RESUMO
The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a worldwide threat in the past 3 years. Although it has been widely and intensively investigated, the mechanism underlying the coronavirus-host interaction requires further elucidation, which may contribute to the development of new antiviral strategies. Here, we demonstrated that the host cAMP-responsive element-binding protein (CREB1) interacts with the non-structural protein 13 (nsp13) of SARS-CoV-2, a conserved helicase for coronavirus replication, both in cells and in lung tissues subjected to SARS-CoV-2 infection. The ATPase and helicase activity of viral nsp13 were shown to be potentiated by CREB1 association, as well as by Protein kinase A (PKA)-mediated CREB1 activation. SARS-CoV-2 replication is significantly suppressed by PKA Cα, cAMP-activated protein kinase catalytic subunit alpha (PRKACA), and CREB1 knockdown or inhibition. Consistently, the CREB1 inhibitor 666-15 has shown significant antiviral effects against both the WIV04 strain and the Omicron strain of the SARS-CoV-2. Our findings indicate that the PKA-CREB1 signaling axis may serve as a novel therapeutic target against coronavirus infection. IMPORTANCE: In this study, we provide solid evidence that host transcription factor cAMP-responsive element-binding protein (CREB1) interacts directly with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) helicase non-structural protein 13 (nsp13) and potentiate its ATPase and helicase activity. And by live SARS-CoV-2 virus infection, the inhibition of CREB1 dramatically impairs SARS-CoV-2 replication in vivo. Notably, the IC50 of CREB1 inhibitor 666-15 is comparable to that of remdesivir. These results may extend to all highly pathogenic coronaviruses due to the conserved nsp13 sequences in the virus.
Assuntos
RNA-Polimerase RNA-Dependente de Coronavírus , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico , Interações entre Hospedeiro e Microrganismos , SARS-CoV-2 , Proteínas não Estruturais Virais , Replicação Viral , Humanos , Adenosina Trifosfatases/metabolismo , Antivirais/farmacologia , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , COVID-19/virologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/antagonistas & inibidores , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/deficiência , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , DNA Helicases/metabolismo , Concentração Inibidora 50 , RNA Helicases/metabolismo , SARS-CoV-2/classificação , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , SARS-CoV-2/crescimento & desenvolvimento , Transdução de Sinais/efeitos dos fármacos , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos , Feminino , Animais , CamundongosRESUMO
Pearl of Csaba (PC) is a valuable backbone parent for early-ripening grapevine (Vitis vinifera) breeding, from which many excellent early ripening varieties have been bred. However, the genetic basis of the stable inheritance of its early ripening trait remains largely unknown. Here, the pedigree, consisting of 40 varieties derived from PC, was re-sequenced for an average depth of â¼30×. Combined with the resequencing data of 24 other late-ripening varieties, 5,795,881 high-quality single nucleotide polymorphisms (SNPs) were identified following a strict filtering pipeline. The population genetic analysis showed that these varieties could be distinguished clearly, and the pedigree was characterized by lower nucleotide diversity and stronger linkage disequilibrium than the non-pedigree varieties. The conserved haplotypes (CHs) transmitted in the pedigree were obtained via identity-by-descent analysis. Subsequently, the key genomic segments were identified based on the combination analysis of haplotypes, selective signatures, known ripening-related quantitative trait loci (QTLs), and transcriptomic data. The results demonstrated that varieties with a superior haplotype, H1, significantly (one-way ANOVA, P < 0.001) exhibited early grapevine berry development. Further analyses indicated that H1 encompassed VIT_16s0039g00720 encoding a folate/biopterin transporter protein (VvFBT) with a missense mutation. VvFBT was specifically and highly expressed during grapevine berry development, particularly at veraison. Exogenous folate treatment advanced the veraison of "Kyoho". This work uncovered core haplotypes and genomic segments related to the early ripening trait of PC and provided an important reference for the molecular breeding of early-ripening grapevine varieties.
Assuntos
Vitis , Vitis/metabolismo , Melhoramento Vegetal , Perfilação da Expressão Gênica/métodos , Transcriptoma , Frutas/metabolismo , GenômicaRESUMO
Cytoskeletal microtubules (MTs) are nucleated from γ-tubulin ring complexes (γTuRCs) located at MT organizing centers (MTOCs), such as the centrosome. However, the exact regulatory mechanism of γTuRC assembly is not fully understood. Here, we showed that the nonreceptor tyrosine kinase c-Abl was associated with and phosphorylated γ-tubulin, the essential component of the γTuRC, mainly on the Y443 residue by in vivo (immunofluorescence and immunoprecipitation) or in vitro (surface plasmon resonance) detection. We further demonstrated that phosphorylation deficiency significantly impaired γTuRC assembly, centrosome construction, and MT nucleation. c-Abl/Arg deletion and γ-tubulin Y443F mutation resulted in an abnormal morphology and compromised spindle function during mitosis, eventually causing uneven chromosome segregation. Our findings reveal that γTuRC assembly and nucleation function are regulated by Abl kinase-mediated γ-tubulin phosphorylation, revealing a fundamental mechanism that contributes to the maintenance of MT function.
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Centro Organizador dos Microtúbulos , Microtúbulos , Proteínas Proto-Oncogênicas c-abl , Tubulina (Proteína) , Centrossomo/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Proto-Oncogênicas c-abl/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
Cucurbits are a diverse plant family that includes economically important crops, such as cucumber, watermelon, melon, and pumpkin. Knowledge of the roles that long terminal repeat retrotransposons (LTR-RTs) have played in diversification of cucurbit species is limited; to add to understanding of the roles of LTR-RTs, we assessed their distributions in four cucurbit species. We identified 381, 578, 1086, and 623 intact LTR-RTs in cucumber (Cucumis sativus L. var. sativus cv. Chinese Long), watermelon (Citrullus lanatus subsp. vulgaris cv. 97103), melon (Cucumis melo cv. DHL92), and Cucurbita (Cucurbita moschata var. Rifu), respectively. Among these LTR-RTs, the Ale clade of the Copia superfamily was the most abundant in all the four cucurbit species. Insertion time and copy number analysis revealed that an LTR-RT burst occurred approximately 2 million years ago in cucumber, watermelon, melon, and Cucurbita, and may have contributed to their genome size variation. Phylogenetic and nucleotide polymorphism analyses suggested that most LTR-RTs were formed after species diversification. Analysis of gene insertions by LTR-RTs revealed that the most frequent insertions were of Ale and Tekay and that genes related to dietary fiber synthesis were the most commonly affected by LTR-RTs in Cucurbita. These results increase our understanding of LTR-RTs and their roles in genome evolution and trait characterization in cucurbits.
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Cucurbita , Retroelementos , Produtos Agrícolas , Fenótipo , Filogenia , Retroelementos/genética , Cucurbita/genéticaRESUMO
SARS-CoV-2 is the causative agent of the ongoing pandemic of coronavirus disease 2019 (COVID-19) and poses a significant threat to global health. N protein (NP), which is a major pathogenic protein among betacoronaviruses, binds to the viral RNA genome to allow viral genome packaging and viral particle release. Recent studies showed that NP antagonizes interferon (IFN) induction and mediates phase separation. Using live SARS-CoV-2 viruses, this study provides solid evidence showing that SARS-CoV-2 NP associates with G3BP1 and G3BP2 in vitro and in vivo. NPSARS-CoV-2 could efficiently suppress G3BP-mediated SG formation and potentiate viral infection by overcoming G3BP1-mediated antiviral innate immunity. G3BP1 conditional knockout mice (g3bp1fl/fL, Sftpc-Cre) exhibit significantly higher lung viral loads after SARS-CoV-2 infection than wild-type mice. Our findings contribute to the growing body of knowledge regarding the pathogenicity of NPSARS-CoV-2 and provide insight into new therapeutics targeting NPSARS-CoV-2. IMPORTANCE In this study, by in vitro assay and live SARS-CoV-2 virus infection, we provide solid evidence that the SARS-CoV-2 NP associates with G3BP1 and G3BP2 in vitro and in vivo. NPSARS-CoV-2 could efficiently suppress G3BP-mediated SG formation and potentiate viral infection by overcoming antiviral innate immunity mediated by G3BP1 in A549 cell lines and G3BP1 conditional knockout mice (g3bp1-cKO) mice, which provide in-depth evidence showing the mechanism underlying NP-related SARS-CoV-2 pathogenesis through G3BPs.
Assuntos
COVID-19 , Proteínas do Nucleocapsídeo de Coronavírus , Proteínas de Ligação a Poli-ADP-Ribose , SARS-CoV-2 , Replicação Viral , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , COVID-19/imunologia , COVID-19/virologia , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , DNA Helicases/metabolismo , Interações entre Hospedeiro e Microrganismos/imunologia , Camundongos , Fosfoproteínas/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Grânulos de Estresse , Replicação Viral/genéticaRESUMO
BACKGROUND: The ClaH3K4s and ClaH3K27s gene families are subfamilies of the SET family, each with a highly conserved SET structure domain and a PHD structural domain. Both participate in histone protein methylation, which affects the chromosome structure and gene expression, and is essential for fruit growth and development. METHODS AND RESULTS: In order to demonstrate the structure and expression characteristics of ClaH3K4s and ClaH3K27s in watermelon, members of the watermelon H3K4 and H3K27 gene families were identified, and their chromosomal localization, gene structure, and protein structural domains were analyzed. The phylogeny and covariance of the gene families with other species were subsequently determined, and the expression profiles were obtained by performing RNA-Seq and qRT-PCR. The watermelon genome had five H3K4 genes with 3207-8043 bp nucleotide sequence lengths and four H3K27 genes with a 1107-5499 bp nucleotide sequence. Synteny analysis revealed the close relationship between watermelon and cucumber, with the majority of members displaying a one-to-one covariance. Approximately half of the 'Hua-Jing 13 watermelon' ClaH3K4s and ClaH3K27s genes were expressed more in the late fruit development stages, while the changes were minimal for the remaining half. H3K4-2 expression was observed to be slightly greater on day 21 compared to other periods. Moreover, ClaH3K27-1 and ClaH3K27-2 were hardly expressed throughout the developing period, and ClaH3K27-4 exhibited the highest expression. CONCLUSION: These results serve as a basis for further functional characterization of the H3K4 and H3K27 genes in the fruit development of watermelon.
Assuntos
Citrullus , Citrullus/genética , Frutas/metabolismo , Sequência de Bases , Reação em Cadeia da Polimerase , Sintenia , Regulação da Expressão Gênica de Plantas/genética , FilogeniaRESUMO
Fruit development is modified by different types of epigenetics. Histone methylation is an important way of epigenetic modification. Eight genes related to H3K4 methyltransferase, named VvH3K4s, were identified and isolated from the grape genome based on conserved domain analysis, which could be divided into 3 categories by the phylogenetic relationship. Transcriptome data showed that VvH3K4-5 was obviously up-regulated during fruit ripe, and its expression level was significantly different between 'Kyoho' and 'Fengzao'. The VvH3K4s promoters contains cis-acting elements of in response to stress, indicating that they may be involved in the metabolic pathways regulated by ROS signaling. The subcellular localization experiment and promoter activity analysis experiment on VvH3K4-5 showed that VvH3K4s may be regulated by H2O2. With H2O2 and Hypotaurine treatment, it was found that the expression pattern of most genes was opposite, and the expression level showed different expression trend with the extension of treatment time.
Assuntos
Vitis , Frutas , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Vitis/genéticaRESUMO
The nonreceptor tyrosine kinase c-Abl plays important roles in T cell development and immune responses; however, the mechanism is poorly understood. IFN regulatory factor 3 (IRF3) is a key transcriptional regulator of type I IFN-dependent immune responses against DNA and RNA viruses. The data in this study show that IRF3 is physically associated with c-Abl in vivo and directly binds to c-Abl in vitro. IRF3 is phosphorylated by c-Abl and c-Abl-related kinase, Arg, mainly at Y292. The inhibitor AMN107 inhibits IFN-ß production induced by poly(dA:dT), poly(I:C), and Sendai virus in THP-1 and mouse bone marrow-derived macrophage cells. IRF3-induced transcription of IFN-ß is significantly reduced by the mutation of Y292 to F. Moreover, AMN107 suppresses gene expression of absent in melanoma 2 (AIM2) and subsequently reduces inflammasome activation induced by cytosolic bacteria, dsDNA, and DNA viruses. Consistent with this finding, Francisella tularensis subsp. holarctica live vaccine strain (Ft LVS), which is known as an activator of AIM2 inflammasome, induces death in significantly more C57BL/6 mice treated with the Abl inhibitor AMN107 or c-Abl/Arg small interfering RNA than in untreated mice. This study provides new insight into the function of c-Abl and Arg in regulating immune responses and AIM2 inflammasome activation, especially against Ft LVS infection.
Assuntos
Regulação da Expressão Gênica/imunologia , Imunidade Inata , Fator Regulador 3 de Interferon/imunologia , Interferon beta/imunologia , Proteínas Proto-Oncogênicas c-abl/imunologia , Animais , Arginina/imunologia , Proteínas de Ligação a DNA/imunologia , Francisella/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamassomos/imunologia , Camundongos , Fosforilação/efeitos dos fármacos , Pirimidinas/farmacologia , Vírus Sendai/imunologia , Células THP-1RESUMO
BACKGROUND: The lateral organ boundaries domain (LBD) gene is a plant-specific transcription factor that plays a critical role in diverse biological processes. However, the evolution and functional divergence of the LBD gene family has not yet been characterized for the Chinese White Pear. RESULTS: In our study, a total of 60 PbrLBDs were identified in the pear genome. The PbrLBD gene family was divided into two classes based on gene structure and phylogenetic analysis: class I (53) and class II (7). Cis-acting element analysis results suggested that PbrLBDs may participate in various biological processes, such as flavonoid biosynthetic and stress response. Synteny analysis results indicated that segmental duplication played a key role in the expansion of the PbrLBD gene family. The mean Ks and 4DTv values showed that the PbrLBD gene family had undergone only one recent whole-genome duplication event occurring at 30-45 MYA. Purifying selection was a primary force during the PbrLBD gene family evolution process. Transcriptome data analysis revealed that 10 PbrLBDs were expressed in all six examined tissues, and 73.33% of members in the PbrLBD gene family were expressed in pear sepal. qRT-PCR was conducted to verify the expression levels of 11 PbrLBDs in these six tissues. Specifically, PbrLBD20, PbrLBD35 and PbrLBD53 genes were down-regulated when anthocyanin concentrations were high, whereas PbrLBD33 was significantly up-regulated in pear when anthocyanin concentrations were high. Furthermore, PbrLBD20, one of the candidate genes related to anthocyanins was localized in the nucleus. CONCLUSIONS: Our analysis provides valuable information for understanding the evolution of the PbrLBD gene family, and provides new insights into the regulation of pear pigment metabolism and lays a foundation for the future disclosure of the molecular mechanism of LBD gene regulating flavonoid metabolism.
Assuntos
Evolução Molecular , Proteínas de Plantas/genética , Pyrus/genética , Fatores de Transcrição/genética , Antocianinas/genética , Antocianinas/metabolismo , Duplicação Gênica , Família MultigênicaRESUMO
The transcription factor Runx1 is an essential regulator of definitive hematopoiesis, megakaryocyte (MK) maturation, and lymphocyte differentiation. Runx1 mutations that interfere with its transcriptional activity are often present in leukemia patients. Recent work demonstrated that the transcriptional activity of Runx1 is regulated by kinase-mediated phosphorylation. In this study, we showed that c-Abl, but not Arg tyrosine kinase, associated with Runx1 both in cultured cells and in vitro. c-Abl-mediated tyrosine phosphorylation in the Runx1 transcription inhibition domain negatively regulated the transcriptional activity of Runx1 and inhibited Runx1-mediated MK maturation. Consistent with these findings, increased numbers of MKs were detected in the spleens and bone marrow of abl gene conditional knockout mice. Our findings demonstrate an important role of c-Abl kinase in Runx1-mediated MK maturation and platelet formation and provide a potential mechanism of Abl kinase-regulated hematopoiesis.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Megacariócitos/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Animais , Sítios de Ligação , Plaquetas/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/química , Regulação da Expressão Gênica , Células HEK293 , Humanos , Células K562 , Camundongos , Fosforilação , Proteínas Proto-Oncogênicas c-abl/genética , Transcrição GênicaRESUMO
Cadmium (Cd) contamination of soil may lead to Cd stress for plants, which significantly hinders plant growth and development, posing a risk to human health through the consumption of Cd-contaminated foods. Watermelon (Citrullus lanatus), a widely consumed fruit, is particularly affected by Cd stress globally, yet the mechanisms underlying its response are not well understood. Here, we subjected watermelon seedlings to simulated Cd stress treatment and explored the physiological, transcriptomic, and metabolic response. Our findings revealed that Cd stress treatment led to increased accumulation of reactive oxygen species (ROS) in watermelon leaves. Transcriptome sequencing unveiled a multitude of osmotic and oxidative stress-responsive genes, including peroxidase (POD), MYB, voltage-dependent anion channel (SLAC1), and ABC transporter. KEGG enrichment analysis highlighted the predominant enrichment of Cd stress-responsive genes in pathways such as glutathione (GSH) metabolism, MAPK signaling, and biosynthesis of secondary metabolites. Within the GSH metabolism pathway, several glutathione S-transferase (GST) genes were up-regulated, alongside phytochelatin synthetase (PCS) genes involved in phytochelatin synthesis. In the MAPK signaling pathway, genes associated with ABA and ethylene signal transduction showed up-regulation following Cd stress. Metabolomic analysis demonstrated that Cd stress enhanced the production of amino acids, phenolamines, and esters. Overall, our study elucidates that watermelon responds to Cd stress by activating its antioxidant system, GSH metabolism pathway, MAPK signal pathway, and biosynthesis of key metabolites. These findings offer valuable insights for the remediation of heavy metal pollution in soil affecting plant life.
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BACKGROUND: DNA methylation is an essential epigenetic modification. However, its contribution to trait changes and diversity in the domestication of perennial fruit trees remains unknown. RESULTS: Here, we investigate the variation in DNA methylation during pear domestication and improvement using whole-genome bisulfite sequencing in 41 pear accessions. Contrary to the significant decrease during rice domestication, we detect a global increase in DNA methylation during pear domestication and improvement. We find this specific increase in pear is significantly correlated with the downregulation of Demeter-like1 (DML1, encoding DNA demethylase) due to human selection. We identify a total of 5591 differentially methylated regions (DMRs). Methylation in the CG and CHG contexts undergoes co-evolution during pear domestication and improvement. DMRs have higher genetic diversity than selection sweep regions, especially in the introns. Approximately 97% of DMRs are not associated with any SNPs, and these DMRs are associated with starch and sucrose metabolism and phenylpropanoid biosynthesis. We also perform correlation analysis between DNA methylation and gene expression. We find genes close to the hypermethylated DMRs that are significantly associated with fruit ripening. We further verify the function of a hyper-DMR-associated gene, CAMTA2, and demonstrate that overexpression of CAMTA2 in tomato and pear callus inhibits fruit ripening. CONCLUSIONS: Our study describes a specific pattern of DNA methylation in the domestication and improvement of a perennial pear tree and suggests that increased DNA methylation plays an essential role in the early ripening of pear fruits.
Assuntos
Metilação de DNA , Pyrus , Humanos , Frutas/genética , Frutas/metabolismo , Pyrus/genética , Domesticação , Epigênese Genética , Proteínas de Ligação ao Cálcio/genética , Transativadores/genéticaRESUMO
Viral inclusion bodies (IBs) commonly form during the replication of Ebola virus (EBOV) in infected cells, but their role in viral immune evasion has rarely been explored. Here, we found that interferon regulatory factor 3 (IRF3), but not TANK-binding kinase 1 (TBK1) or IκB kinase epsilon (IKKε), was recruited and sequestered in viral IBs when the cells were infected by EBOV transcription- and replication-competent virus-like particles (trVLPs). Nucleoprotein/virion protein 35 (VP35)-induced IBs formation was critical for IRF3 recruitment and sequestration, probably through interaction with STING. Consequently, the association of TBK1 and IRF3, which plays a vital role in type I interferon (IFN-I) induction, was blocked by EBOV trVLPs infection. Additionally, IRF3 phosphorylation and nuclear translocation induced by Sendai virus or poly(I:C) stimulation were suppressed by EBOV trVLPs. Furthermore, downregulation of STING significantly attenuated VP35-induced IRF3 accumulation in IBs. Coexpression of the viral proteins by which IB-like structures formed was much more potent in antagonizing IFN-I than expression of the IFN-I antagonist VP35 alone. These results suggested a novel immune evasion mechanism by which EBOV evades host innate immunity.
Assuntos
Doença pelo Vírus Ebola , Evasão da Resposta Imune , Corpos de Inclusão Viral , Fator Regulador 3 de Interferon , Interferon Tipo I , Humanos , Ebolavirus , Doença pelo Vírus Ebola/imunologiaRESUMO
Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is an epigenetic regulator that plays critical roles in tumours. However, the DNA methylation alteration patterns driven by UHRF1 and the related differentially expressed tumour-related genes remain unclear. In this study, a UHRF1-shRNA MCF-7 cell line was constructed, and whole-genome bisulfite sequencing and RNA sequencing were performed. The DNA methylation alteration landscape was elucidated, and DNA methylation-altered regions (DMRs) were found to be distributed in both gene bodies and adjacent regions. The DMRs were annotated and categorized into 488 hypermethylated/1696 hypomethylated promoters and 1149 hypermethylated/5501 hypomethylated gene bodies. Through an integrated analysis with the RNA sequencing data, 217 methylation-regulated upregulated genes and 288 downregulated genes were identified, and these genes were primarily enriched in nervous system development and cancer signalling pathways. Further analysis revealed 21 downregulated oncogenes and 15 upregulated TSGs. We also showed that UHRF1 silencing inhibited cell proliferation and migration and suppressed tumour growth in vivo. Our study suggested that UHRF1 and the oncogenes or TSGs it regulates might serve as biomarkers and targets for breast cancer treatment.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Ubiquitina-Proteína Ligases , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Humanos , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Células MCF-7 , Feminino , Proliferação de Células/genética , Animais , Regiões Promotoras Genéticas , Camundongos , Epigênese Genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Movimento Celular/genéticaRESUMO
The transcription factor FOXM1, which plays critical roles in cell cycle progression and tumorigenesis, is highly expressed in rapidly proliferating cells and various tumor tissues, and high FOXM1 expression is related to a poor prognosis. However, the mechanism responsible for FOXM1 dysregulation is not fully understood. Here, we show that ABL1, a nonreceptor tyrosine kinase, contributes to the high expression of FOXM1 and FOXM1-dependent tumor development. Mechanistically, ABL1 directly binds FOXM1 and mediates FOXM1 phosphorylation at multiple tyrosine (Y) residues. Among these phospho-Y sites, pY575 is indispensable for FOXM1 stability as phosphorylation at this site protects FOXM1 from ubiquitin-proteasomal degradation. The interaction of FOXM1 with CDH1, a coactivator of the E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C), which is responsible for FOXM1 degradation, is significantly inhibited by Y575 phosphorylation. The phospho-deficient FOXM1(Y575F) mutant exhibited increased ubiquitination, a shortened half-life, and consequently a substantially decreased abundance. Compared to wild-type cells, a homozygous Cr-Y575F cell line expressing endogenous FOXM1(Y575F) that was generated by CRISPR/Cas9 showed obviously delayed mitosis progression, impeded colony formation and inhibited xenotransplanted tumor growth. Overall, our study demonstrates that ABL1 kinase is involved in high FOXM1 expression, providing clear evidence that ABL1 may act as a therapeutic target for the treatment of tumors with high FOXM1 expression.
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To gain a comprehensive understanding of non-histone methylation during berry ripening in grape (Vitis vinifera L.), the methylation of non-histone lysine residues was studied using a 4D label-free quantitative proteomics approach. In total, 822 methylation sites in 416 methylated proteins were identified, with xxExxx_K_xxxxxx as the conserved motif. Functional annotation of non-histone proteins with methylated lysine residues indicated that these proteins were mostly associated with "ripening and senescence", "energy metabolism", "oxidation-reduction process", and "stimulus response". Most of the genes encoding proteins subjected to methylation during grape berry ripening showed a significant increase in expression during maturation at least at one developmental stage. The correlation of methylated proteins with QTLs, SNPs, and selective regions associated with fruit quality and development was also investigated. This study reports the first proteomic analysis of non-histone lysine methylation in grape berry and indicates that non-histone methylation plays an important role in grape berry ripening.
Assuntos
Vitis , Vitis/anatomia & histologia , Vitis/química , Vitis/metabolismo , Proteoma/metabolismo , Histonas/química , Metilação , Lisina/química , Peptídeos/química , Mapas de Interação de Proteínas , Perfilação da Expressão GênicaRESUMO
DNA methylation, an epigenetic mark, is proposed to regulate plant anthocyanin biosynthesis. It well known that light induces anthocyanin accumulation, with bagging treatments commonly used to investigate light-controlled anthocyanin biosynthesis. We studied the DNA methylome landscape during pear skin coloration under various conditions (fruits re-exposed to sunlight after bag removal). The DNA methylation level in gene body/TE and its flanking sequence was generally similar between debagged and bagged treatments, however differentially methylated regions (DMRs) were re-modelled after light-exposure. Both DNA demethylase homologs and the RNA-directed DNA methylation (RdDM) pathways contributed to this re-distribution. A total of 310 DEGs were DMR-associated during light-induced anthocyanin biosynthesis between debagged and bagged treatments. The hypomethylated mCHH context was seen within the promoter of PyUFGT, together with other anthocyanin biosynthesis genes (PyPAL, PyDFR and PyANS). This enhanced transcriptional activation and promoted anthocyanin accumulation after light re-exposure. Unlike previous reports on bud sports, we did not detect DMRs within the MYB10 promoter. Instead, we observed the genome-wide re-distribution of methylation patterns, suggesting different mechanisms underlying methylation patterns of differentially accumulated anthocyanins caused by either bud mutation or environment change. We investigate the dynamic landscape of genome-scale DNA methylation, which is the combined effect of DNA demethylation and RdDM pathway, in the process of light-induced fruit colour formation in pear. This process is regulated by methylation changes on promoter regions of several DEGs. These results provide a DMR-associated DEGs set and new insight into the mechanism of DNA methylation involved in light-induced anthocyanin biosynthesis.
Assuntos
Pyrus , Pyrus/genética , Pyrus/metabolismo , Antocianinas/metabolismo , Metilação de DNA , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Frutas/genética , Frutas/metabolismoRESUMO
Infectious diseases caused by waterborne viruses have attracted researchers' great attention. To ensure a safe water environment, it is important to advance water treatment and disinfection technology. Photocatalytic technology offers an efficient and practical approach for achieving this goal. This paper reviews the latest studies on visible-light composite catalysts for bacteriophage inactivation, with a main focus on three distinct categories: modified UV materials, direct visible-light materials and carbon-based materials. This review gives an insight into the progress in photocatalytic material development and offers a promising solution for bacteriophage inactivation.
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Grape is an economically important crop but recalcitrant to Agrobacterium-mediated genetic transformation and in vitro regeneration. Here, we have developed a protocol for transient transformation of grapes by investigating the effects of explant pre-culture and duration of vacuum infiltration on transformation efficiency. Using sliced grape berries of "Shine-Muscat" (Vitis labrusca × Vitis vinifera) between the end of fruit expansion phase and the mature stage as explants, we firstly compared the effect of pre-culture explants into a susceptible state (incubation on Murashige and Skoog (MS) agar plate in the dark at 25 ± 1 °C for 48 h) with no pre-culture and then tested different vacuum infiltration times on transformation efficiency using ß-glucuronidase (GUS) reporter system. Pre-culture increased the susceptibility of explants to the agrobacteria infection and increased transient transformation efficiency as assessed by histochemical GUS activity, with intense blue coloration compared with the faint staining observed in the non-susceptible explants. Using a Circulating Water Vacuum Pump system to facilitate agrobacteria entry into berry cells, we tested vacuum durations of 5, 10, and 15 min and observed that transformation efficiency increased with vacuum duration of infiltration. These results were confirmed by relative gene expression of GUS transgene as assessed by RT-qPCR and GUS activity assay. To further confirm the usefulness of our protocol, we transiently transformed grape berries with the hydrogen peroxide sensor gene VvHPCA3, and this was confirmed by gene expression analysis as well as increased sensitivity of the explants to hydrogen peroxide treatment. Overall, this study has resulted in a simple but efficient transient transformation protocol for grape berries and would be a valuable tool for the rapid testing of gene function and the study of key regulatory networks in this important crop.