RESUMEN
Nasopharyngeal carcinoma (NPC) most frequently occurs in southern China and southeast Asia. Epidemiology studies link NPC to genetic predisposition, Epstein-Barr virus (EBV) infection, and environmental factors. Genetic studies indicate that mutations in chromatin-modifying enzymes are the most frequent genetic alterations in NPC. Here, we used H3K27ac chromatin immune precipitation followed by deep sequencing (ChIP-seq) to define the NPC epigenome in primary NPC biopsies, NPC xenografts, and an NPC cell line, and compared them to immortalized normal nasopharyngeal or oral epithelial cells. We identified NPC-specific enhancers and found these enhancers were enriched with nuclear factor κB (NF-κB), IFN-responsive factor 1 (IRF1) and IRF2, and ETS family members ETS1 motifs. Normal cell-specific enhancers were enriched with basic leucine zipper family members and TP53 motifs. NPC super-enhancers with extraordinarily broad and high H3K27ac signals were also identified, and they were linked to genes important for oncogenesis including ETV6. ETV6 was also highly expressed in NPC biopsies by immunohistochemistry. High ETV6 expression correlated with a poor prognosis. Furthermore, we defined the EBV episome epigenetic landscapes in primary NPC tissue.
Asunto(s)
Carcinoma/genética , Elementos de Facilitación Genéticos , Neoplasias Nasofaríngeas/genética , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Represoras/genética , Adolescente , Adulto , Anciano , Animales , Azepinas/farmacología , Carcinoma/etiología , Carcinoma/metabolismo , Proteínas de Ciclo Celular , Línea Celular Tumoral , Elementos de Facilitación Genéticos/efectos de los fármacos , Epigénesis Genética , Infecciones por Virus de Epstein-Barr/complicaciones , Femenino , Genoma Viral , Xenoinjertos , Secuenciación de Nucleótidos de Alto Rendimiento , Código de Histonas/genética , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/etiología , Neoplasias Nasofaríngeas/metabolismo , Proteínas Nucleares/antagonistas & inhibidores , Pronóstico , Proteínas Proto-Oncogénicas c-ets/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Triazoles/farmacología , Adulto Joven , Proteína ETS de Variante de Translocación 6RESUMEN
Epstein-Barr virus (EBV) is a major cause of immunosuppression-related B-cell lymphomas and Hodgkin lymphoma (HL). In these malignancies, EBV latent membrane protein 1 (LMP1) and LMP2A provide infected B cells with surrogate CD40 and B-cell receptor growth and survival signals. To gain insights into their synergistic in vivo roles in germinal center (GC) B cells, from which most EBV-driven lymphomas arise, we generated a mouse model with conditional GC B-cell LMP1 and LMP2A coexpression. LMP1 and LMP2A had limited effects in immunocompetent mice. However, upon T- and NK-cell depletion, LMP1/2A caused massive plasmablast outgrowth, organ damage, and death. RNA-sequencing analyses identified EBV oncoprotein effects on GC B-cell target genes, including up-regulation of multiple proinflammatory chemokines and master regulators of plasma cell differentiation. LMP1/2A coexpression also up-regulated key HL markers, including CD30 and mixed hematopoietic lineage markers. Collectively, our results highlight synergistic EBV membrane oncoprotein effects on GC B cells and provide a model for studies of their roles in immunosuppression-related lymphoproliferative diseases.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/inmunología , Regulación Viral de la Expresión Génica/inmunología , Herpesvirus Humano 4/inmunología , Enfermedad de Hodgkin/inmunología , Linfoma de Células B/inmunología , Neoplasias Experimentales/inmunología , Proteínas de la Matriz Viral/inmunología , Animales , Linfocitos B/inmunología , Linfocitos B/patología , Centro Germinal/inmunología , Centro Germinal/patología , Herpesvirus Humano 4/genética , Enfermedad de Hodgkin/genética , Enfermedad de Hodgkin/patología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Linfoma de Células B/genética , Linfoma de Células B/patología , Ratones , Ratones Mutantes , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Linfocitos T/inmunología , Linfocitos T/patología , Proteínas de la Matriz Viral/genéticaRESUMEN
Epstein-Barr virus (EBV) super-enhancers (ESEs) are essential for lymphoblastoid cell (LCL) growth and survival. Reanalyses of LCL global run-on sequencing (Gro-seq) data found abundant enhancer RNAs (eRNAs) being transcribed at ESEs. Inactivation of ESE components, EBV nuclear antigen 2 (EBNA2) and bromodomain-containing protein 4 (BRD4), significantly decreased eRNAs at ESEs -428 and -525 kb upstream of the MYC oncogene transcription start site (TSS). shRNA knockdown of the MYC -428 and -525 ESE eRNA caused LCL growth arrest and reduced cell growth. Furthermore, MYC ESE eRNA knockdown also significantly reduced MYC expression, ESE H3K27ac signals, and MYC ESEs looping to MYC TSS. These data indicate that ESE eRNAs strongly affect cell gene expression and enable LCL growth.
Asunto(s)
Elementos de Facilitación Genéticos , Herpesvirus Humano 4/fisiología , Interacciones Huésped-Patógeno , Trastornos Linfoproliferativos/virología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas de Ciclo Celular , Línea Celular , Endonucleasas , Humanos , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Epstein-Barr Virus (EBV) Nuclear Antigen 1 (EBNA1)-mediated origin of plasmid replication (oriP) DNA episome maintenance is essential for EBV-mediated tumorigenesis. We have now found that EBNA1 binds to Ribosome Protein L4 (RPL4). RPL4 shRNA knockdown decreased EBNA1 activation of an oriP luciferase reporter, EBNA1 DNA binding in lymphoblastoid cell lines, and EBV genome number per lymphoblastoid cell line. EBV infection increased RPL4 expression and redistributed RPL4 to cell nuclei. RPL4 and Nucleolin (NCL) were a scaffold for an EBNA1-induced oriP complex. The RPL4 N terminus cooperated with NCL-K429 to support EBNA1 and oriP-mediated episome binding and maintenance, whereas the NCL C-terminal K380 and K393 induced oriP DNA H3K4me2 modification and promoted EBNA1 activation of oriP-dependent transcription. These observations provide new insights into the mechanisms by which EBV uses NCL and RPL4 to establish persistent B-lymphoblastoid cell infection.
Asunto(s)
Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Proteínas Ribosómicas/metabolismo , Linfocitos B/metabolismo , Linfocitos B/virología , Línea Celular , ADN Viral/genética , ADN Viral/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/genética , Técnicas de Silenciamiento del Gen , Genoma Viral , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/patogenicidad , Interacciones Huésped-Patógeno , Humanos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Plásmidos/genética , Plásmidos/metabolismo , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Origen de Réplica , Proteínas Ribosómicas/antagonistas & inhibidores , Proteínas Ribosómicas/genética , Activación Transcripcional , NucleolinaRESUMEN
Genotypic differences greatly influence susceptibility and resistance to disease. Understanding genotype-phenotype relationships requires that phenotypes be viewed as manifestations of network properties, rather than simply as the result of individual genomic variations. Genome sequencing efforts have identified numerous germline mutations, and large numbers of somatic genomic alterations, associated with a predisposition to cancer. However, it remains difficult to distinguish background, or 'passenger', cancer mutations from causal, or 'driver', mutations in these data sets. Human viruses intrinsically depend on their host cell during the course of infection and can elicit pathological phenotypes similar to those arising from mutations. Here we test the hypothesis that genomic variations and tumour viruses may cause cancer through related mechanisms, by systematically examining host interactome and transcriptome network perturbations caused by DNA tumour virus proteins. The resulting integrated viral perturbation data reflects rewiring of the host cell networks, and highlights pathways, such as Notch signalling and apoptosis, that go awry in cancer. We show that systematic analyses of host targets of viral proteins can identify cancer genes with a success rate on a par with their identification through functional genomics and large-scale cataloguing of tumour mutations. Together, these complementary approaches increase the specificity of cancer gene identification. Combining systems-level studies of pathogen-encoded gene products with genomic approaches will facilitate the prioritization of cancer-causing driver genes to advance the understanding of the genetic basis of human cancer.
Asunto(s)
Genes Relacionados con las Neoplasias/genética , Genoma Humano/genética , Interacciones Huésped-Patógeno , Neoplasias/genética , Neoplasias/metabolismo , Virus Oncogénicos/patogenicidad , Proteínas Virales/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Adenoviridae/patogenicidad , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Herpesvirus Humano 4/patogenicidad , Interacciones Huésped-Patógeno/genética , Humanos , Neoplasias/patología , Virus Oncogénicos/genética , Virus Oncogénicos/metabolismo , Sistemas de Lectura Abierta/genética , Papillomaviridae/genética , Papillomaviridae/metabolismo , Papillomaviridae/patogenicidad , Poliomavirus/genética , Poliomavirus/metabolismo , Poliomavirus/patogenicidad , Receptores Notch/metabolismo , Transducción de Señal , Técnicas del Sistema de Dos Híbridos , Proteínas Virales/genéticaRESUMEN
Epstein-Barr Virus (EBV) conversion of B-lymphocytes to Lymphoblastoid Cell Lines (LCLs) requires four EBV nuclear antigen (EBNA) oncoproteins: EBNA2, EBNALP, EBNA3A, and EBNA3C. EBNA2 and EBNALP associate with EBV and cell enhancers, up-regulate the EBNA promoter, MYC, and EBV Latent infection Membrane Proteins (LMPs), which up-regulate BCL2 to protect EBV-infected B-cells from MYC proliferation-induced cell death. LCL proliferation induces p16(INK4A) and p14(ARF)-mediated cell senescence. EBNA3A and EBNA3C jointly suppress p16(INK4A) and p14(ARF), enabling continuous cell proliferation. Analyses of the EBNA3A human genome-wide ChIP-seq landscape revealed 37% of 10,000 EBNA3A sites to be at strong enhancers; 28% to be at weak enhancers; 4.4% to be at active promoters; and 6.9% to be at weak and poised promoters. EBNA3A colocalized with BATF-IRF4, ETS-IRF4, RUNX3, and other B-cell Transcription Factors (TFs). EBNA3A sites clustered into seven unique groups, with differing B-cell TFs and epigenetic marks. EBNA3A coincidence with BATF-IRF4 or RUNX3 was associated with stronger EBNA3A ChIP-Seq signals. EBNA3A was at MYC, CDKN2A/B, CCND2, CXCL9/10, and BCL2, together with RUNX3, BATF, IRF4, and SPI1. ChIP-re-ChIP revealed complexes of EBNA3A on DNA with BATF. These data strongly support a model in which EBNA3A is tethered to DNA through a BATF-containing protein complexes to enable continuous cell proliferation.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , ADN/genética , ADN/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/genética , Genoma Viral , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/patogenicidad , Linfocitos B/metabolismo , Linfocitos B/virología , Sitios de Unión/genética , Línea Celular , Quimiocina CXCL10/genética , Quimiocina CXCL9/genética , Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , Ciclina D2/genética , Elementos de Facilitación Genéticos , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Genes bcl-2 , Genes myc , Genes p16 , Genoma Humano , Herpesvirus Humano 4/fisiología , Interacciones Huésped-Patógeno/genética , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Factores Reguladores del Interferón/metabolismo , Regiones Promotoras Genéticas , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Epstein-Barr virus (EBV) infects germinal center (GC) B cells and establishes persistent infection in memory B cells. EBV-infected B cells can cause B-cell malignancies in humans with T- or natural killer-cell deficiency. We now find that EBV-encoded latent membrane protein 2A (LMP2A) mimics B-cell antigen receptor (BCR) signaling in murine GC B cells, causing altered humoral immune responses and autoimmune diseases. Investigation of the impact of LMP2A on B-cell differentiation in mice that conditionally express LMP2A in GC B cells or all B-lineage cells found LMP2A expression enhanced not only BCR signals but also plasma cell differentiation in vitro and in vivo. Conditional LMP2A expression in GC B cells resulted in preferential selection of low-affinity antibody-producing B cells despite apparently normal GC formation. GC B-cell-specific LMP2A expression led to systemic lupus erythematosus-like autoimmune phenotypes in an age-dependent manner. Epigenetic profiling of LMP2A B cells found increased H3K27ac and H3K4me1 signals at the zinc finger and bric-a-brac, tramtrack domain-containing protein 20 locus. We conclude that LMP2A reduces the stringency of GC B-cell selection and may contribute to persistent EBV infection and pathogenesis by providing GC B cells with excessive prosurvival effects.
Asunto(s)
Centro Germinal/metabolismo , Herpesvirus Humano 4/metabolismo , Proteínas de la Matriz Viral/metabolismo , Animales , Autoanticuerpos/química , Enfermedades Autoinmunes/metabolismo , Enfermedades Autoinmunes/virología , Diferenciación Celular , Linaje de la Célula , Cruzamientos Genéticos , Epigénesis Genética , Citometría de Flujo , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Heterocigoto , Inmunidad Humoral , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microscopía Fluorescente , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal , Bazo/citología , Dedos de ZincRESUMEN
EBV causes B lymphomas and undifferentiated nasopharyngeal carcinoma (NPC). Although the mechanisms by which EBV infects B lymphocytes have been extensively studied, investigation of the mechanisms by which EBV infects nasopharyngeal epithelial cells (NPECs) has only recently been enabled by the successful growth of B lymphoma Mo-MLV insertion region 1 homolog (BMI1)-immortalized NPECs in vitro and the discovery that neuropilin 1 expression positively affects EBV glycoprotein B (gB)-mediated infection and tyrosine kinase activations in enhancing EBV infection of BMI1-immortalized NPECs. We have now found that even though EBV infected NPECs grown as a monolayer at extremely low efficiency (<3%), close to 30% of NPECs grown as sphere-like cells (SLCs) were infected by EBV. We also identified nonmuscle myosin heavy chain IIA (NMHC-IIA) as another NPEC protein important for efficient EBV infection. EBV gH/gL specifically interacted with NMHC-IIA both in vitro and in vivo. NMHC-IIA densely aggregated on the surface of NPEC SLCs and colocalized with EBV. EBV infection of NPEC SLCs was significantly reduced by NMHC-IIA siRNA knock-down. NMHC-IIA antisera also efficiently blocked EBV infection. These data indicate that NMHC-IIA is an important factor for EBV NPEC infection.
Asunto(s)
Infecciones por Virus de Epstein-Barr/fisiopatología , Cadenas Pesadas de Miosina/fisiología , Nasofaringe/virología , Secuencia de Aminoácidos , Línea Celular Transformada , Humanos , Datos de Secuencia Molecular , Cadenas Pesadas de Miosina/química , Nasofaringe/patologíaRESUMEN
Epstein-Barr virus nuclear antigen 3C (EBNA3C) repression of CDKN2A p14(ARF) and p16(INK4A) is essential for immortal human B-lymphoblastoid cell line (LCL) growth. EBNA3C ChIP-sequencing identified >13,000 EBNA3C sites in LCL DNA. Most EBNA3C sites were associated with active transcription; 64% were strong H3K4me1- and H3K27ac-marked enhancers and 16% were active promoters marked by H3K4me3 and H3K9ac. Using ENCODE LCL transcription factor ChIP-sequencing data, EBNA3C sites coincided (±250 bp) with RUNX3 (64%), BATF (55%), ATF2 (51%), IRF4 (41%), MEF2A (35%), PAX5 (34%), SPI1 (29%), BCL11a (28%), SP1 (26%), TCF12 (23%), NF-κB (23%), POU2F2 (23%), and RBPJ (16%). EBNA3C sites separated into five distinct clusters: (i) Sin3A, (ii) EBNA2/RBPJ, (iii) SPI1, and (iv) strong or (v) weak BATF/IRF4. EBNA3C signals were positively affected by RUNX3, BATF/IRF4 (AICE) and SPI1/IRF4 (EICE) cooccupancy. Gene set enrichment analyses correlated EBNA3C/Sin3A promoter sites with transcription down-regulation (P < 1.6 × 10(-4)). EBNA3C signals were strongest at BATF/IRF4 and SPI1/IRF4 composite sites. EBNA3C bound strongly to the p14(ARF) promoter through SPI1/IRF4/BATF/RUNX3, establishing RBPJ-, Sin3A-, and REST-mediated repression. EBNA3C immune precipitated with Sin3A and conditional EBNA3C inactivation significantly decreased Sin3A binding at the p14(ARF) promoter (P < 0.05). These data support a model in which EBNA3C binds strongly to BATF/IRF4/SPI1/RUNX3 sites to enhance transcription and recruits RBPJ/Sin3A- and REST/NRSF-repressive complexes to repress p14(ARF) and p16(INK4A) expression.
Asunto(s)
Antígenos Virales/química , Linfocitos B/virología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Factores Reguladores del Interferón/química , Proteínas Proto-Oncogénicas/química , Proteínas Represoras/metabolismo , Transactivadores/química , Secuencias de Aminoácidos , Linfocitos B/citología , Sitios de Unión , Proliferación Celular , Inmunoprecipitación de Cromatina , Antígenos Nucleares del Virus de Epstein-Barr , Regulación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Herpesvirus Humano 4/metabolismo , Histonas/química , Humanos , Linfoma/metabolismo , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Recombinantes/metabolismo , Complejo Correpresor Histona Desacetilasa y Sin3 , Proteína p14ARF Supresora de Tumor/metabolismoRESUMEN
Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is essential for EBV episome maintenance, replication, and transcription. These effects are mediated by EBNA1 binding to cognate oriP DNA, which comprise 20 imperfect copies of a 30-bp dyad symmetry enhancer and an origin for DNA replication. To identify cell proteins essential for these EBNA1 functions, EBNA1 associated cell proteins were immune precipitated and analyzed by liquid chromatography-tandem mass spectrometry. Nucleolin (NCL) was identified to be EBNA1 associated. EBNA1's N-terminal 100 aa and NCL's RNA-binding domains were critical for EBNA1/NCL interaction. Lentivirus shRNA-mediated NCL depletion substantially reduced EBNA1 recruitment to oriP DNA, EBNA1-dependent transcription of an EBV oriP luciferase reporter, and EBV genome maintenance in lymphoblastoid cell lines. NCL RNA-binding domain K429 was critical for ATP and EBNA1 binding. NCL overexpression increased EBNA1 binding to oriP and transcription, whereas NCL K429A was deficient. Moreover, NCL silencing impaired lymphoblastoid cell line growth. These experiments reveal a surprisingly critical role for NCL K429 in EBNA1 episome maintenance and transcription, which may be a target for therapeutic intervention.
Asunto(s)
Antígenos Nucleares del Virus de Epstein-Barr/química , Antígenos Nucleares del Virus de Epstein-Barr/fisiología , Fosfoproteínas/química , Fosfoproteínas/fisiología , Plásmidos/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/fisiología , Transcripción Genética , Adenosina Trifosfato/química , Sitios de Unión , Línea Celular Tumoral , Núcleo Celular/metabolismo , Cromatografía Liquida , Replicación del ADN , Epítopos/química , Regulación de la Expresión Génica , Silenciador del Gen , Genoma , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunoprecipitación , Luciferasas/metabolismo , Espectrometría de Masas , Microscopía Confocal , Unión Proteica , Estructura Terciaria de Proteína , Origen de Réplica , Replicación Viral , NucleolinaRESUMEN
Epstein-Barr virus (EBV) nuclear antigens EBNALP (LP) and EBNA2 (E2) are coexpressed in EBV-infected B lymphocytes and are critical for lymphoblastoid cell line outgrowth. LP removes NCOR and RBPJ repressive complexes from promoters, enhancers, and matrix-associated deacetylase bodies, whereas E2 activates transcription from distal enhancers. LP ChIP-seq analyses identified 19,224 LP sites of which ~50% were ± 2 kb of a transcriptional start site. LP sites were enriched for B-cell transcription factors (TFs), YY1, SP1, PAX5, BATF, IRF4, ETS1, RAD21, PU.1, CTCF, RBPJ, ZNF143, SMC3, NFκB, TBLR, and EBF. E2 sites were also highly enriched for LP-associated cell TFs and were more highly occupied by RBPJ and EBF. LP sites were highly marked by H3K4me3, H3K27ac, H2Az, H3K9ac, RNAPII, and P300, indicative of activated transcription. LP sites were 29% colocalized with E2 (LP/E2). LP/E2 sites were more similar to LP than to E2 sites in associated cell TFs, RNAPII, P300, and histone H3K4me3, H3K9ac, H3K27ac, and H2Az occupancy, and were more highly transcribed than LP or E2 sites. Gene affected by CTCF and LP cooccupancy were more highly expressed than genes affected by CTCF alone. LP was at myc enhancers and promoters and of MYC regulated ccnd2, 23 med complex components, and MYC regulated cell survival genes, igf2r and bcl2. These data implicate LP and associated TFs and DNA looping factors CTCF, RAD21, SMC3, and YY1/INO80 chromatin-remodeling complexes in repressor depletion and gene activation necessary for lymphoblastoid cell line growth and survival.
Asunto(s)
Elementos de Facilitación Genéticos/fisiología , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Regulación de la Expresión Génica/inmunología , Regiones Promotoras Genéticas/fisiología , Factores de Transcripción/metabolismo , Proteínas Virales/metabolismo , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Callithrix , Línea Celular , Inmunoprecipitación de Cromatina , Análisis por Conglomerados , Elementos de Facilitación Genéticos/genética , Perfilación de la Expresión Génica , Regiones Promotoras Genéticas/genética , Análisis de Secuencia de ADNRESUMEN
The ubiquitous Epstein Barr virus (EBV) exploits human B-cell development to establish a persistent infection in â¼90% of the world population. Constitutive activation of NF-κB by the viral oncogene latent membrane protein 1 (LMP1) has an important role in persistence, but is a risk factor for EBV-associated lymphomas. Here, we demonstrate that endogenous LMP1 escapes degradation upon accumulation within intraluminal vesicles of multivesicular endosomes and secretion via exosomes. LMP1 associates and traffics with the intracellular tetraspanin CD63 into vesicles that lack MHC II and sustain low cholesterol levels, even in 'cholesterol-trapping' conditions. The lipid-raft anchoring sequence FWLY, nor ubiquitylation of the N-terminus, controls LMP1 sorting into exosomes. Rather, C-terminal modifications that retain LMP1 in Golgi compartments preclude assembly within CD63-enriched domains and/or exosomal discharge leading to NF-κB overstimulation. Interference through shRNAs further proved the antagonizing role of CD63 in LMP1-mediated signalling. Thus, LMP1 exploits CD63-enriched microdomains to restrain downstream NF-κB activation by promoting trafficking in the endosomal-exosomal pathway. CD63 is thus a critical mediator of LMP1 function in- and outside-infected (tumour) cells.
Asunto(s)
Antígenos CD/metabolismo , Endosomas/metabolismo , Exosomas/metabolismo , Herpesvirus Humano 4/inmunología , FN-kappa B/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Proteínas de la Matriz Viral/metabolismo , Línea Celular , Herpesvirus Humano 4/patogenicidad , Humanos , Unión Proteica , Transporte de Proteínas , Tetraspanina 30RESUMEN
Germinal centers (GCs) are specialized microenvironments in secondary lymphoid organs where high-affinity antibody-producing B cells are selected based on B-cell antigen receptor (BCR) signal strength. BCR signaling required for normal GC selection is uncertain. We have found that protein kinase N1 (PKN1, also known as PRK1) negatively regulates Akt kinase downstream of the BCR and that this regulation is necessary for normal GC development. PKN1 interacted with and inhibited Akt1 kinase and transforming activities. Pkn1(-/-) B cells were hyperresponsive and had increased phosphorylated Akt1 levels upon BCR stimulation. In the absence of immunization or infection, Pkn1(-/-) mice spontaneously formed GCs and developed an autoimmune-like disease with age, which was characterized by autoantibody production and glomerulonephritis. More B cells, with fewer somatic BCR gene V region hypermutations were selected in Pkn1(-/-) GCs. These results indicate that PKN1 down-regulation of BCR-activated Akt activity is critical for normal GC B-cell survival and selection.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Centro Germinal/inmunología , Proteína Quinasa C/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Autoanticuerpos/química , Linfocitos B/metabolismo , Proliferación Celular , Regulación hacia Abajo , Glomerulonefritis/metabolismo , Inmunidad Humoral , Inmunoglobulinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosforilación , Transducción de SeñalRESUMEN
Although canonical NFκB is frequently critical for cell proliferation, survival, or differentiation, NFκB hyperactivation can cause malignant, inflammatory, or autoimmune disorders. Despite intensive study, mammalian NFκB pathway loss-of-function RNAi analyses have been limited to specific protein classes. We therefore undertook a human genome-wide siRNA screen for novel NFκB activation pathway components. Using an Epstein Barr virus latent membrane protein (LMP1) mutant, the transcriptional effects of which are canonical NFκB-dependent, we identified 155 proteins significantly and substantially important for NFκB activation in HEK293 cells. These proteins included many kinases, phosphatases, ubiquitin ligases, and deubiquinating enzymes not previously known to be important for NFκB activation. Relevance to other canonical NFκB pathways was extended by finding that 118 of the 155 LMP1 NF-κB activation pathway components were similarly important for IL-1ß-, and 79 for TNFα-mediated NFκB activation in the same cells. MAP3K8, PIM3, and six other enzymes were uniquely relevant to LMP1-mediated NFκB activation. Most novel pathway components functioned upstream of IκB kinase complex (IKK) activation. Robust siRNA knockdown effects were confirmed for all mRNAs or proteins tested. Although multiple ZC3H-family proteins negatively regulate NFκB, ZC3H13 and ZC3H18 were activation pathway components. ZC3H13 was critical for LMP1, TNFα, and IL-1ß NFκB-dependent transcription, but not for IKK activation, whereas ZC3H18 was critical for IKK activation. Down-modulators of LMP1 mediated NFκB activation were also identified. These experiments identify multiple targets to inhibit or stimulate LMP1-, IL-1ß-, or TNFα-mediated canonical NFκB activation.
Asunto(s)
Genoma Humano , FN-kappa B/metabolismo , ARN Interferente Pequeño , Línea Celular , Humanos , Interleucina-1beta/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Epstein-Barr Virus (EBV) is an oncogenic γ-herpesvirus that capably establishes both latent and lytic modes of infection in host cells and causes malignant diseases in humans. Nuclear antigen 2 (EBNA2)-mediated transcription of both cellular and viral genes is essential for the establishment and maintenance of the EBV latency program in B lymphocytes. Here, we employed a protein affinity pull-down and LC-MS/MS analysis to identify nucleophosmin (NPM1) as one of the cellular proteins bound to EBNA2. Additionally, the specific domains that are responsible for protein-protein interactions were characterized as EBNA2 residues 300 to 360 and the oligomerization domain (OD) of NPM1. As in c-MYC, dramatic NPM1 expression was induced in EBV positively infected B cells after three days of viral infection, and both EBNA2 and EBNALP were implicated in the transactivation of the NPM1 promoter. Depletion of NPM1 with the lentivirus-expressed short-hairpin RNAs (shRNAs) effectively abrogated EBNA2-dependent transcription and transformation outgrowth of lymphoblastoid cells. Notably, the ATP-bound state of NPM1 was required to induce assembly of a protein complex containing EBNA2, RBP-Jκ, and NPM1 by stabilizing the interaction of EBNA2 with RBP-Jκ. In a NPM1-knockdown cell line, we demonstrated that an EBNA2-mediated transcription defect was fully restored by the ectopic expression of NPM1. Our findings highlight the essential role of NPM1 in chaperoning EBNA2 onto the latency-associated membrane protein 1 (LMP1) promoters, which is coordinated with the subsequent activation of transcriptional cascades through RBP-Jκ during EBV infection. These data advance our understanding of EBV pathology and further imply that NPM1 can be exploited as a therapeutic target for EBV-associated diseases.
Asunto(s)
Linfocitos B/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/fisiología , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Transcripción Genética , Proteínas Virales/metabolismo , Latencia del Virus/fisiología , Linfocitos B/virología , Línea Celular Tumoral , Antígenos Nucleares del Virus de Epstein-Barr/genética , Humanos , Chaperonas Moleculares/genética , Proteínas Nucleares/genética , Nucleofosmina , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-myc , Proteínas de la Matriz Viral/biosíntesis , Proteínas de la Matriz Viral/genética , Proteínas Virales/genética , Ensamble de Virus/fisiologíaRESUMEN
Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) and EBNA3A are each essential for EBV conversion of primary human B lymphocytes into continuously proliferating lymphoblast cell lines (LCLs) and for maintaining LCL growth. We now find that EBNA3C and EBNA3A's essential roles are to repress p16(INK4A) and p14(ARF). In the absence of EBNA3C or EBNA3A, p16(INK4A) and p14(ARF) expression increased and cell growth ceased. EBNA3C inactivation did not alter p16(INK4A) promoter CpG methylation, but reduced already low H3K27me3, relative to resting B cells, and increased H3K4me3 and H3-acetylation, linking EBNA3C inactivation to histone modifications associated with increased transcription. Importantly, knockdown of p16(INK4A) or p14(ARF) partially rescued LCLs from EBNA3C or EBNA3A inactivation-induced growth arrest and knockdown of both rescued LCL growth, confirming central roles for p16(INK4A) and p14(ARF) in LCL growth arrest following EBNA3C or EBNA3A inactivation. Moreover, blockade of p16(INK4A) and p14(ARF) effects on pRb and p53 by human papilloma virus type 16 E7 and E6 expression, sustained LCL growth after EBNA3C or EBNA3A inactivation. These data indicate that EBNA3C and EBNA3A joint repression of CDKN2A p16(INK4A) and p14(ARF) is essential for LCL growth.
Asunto(s)
División Celular/fisiología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/fisiología , Antígenos Nucleares del Virus de Epstein-Barr/fisiología , Proteína p14ARF Supresora de Tumor/fisiología , Línea Celular , Islas de CpG , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Metilación de ADN , Técnicas de Silenciamiento del Gen , Humanos , Regiones Promotoras Genéticas , Proteína p14ARF Supresora de Tumor/genéticaRESUMEN
EBV nuclear antigen 2 (EBNA2) and EBV nuclear antigen LP (EBNALP) are critical for B-lymphocyte transformation to lymphoblastoid cell lines (LCLs). EBNA2 activates transcription through recombination signal-binding immunoglobulin κJ region (RBPJ), a transcription factor associated with NCoR repressive complexes, and EBNALP is implicated in repressor relocalization. EBNALP coactivation with EBNA2 was found to dominate over NCoR repression. EBNALP associated with NCoR and dismissed NCoR, NCoR and RBPJ, or NCoR, RBPJ, and EBNA2 from matrix-associated deacetylase (MAD) bodies. In non-EBV-infected BJAB B lymphoma cells that stably express EBNA2, EBNALP, or EBNA2 and EBNALP, EBNALP was associated with hairy and enhancer of split 1 (hes1), cd21, cd23, and arginine and glutamate-rich 1 (arglu1) enhancer or promoter DNA and was associated minimally with coding DNA. With the exception of RBPJ at the arglu1 enhancer, NCoR and RBPJ were significantly decreased at enhancer and promoter sites in EBNALP or EBNA2 and EBNALP BJAB cells. EBNA2 DNA association was unaffected by EBNALP, and EBNALP was unaffected by EBNA2. EBNA2 markedly increased RBPJ at enhancer sites without increasing NCoR. EBNALP further increased hes1 and arglu1 RNA levels with EBNA2 but did not further increase cd21 or cd23 RNA levels. EBNALP in which the 45 C-terminal residues critical for transformation and transcriptional activation were deleted associated with NCoR but was deficient in dismissing NCoR from MAD bodies and from enhancer and promoter sites. These data strongly support a model in which EBNA2 association with NCoR-deficient RBPJ enhances transcription and EBNALP dismisses NCoR and RBPJ repressive complexes from enhancers to coactivate hes1 and arglu1 but not cd21 or cd23.
Asunto(s)
Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Co-Represor 1 de Receptor Nuclear/metabolismo , Co-Represor 2 de Receptor Nuclear/metabolismo , Proteínas Virales/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Línea Celular , Transformación Celular Viral/genética , ADN/genética , ADN/metabolismo , Elementos de Facilitación Genéticos , Antígenos Nucleares del Virus de Epstein-Barr/genética , Regulación Viral de la Expresión Génica , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/patogenicidad , Proteínas de Homeodominio/genética , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Modelos Biológicos , Complejos Multiproteicos/metabolismo , Co-Represor 1 de Receptor Nuclear/genética , Co-Represor 2 de Receptor Nuclear/genética , Regiones Promotoras Genéticas , Receptores de Complemento 3d/genética , Receptores de IgE/genética , Factor de Transcripción HES-1 , Proteínas Virales/genéticaRESUMEN
EBV nuclear antigen 3C (EBNA3C) is an essential transcription factor for EBV transformed lymphoblast cell line (LCL) growth. To identify EBNA3C-regulated genes in LCLs, microarrays were used to measure RNA abundances in each of three different LCLs that conditionally express EBNA3C fused to a 4-OH-Tamoxifen-dependent estrogen receptor hormone binding domain (EBNA3CHT). At least three RNAs were assayed for each EBNA3CHT LCL under nonpermissive conditions, permissive conditions, and nonpermissive conditions with wild-type EBNA3C transcomplementation. Using a two-way ANOVA model of EBNA3C levels, we identified 550 regulated genes that were at least 1.5-fold up- or down-regulated with false discovery rates < 0.01. EBNA3C-regulated genes overlapped significantly with genes regulated by EBNA2 and EBNA3A consistent with coordinated effects on cell gene transcription. Of the 550 EBNA3C-regulated genes, 106 could be placed in protein networks. A seeded Bayesian network analysis of the 80 most significant EBNA3C-regulated genes suggests that RAC1, LYN, and TNF are upstream of other EBNA3C-regulated genes. Gene set enrichment analysis found enrichment for MAP kinase signaling, cytokine-cytokine receptor interactions, JAK-STAT signaling, and cell adhesion molecules, implicating these pathways in EBNA3C effects on LCL growth or survival. EBNA3C significantly up-regulated the CXCL12 ligand and its CXCR4 receptor and increased LCL migration. CXCL12 up-regulation depended on EBNA3C's interaction with the cell transcription factor, RBPJ, which is essential for LCL growth. EBNA3C also up-regulated MYC 1.3-fold and down-regulated CDKN2A exons 2 and 3, shared by p16 and p14, 1.4-fold, with false discovery rates < 5 × 10(-4).
Asunto(s)
Antígenos Virales/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Genes/genética , Factores de Transcripción/metabolismo , Análisis de Varianza , Teorema de Bayes , Línea Celular Tumoral , Análisis por Conglomerados , Antígenos Nucleares del Virus de Epstein-Barr , Perfilación de la Expresión Génica , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Activación de Linfocitos , Análisis por Micromatrices , Receptores de Estrógenos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Epstein-Barr virus nuclear antigen 2 (EBNA2) regulation of transcription through the cell transcription factor RBPJ is essential for resting B-lymphocyte (RBL) conversion to immortal lymphoblast cell lines (LCLs). ChIP-seq of EBNA2 and RBPJ sites in LCL DNA found EBNA2 at 5,151 and RBPJ at 10,529 sites. EBNA2 sites were enriched for RBPJ (78%), early B-cell factor (EBF, 39%), RUNX (43%), ETS (39%), NFκB (22%), and PU.1 (22%) motifs. These motif associations were confirmed by LCL RBPJ ChIP-seq finding 72% RBPJ occupancy and Encyclopedia Of DNA Elements LCL ChIP-seq finding EBF, NFκB RELA, and PU.1 at 54%, 31%, and 17% of EBNA2 sites. EBNA2 and RBPJ were predominantly at intergene and intron sites and only 14% at promoter sites. K-means clustering of EBNA2 site transcription factors identified RELA-ETS, EBF-RUNX, EBF, ETS, RBPJ, and repressive RUNX clusters, which ranked from highest to lowest in H3K4me1 signals and nucleosome depletion, indicative of active chromatin. Surprisingly, although quantitatively less, the same genome sites in RBLs exhibited similar high-level H3K4me1 signals and nucleosome depletion. The EBV genome also had an LMP1 promoter EBF site, which proved critical for EBNA2 activation. LCL HiC data mapped intergenic EBNA2 sites to EBNA2 up-regulated genes. FISH and chromatin conformation capture linked EBNA2/RBPJ enhancers 428 kb 5' of MYC to MYC. These data indicate that EBNA2 evolved to target RBL H3K4me1 modified, nucleosome-depleted, nonpromoter sites to drive B-lymphocyte proliferation in primary human infection. The primed RBL program likely supports antigen-induced proliferation.
Asunto(s)
Linfocitos B/metabolismo , Proliferación Celular , Infecciones por Virus de Epstein-Barr/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Elementos de Respuesta , Transcripción Genética , Proteínas Virales/metabolismo , Linfocitos B/virología , Línea Celular Tumoral , Subunidades alfa del Factor de Unión al Sitio Principal/genética , Subunidades alfa del Factor de Unión al Sitio Principal/metabolismo , Infecciones por Virus de Epstein-Barr/genética , Antígenos Nucleares del Virus de Epstein-Barr/genética , Genoma Viral/genética , Herpesvirus Humano 4/genética , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Nucleosomas/genética , Nucleosomas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Proto-Oncogénicas c-ets/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Proteínas Virales/genéticaRESUMEN
Notch1 regulates gene expression by associating with the DNA-binding factor RBPJ and is oncogenic in murine and human T-cell progenitors. Using ChIP-Seq, we find that in human and murine T-lymphoblastic leukemia (TLL) genomes Notch1 binds preferentially to promoters, to RBPJ binding sites, and near imputed ZNF143, ETS, and RUNX sites. ChIP-Seq confirmed that ZNF143 binds to â¼40% of Notch1 sites. Notch1/ZNF143 sites are characterized by high Notch1 and ZNF143 signals, frequent cobinding of RBPJ (generally through sites embedded within ZNF143 motifs), strong promoter bias, and relatively low mean levels of activating chromatin marks. RBPJ and ZNF143 binding to DNA is mutually exclusive in vitro, suggesting RBPJ/Notch1 and ZNF143 complexes exchange on these sites in cells. K-means clustering of Notch1 binding sites and associated motifs identified conserved Notch1-RUNX, Notch1-ETS, Notch1-RBPJ, Notch1-ZNF143, and Notch1-ZNF143-ETS clusters with different genomic distributions and levels of chromatin marks. Although Notch1 binds mainly to gene promoters, â¼75% of direct target genes lack promoter binding and are presumably regulated by enhancers, which were identified near MYC, DTX1, IGF1R, IL7R, and the GIMAP cluster. Human and murine TLL genomes also have many sites that bind only RBPJ. Murine RBPJ-only sites are highly enriched for imputed REST (a DNA-binding transcriptional repressor) sites, whereas human RPBJ-only sites lack REST motifs and are more highly enriched for imputed CREB sites. Thus, there is a conserved network of cis-regulatory factors that interacts with Notch1 to regulate gene expression in TLL cells, as well as unique classes of divergent RBPJ-only sites that also likely regulate transcription.