RESUMO
Cytosine methylation contributes to the regulation of gene expression and normal hematopoiesis in mammals. It is catalyzed by the family of DNA methyltransferases that include DNMT1, DNMT3A, and DNMT3B. Peripheral T-cell lymphomas (PTCLs) represent aggressive mature T-cell malignancies exhibiting a broad spectrum of clinical features with poor prognosis and inadequately understood molecular pathobiology. To better understand the molecular landscape and identify candidate genes involved in disease maintenance, we profiled DNA methylation and gene expression of PTCLs. We found that the methylation patterns in PTCLs are deregulated and heterogeneous but share 767 hypo- and 567 hypermethylated differentially methylated regions (DMRs) along with 231 genes up- and 91 genes downregulated in all samples, suggesting a potential association with tumor development. We further identified 39 hypomethylated promoters associated with increased gene expression in the majority of PTCLs. This putative oncogenic signature included the TRIP13 (thyroid hormone receptor interactor 13) gene whose genetic and pharmacologic inactivation inhibited the proliferation of T-cell lines by inducing G2-M arrest and apoptosis. Our data thus show that human PTCLs have a significant number of recurrent methylation alterations that may affect the expression of genes critical for proliferation whose targeting might be beneficial in anti-lymphoma treatments.
RESUMO
Influenza type A virus (IAV) infection is a major cause of morbidity and mortality during influenza epidemics. Recently, a specific link between IAV infection and neurodegenerative disease progression has been established. The non-structural NS1 protein of IAV regulates viral replication during infection and antagonizes host antiviral responses, contributing to influenza virulence. In the present study, we have prepared a mouse lung-to-lung adapted to the NS1-truncated virus (NS80ad). Transcriptome analysis of the gene expression in the lungs revealed that infection with wild-type A/WSN/33 (WSN), NS80, and NS80ad viruses resulted in different regulation of genes involved in signaling pathways associated with the cell proliferation, inflammatory response, and development of neurodegenerative diseases. NS1 protein did not influence the genes involved in the RIG-I-like receptor signaling pathway in the brains. Lethal infection with IAVs dysregulated expression of proteins associated with the development of neurodegenerative diseases (CX3CL1/Fractalkine, Coagulation factor III, and CD105/Endoglin, CD54/ICAM-1, insulin-like growth factor-binding protein (IGFBP)-2, IGFBP-5, IGFBP-6, chitinase 3-like 1 (CHI3L1), Myeloperoxidase (MPO), Osteopontin (OPN), cystatin C, and LDL R). Transcription of GATA3 mRNA was decreased, and expression of MPO was inhibited in the brain infected with NS80 and NS80ad viruses. In addition, the truncation of NS1 protein led to reduced expression of IGFBP-2, CHI3L1, MPO, and LDL-R proteins in the brains. Our results indicate that the influenza virus influences the expression of proteins involved in brain function, and this might occur mostly through the NS1 protein. These findings suggest that the abovementioned proteins represent a promising target for the development of potentially effective immunotherapy against neurodegeneration.
Assuntos
Vírus da Influenza A , Influenza Humana , Doenças Neurodegenerativas , Animais , Camundongos , Humanos , Vírus da Influenza A/genética , Imunidade Inata , Interações Hospedeiro-Patógeno/genética , EncéfaloRESUMO
Cytosine methylation of genomic DNA contributes to the regulation of gene expression and is involved in normal development including hematopoiesis in mammals. It is catalyzed by the family of DNA methyltransferases (DNMTs) that include DNMT1, DNMT3A, and DNMT3B. Peripheral T-cell lymphomas (PTCLs) represent a diverse group of aggressive mature T-cell malignancies accounting for approximately 10-15% of non-Hodgkin lymphoma cases in the US. PTCLs exhibit a broad spectrum of clinical, histological, and immunophenotypic features with poor prognosis and inadequately understood molecular pathobiology. To better understand the molecular landscape and identify candidate genes involved in disease maintenance, we used high-resolution Whole Genome Bisulfite Sequencing (WGBS) and RNA-seq to profile DNA methylation and gene expression of PTCLs and normal T-cells. We found that the methylation patterns in PTCLs are deregulated and heterogeneous but share 767 hypo- and 567 hypermethylated differentially methylated regions (DMRs) along with 231 genes up- and 91 genes downregulated in all samples suggesting a potential association with tumor development. We further identified 39 hypomethylated promoters associated with increased gene expression in the majority of PTCLs. This putative oncogenic signature included the TRIP13 (thyroid hormone receptor interactor 13) gene whose both genetic and pharmacologic inactivation, inhibited cellular growth of PTCL cell lines by inducing G2-M arrest accompanied by apoptosis suggesting that such an approach might be beneficial in human lymphoma treatment. Altogether we show that human PTCLs are characterized by a large number of recurrent methylation alterations, and demonstrated that TRIP13 is critical for PTCL maintenance in vitro.
RESUMO
The viral genome of the SARS-CoV-2 coronavirus, the aetiologic agent of COVID-19, encodes structural, non-structural, and accessory proteins. Most of these components undergo rapid genetic variations, though to a lesser extent the essential viral proteases. Consequently, the protease and/or deubiquitinase activities of the cysteine proteases Mpro and PLpro became attractive targets for the design of antiviral agents. Here, we develop and evaluate new bis(benzylidene)cyclohexanones (BBC) and identify potential antiviral compounds. Three compounds were found to be effective in reducing the SARS-CoV-2 load, with EC50 values in the low micromolar concentration range. However, these compounds also exhibited inhibitory activity IC50 against PLpro at approximately 10-fold higher micromolar concentrations. Although originally developed as PLpro inhibitors, the comparison between IC50 and EC50 of BBC indicates that the mechanism of their in vitro antiviral activity is probably not directly related to inhibition of viral cysteine proteases. In conclusion, our study has identified new potential noncytotoxic antiviral compounds suitable for in vivo testing and further improvement.
Assuntos
COVID-19 , Cisteína Proteases , Humanos , SARS-CoV-2 , Cisteína Endopeptidases/metabolismo , Proteínas não Estruturais Virais/química , Antivirais/farmacologia , Antivirais/química , Inibidores de Proteases/farmacologia , Inibidores de Proteases/química , Simulação de Acoplamento MolecularRESUMO
DNA methylation regulates gene transcription and is involved in various physiological processes in mammals, including development and hematopoiesis. It is catalyzed by DNA methyltransferases including Dnmt1, Dnmt3a, and Dnmt3b. For Dnmt3b, its effects on transcription can result from its own DNA methylase activity, the recruitment of other Dnmts to mediate methylation, or transcription repression in a methylation-independent manner. Low-frequency mutations in human DNMT3B are found in hematologic malignancies including cutaneous T-cell lymphomas, hairy cell leukemia, and diffuse large B-cell lymphomas. Moreover, Dnmt3b is a tumor suppressor in oncogene-driven lymphoid and myeloid malignancies in mice. However, it is poorly understood how the different Dnmt3b activities contribute to these outcomes. We modulated Dnmt3b activity in vivo by generating Dnmt3b+/- mice expressing one wild-type allele as well as Dnmt3b+/CI and Dnmt3bCI/CI mice where one or both alleles express catalytically inactive Dnmt3bCI. We show that 43% of Dnmt3b+/- mice developed T-cell lymphomas, chronic lymphocytic leukemia, and myeloproliferation over 18 months, thus resembling phenotypes previously observed in Dnmt3a+/- mice, possibly through regulation of shared target genes. Interestingly, Dnmt3b+/CI and Dnmt3bCI/CI mice survived postnatal development and were affected by B-cell rather than T-cell malignancies with decreased penetrance. Genome-wide hypomethylation, increased expression of oncogenes such as Jdp2, STAT1, and Trip13, and p53 downregulation were major events contributing to Dnmt3b+/- lymphoma development. We conclude that Dnmt3b catalytic activity is critical to prevent B-cell transformation in vivo, whereas accessory and methylation-independent repressive functions are important to prevent T-cell transformation.
Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Leucemia Linfocítica Crônica de Células B/genética , Linfoma de Células B/genética , Linfoma de Células T/genética , Transtornos Mieloproliferativos/genética , Neoplasias Experimentais/genética , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA (Citosina-5-)-Metiltransferases/deficiência , Metilação de DNA , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Heterozigoto , Homozigoto , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Leucemia Linfocítica Crônica de Células B/enzimologia , Leucemia Linfocítica Crônica de Células B/patologia , Linfoma de Células B/enzimologia , Linfoma de Células B/patologia , Linfoma de Células T/enzimologia , Linfoma de Células T/patologia , Masculino , Camundongos , Camundongos Knockout , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/patologia , Neoplasias Experimentais/enzimologia , Neoplasias Experimentais/patologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , DNA Metiltransferase 3BRESUMO
BACKGROUND: DNA methylation regulates gene transcription in many physiological processes in mammals including development and haematopoiesis. It is catalysed by several DNA methyltransferases, including Dnmt3b that mediates both methylation-dependant and independent gene repression. Dnmt3b is critical for mouse embryogenesis and functions as a tumour suppressor in haematologic malignancies in mice. However, the extent to which Dnmt3b's catalytic activity (CA) is involved in development and cancer is unclear. METHODS: We used a mouse model expressing catalytically inactive Dnmt3b (Dnmt3bCI) to study a role of Dnmt3b's CA in development and cancer. We utilized global approaches including Whole-genome Bisulfite sequencing and RNA-seq to analyse DNA methylation and gene expression to identify putative targets of Dnmt3b's CA. To analyse postnatal development and haematopoiesis, we used tissue staining, histological and FACS analysis. To determine potential involvement of selected genes in lymphomagenesis, we used overexpression and knock down approaches followed by in vitro growth assays. FINDINGS: We show that mice expressing Dnmt3bCI only, survive postnatal development and develop ICF (the immunodeficiency-centromeric instability-facial anomalies) -like syndrome. The lack of Dnmt3b's CA promoted fibroblasts transformation in vitro, accelerated MLL-AF9 driven Acute Myeloid Leukaemia and MYC-induced T-cell lymphomagenesis in vivo. The elimination of Dnmt3b's CA resulted in decreased methylation of c-Met promoter and its upregulation, activated oncogenic Met signalling, Stat3 phosphorylation and up-regulation of Lin28b promoting lymphomagenesis. INTERPRETATION: Our data demonstrates that Dnmt3b's CA is largely dispensable for mouse development but critical to prevent tumourigenesis by controlling events involved in cellular transformation. FUNDING: This study was supported by Department of Anatomy and Cell Biology and Cancer Centre at the University of Florida start-up funds, NIH/NCI grant 1R01CA188561-01A1 (R.O.).
Assuntos
Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Animais , Catálise , Linhagem Celular , Metilação de DNA , Modelos Animais de Doenças , Ativação Enzimática , Epigênese Genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Linfoma/diagnóstico , Linfoma/etiologia , Linfoma/metabolismo , Camundongos , Camundongos Knockout , Mutação , DNA Metiltransferase 3BRESUMO
DNA methylation regulates gene expression in a variety of processes, including mouse embryonic development. Four catalytically active enzymes function in mice as DNA methyltransferases (Dnmts) and as transcriptional regulators. Inactivation of Dnmt3b results in mouse embryonic lethality, but which activities are involved is unclear. Here we show that catalytically inactive Dnmt3b restores a majority of methylation and expression changes deregulated in the absence of Dnmt3b, and as a result, mice survive embryonic development. Thus, Dnmt3b functions as an accessory cofactor supporting catalytic activities performed by other Dnmts. We further demonstrate that Dnmt3b is linked to a control of major developmental pathways, including Wnt and hedgehog signaling. Dnmt3b directly represses Wnt9b whose aberrant up-regulation contributes to embryonic lethality of Dnmt3b knockout embryos. Our results highlight that Dnmt3b is a multifaceted protein that serves as an enzyme, an accessory factor for other methyltransferases, and as a transcriptional repressor in mouse embryogenesis.
Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Animais , Biocatálise , DNA (Citosina-5-)-Metiltransferases/metabolismo , Feminino , Células HEK293 , Proteínas Hedgehog/genética , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Transdução de Sinais/genética , Proteínas Wnt/genética , DNA Metiltransferase 3BRESUMO
Herpes simplex encephalitis (HSE) is the most common form of acute viral encephalitis in industrialized countries. Type I interferon (IFN) is important for control of herpes simplex virus (HSV-1) in the central nervous system (CNS). Here we show that microglia are the main source of HSV-induced type I IFN expression in CNS cells and these cytokines are induced in a cGAS-STING-dependent manner. Consistently, mice defective in cGAS or STING are highly susceptible to acute HSE. Although STING is redundant for cell-autonomous antiviral resistance in astrocytes and neurons, viral replication is strongly increased in neurons in STING-deficient mice. Interestingly, HSV-infected microglia confer STING-dependent antiviral activities in neurons and prime type I IFN production in astrocytes through the TLR3 pathway. Thus, sensing of HSV-1 infection in the CNS by microglia through the cGAS-STING pathway orchestrates an antiviral program that includes type I IFNs and immune-priming of other cell types.
Assuntos
Encefalite por Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Proteínas de Membrana/deficiência , Microglia/virologia , Nucleotidiltransferases/deficiência , Animais , Antivirais/uso terapêutico , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/virologia , Células Cultivadas , Encefalite por Herpes Simples/tratamento farmacológico , Encefalite por Herpes Simples/genética , Herpesvirus Humano 1/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Interferon Tipo I/metabolismo , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/efeitos dos fármacos , Microglia/metabolismo , Nucleotidiltransferases/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
The innate immune response represents a primary line of defense against invading viral pathogens. Since epithelial cells are the primary site of gammaherpesvirus replication during infection in vivo and there are no information on activity of IFN-III signaling against gammaherpesviruses in this cell type, in present study, we evaluated the expression profile and virus-host interactions in mouse mammary epithelial cell (NMuMG) infected with three strains of murine gammaherpesvirus, MHV-68, MHV-72 and MHV-4556. Studying three strains of murine gammaherpesvirus, which differ in nucleotide sequence of some structural and non-structural genes, allowed us to compare the strain-dependent interactions with host organism. Our results clearly demonstrate that: (i) MHV-68, MHV-72 and MHV-4556 differentially interact with intracellular signaling and dysregulate IFN signal transduction; (ii) MHV-68, MHV-72 and MHV-4556 degrade type I IFN receptor in very early stages of infection (2-4hpi), but not type III IFN receptor; (iii) type III IFN signaling might play a key role in antiviral defense of epithelial cells in early stages of murine gammaherpesvirus replication; (iv) NMuMG cells are an appropriate model for study of not only type I IFN signaling, but also type III IFN signaling pathway. These findings are important for better understanding of individual virus-host interactions in lytic as well as in persistent gammaherpesvirus replication and help us to elucidate IFN-III function in early events of virus infection.