were involved in the expansion of the bZIP family. To evaluate the expression patterns of OebZIP genes, RNA-seq data available in public databases were analyzed. The highly expressed OebZIP genes and several lipid synthesis genes (LPGs) in fruits of two varieties with different oil contents during the fast oil accumulation stage were examined via qRT-PCR. By comparing the dynamic changes of oil accumulation, OebZIP1, OebZIP7, OebZIP22, and OebZIP99 were shown to have a close relationship with fruit development and lipid synthesis. Additionally, some OebZIP had a significant positive correlation with various LPG genes. This study gives insights into the structural features, evolutionary patterns, and expression analysis, laying a foundation to further reveal the function of the 103 OebZIP genes in olive.