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1.
J Virol ; 94(24)2020 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-32999023

RESUMO

The Epstein-Barr virus (EBV) episome is known to interact with the three-dimensional structure of the human genome in infected cells. However, the exact locations of these interactions and their potential functional consequences remain unclear. Recently, high-resolution chromatin conformation capture (Hi-C) assays in lymphoblastoid cells have become available, enabling us to precisely map the contacts between the EBV episome(s) and the human host genome. Using available Hi-C data at a 10-kb resolution, we have identified 15,000 reproducible contacts between EBV episome(s) and the human genome. These contacts are highly enriched in chromatin regions denoted by typical or super enhancers and active markers, including histone H3K27ac and H3K4me1. Additionally, these contacts are highly enriched at loci bound by host transcription factors that regulate B cell growth (e.g., IKZF1 and RUNX3), factors that enhance cell proliferation (e.g., HDGF), or factors that promote viral replication (e.g., NBS1 and NFIC). EBV contacts show nearly 2-fold enrichment in host regions bound by EBV nuclear antigen 2 (EBNA2) and EBNA3 transcription factors. Circular chromosome conformation capture followed by sequencing (4C-seq) using the EBV origin of plasmid replication (oriP) as a "bait" in lymphoblastoid cells further confirmed contacts with active chromatin regions. Collectively, our analysis supports interactions between EBV episome(s) and active regions of the human genome in lymphoblastoid cells.IMPORTANCE EBV is associated with ∼200,000 cancers each year. In vitro, EBV can transform primary human B lymphocytes into immortalized cell lines. EBV-encoded proteins, along with noncoding RNAs and microRNAs, hijack cellular proteins and pathways to control cell growth. EBV nuclear proteins usurp normal transcriptional programs to activate the expression of key oncogenes, including MYC, to provide a proliferation signal. EBV nuclear antigens also repress CDKN2A to suppress senescence. EBV membrane protein activates NF-κB to provide survival signals. EBV genomes are maintained by EBNA1, which tethers EBV episomes to the host chromosomes during mitosis. However, little is known about where EBV episomes are located in interphase cells. In interphase cells, EBV promoters drive the expression of latency genes, while oriP functions as an enhancer for these promoters. In this study, integrative analyses of published lymphoblastoid cell line (LCL) Hi-C data and our 4C-seq experiments position EBV episomes to host genomes with active epigenetic marks. These contact points were significantly enriched for super enhancers. The close proximity of EBV episomes and the super enhancers that are enriched for transcription cofactors or mediators in lymphoblasts may benefit EBV gene expression, suggesting a novel mechanism of transcriptional activation.

2.
Genet Epidemiol ; 2020 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-32924180

RESUMO

Clinical trial results have recently demonstrated that inhibiting inflammation by targeting the interleukin-1ß pathway can offer a significant reduction in lung cancer incidence and mortality, highlighting a pressing and unmet need to understand the benefits of inflammation-focused lung cancer therapies at the genetic level. While numerous genome-wide association studies (GWAS) have explored the genetic etiology of lung cancer, there remains a large gap between the type of information that may be gleaned from an association study and the depth of understanding necessary to explain and drive translational findings. Thus, in this study we jointly model and integrate extensive multiomics data sources, utilizing a total of 40 genome-wide functional annotations that augment previously published results from the International Lung Cancer Consortium (ILCCO) GWAS, to prioritize and characterize single nucleotide polymorphisms (SNPs) that increase risk of squamous cell lung cancer through the inflammatory and immune responses. Our work bridges the gap between correlative analysis and translational follow-up research, refining GWAS association measures in an interpretable and systematic manner. In particular, reanalysis of the ILCCO data highlights the impact of highly associated SNPs from nuclear factor-κB signaling pathway genes as well as major histocompatibility complex mediated variation in immune responses. One consequence of prioritizing likely functional SNPs is the pruning of variants that might be selected for follow-up work by over an order of magnitude, from potentially tens of thousands to hundreds. The strategies we introduce provide informative and interpretable approaches for incorporating extensive genome-wide annotation data in analysis of genetic association studies.

3.
Nat Genet ; 52(9): 969-983, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32839606

RESUMO

Large-scale whole-genome sequencing studies have enabled the analysis of rare variants (RVs) associated with complex phenotypes. Commonly used RV association tests have limited scope to leverage variant functions. We propose STAAR (variant-set test for association using annotation information), a scalable and powerful RV association test method that effectively incorporates both variant categories and multiple complementary annotations using a dynamic weighting scheme. For the latter, we introduce 'annotation principal components', multidimensional summaries of in silico variant annotations. STAAR accounts for population structure and relatedness and is scalable for analyzing very large cohort and biobank whole-genome sequencing studies of continuous and dichotomous traits. We applied STAAR to identify RVs associated with four lipid traits in 12,316 discovery and 17,822 replication samples from the Trans-Omics for Precision Medicine Program. We discovered and replicated new RV associations, including disruptive missense RVs of NPC1L1 and an intergenic region near APOC1P1 associated with low-density lipoprotein cholesterol.


Assuntos
Predisposição Genética para Doença/genética , Variação Genética/genética , Genoma/genética , LDL-Colesterol/genética , Simulação por Computador , Estudo de Associação Genômica Ampla/métodos , Humanos , Modelos Genéticos , Anotação de Sequência Molecular/métodos , Fenótipo , Sequenciamento Completo do Genoma/métodos
4.
Am J Hum Genet ; 104(5): 802-814, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-30982610

RESUMO

Whole-genome sequencing (WGS) studies are being widely conducted in order to identify rare variants associated with human diseases and disease-related traits. Classical single-marker association analyses for rare variants have limited power, and variant-set-based analyses are commonly used by researchers for analyzing rare variants. However, existing variant-set-based approaches need to pre-specify genetic regions for analysis; hence, they are not directly applicable to WGS data because of the large number of intergenic and intron regions that consist of a massive number of non-coding variants. The commonly used sliding-window method requires the pre-specification of fixed window sizes, which are often unknown as a priori, are difficult to specify in practice, and are subject to limitations given that the sizes of genetic-association regions are likely to vary across the genome and phenotypes. We propose a computationally efficient and dynamic scan-statistic method (Scan the Genome [SCANG]) for analyzing WGS data; this method flexibly detects the sizes and the locations of rare-variant association regions without the need to specify a prior, fixed window size. The proposed method controls for the genome-wise type I error rate and accounts for the linkage disequilibrium among genetic variants. It allows the detected sizes of rare-variant association regions to vary across the genome. Through extensive simulated studies that consider a wide variety of scenarios, we show that SCANG substantially outperforms several alternative methods for detecting rare-variant-associations while controlling for the genome-wise type I error rates. We illustrate SCANG by analyzing the WGS lipids data from the Atherosclerosis Risk in Communities (ARIC) study.


Assuntos
Algoritmos , Biologia Computacional/métodos , Variação Genética , Genoma Humano , Estudo de Associação Genômica Ampla , Sequenciamento Completo do Genoma/métodos , Humanos , Desequilíbrio de Ligação , Modelos Genéticos
5.
Artigo em Inglês | MEDLINE | ID: mdl-30258822

RESUMO

In this study, animal experimentation verified that the canonical Wnt/ß-catenin signaling pathway was activated under a reduced activity of p-ß-catenin (Ser33/37/Thr41) and an increased accumulation of ß-catenin in the lungs and kidneys of pigs infected with a highly virulent strain of H. parasuis. In PK-15 and NPTr cells, it was also confirmed that infection with a high-virulence strain of H. parasuis induced cytoplasmic accumulation and nuclear translocation of ß-catenin. H. parasuis infection caused a sharp degradation of E-cadherin and an increase of the epithelial cell monolayer permeability, as well as a broken interaction between ß-catenin and E-cadherin dependent on Wnt/ß-catenin signaling pathway. Moreover, Wnt/ß-catenin signaling pathway also contributed to the initiation of epithelial-mesenchymal transition (EMT) during high-virulence strain of H. parasuis infection with expression changes of epithelial/mesenchymal markers, increased migratory capabilities as well as the morphologically spindle-like switch in PK-15 and NPTr cells. Therefore, we originally speculated that H. parasuis infection activates the canonical Wnt/ß-catenin signaling pathway leading to a disruption of the epithelial barrier, altering cell structure and increasing cell migration, which results in severe acute systemic infection characterized by fibrinous polyserositis during H. parasuis infection.


Assuntos
Junções Aderentes/patologia , Infecções por Haemophilus/patologia , Haemophilus parasuis/crescimento & desenvolvimento , Transdução de Sinais , Doenças dos Suínos/patologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Linhagem Celular , Células Epiteliais/patologia , Interações Hospedeiro-Patógeno , Rim/patologia , Pulmão/patologia , Suínos
6.
J Virol ; 92(9)2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29467311

RESUMO

Epstein-Barr virus nuclear antigen (EBNA) leader protein (EBNALP) is one of the first viral genes expressed upon B-cell infection. EBNALP is essential for EBV-mediated B-cell immortalization. EBNALP is thought to function primarily by coactivating EBNA2-mediated transcription. Chromatin immune precipitation followed by deep sequencing (ChIP-seq) studies highlight that EBNALP frequently cooccupies DNA sites with host cell transcription factors (TFs), in particular, EP300, implicating a broader role in transcription regulation. In this study, we investigated the mechanisms of EBNALP transcription coactivation through EP300. EBNALP greatly enhanced EP300 transcription activation when EP300 was tethered to a promoter. EBNALP coimmunoprecipitated endogenous EP300 from lymphoblastoid cell lines (LCLs). EBNALP W repeat serine residues 34, 36, and 63 were required for EP300 association and coactivation. Deletion of the EP300 histone acetyltransferase (HAT) domain greatly reduced EBNALP coactivation and abolished the EBNALP association. An EP300 bromodomain inhibitor also abolished EBNALP coactivation and blocked the EP300 association with EBNALP. EBNALP sites cooccupied by EP300 had significantly higher ChIP-seq signals for sequence-specific TFs, including SPI1, RelA, EBF1, IRF4, BATF, and PAX5. EBNALP- and EP300-cooccurring sites also had much higher H3K4me1 and H3K27ac signals, indicative of activated enhancers. EBNALP-only sites had much higher signals for DNA looping factors, including CTCF and RAD21. EBNALP coactivated reporters under the control of NF-κB or SPI1. EP300 inhibition abolished EBNALP coactivation of these reporters. Clustered regularly interspaced short palindromic repeat interference targeting of EBNALP enhancer sites significantly reduced target gene expression, including that of EP300 itself. These data suggest a previously unrecognized mechanism by which EBNALP coactivates transcription through subverting of EP300 and thus affects the expression of LCL genes regulated by a broad range of host TFs.IMPORTANCE Epstein-Barr virus was the first human DNA tumor virus discovered over 50 years ago. EBV is causally linked to ∼200,000 human malignancies annually. These cancers include endemic Burkitt lymphoma, Hodgkin lymphoma, lymphoma/lymphoproliferative disease in transplant recipients or HIV-infected people, nasopharyngeal carcinoma, and ∼10% of gastric carcinoma cases. EBV-immortalized human B cells faithfully model key aspects of EBV lymphoproliferative diseases and are useful models of EBV oncogenesis. EBNALP is essential for EBV to transform B cells and transcriptionally coactivates EBNA2 by removing repressors from EBNA2-bound DNA sites. Here, we found that EBNALP can also modulate the activity of the key transcription activator EP300, an acetyltransferase that activates a broad range of transcription factors. Our data suggest that EBNALP regulates a much broader range of host genes than was previously appreciated. A small-molecule inhibitor of EP300 abolished EBNALP coactivation of multiple target genes. These findings suggest novel therapeutic approaches to control EBV-associated lymphoproliferative diseases.


Assuntos
Proteína p300 Associada a E1A/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Proteínas Virais/metabolismo , Linfócitos B/virologia , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Proteína p300 Associada a E1A/antagonistas & inibidores , Proteína p300 Associada a E1A/genética , Antígenos Nucleares do Vírus Epstein-Barr/genética , Células HEK293 , Herpesvirus Humano 4/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Regiões Promotoras Genéticas/genética , Ativação Transcricional/genética , Proteínas Virais/genética
7.
Dev Comp Immunol ; 79: 158-165, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29097236

RESUMO

Haemophilus parasuis, an important swine pathogen, was recently proven able to invade into endothelial or epithelial cell in vitro. NOD1/2 are specialized NLRs that participate in the recognition of pathogens able to invade intracellularly and therefore, we assessed that the contribution of NOD1/2 to inflammation responses during H. parasuis infection. We observed that H. parasuis infection enhanced NOD2 expression and RIP2 phosphorylation in porcine kidney 15 cells. Our results also showed that knock down of NOD1/2 or RIP2 expression respectively significantly decreased H. parasuis-induced NF-κB activity, while the phosphorylation level of p38, JNK or ERK was not changed. Moreover, real-time PCR result showed that NOD1, NOD2 or RIP2 was involved in the expression of CCL4, CCL5 and IL-8. Inhibition of NOD1 and NOD2 significantly reduced CCL5 promoter activity, even in a more effective way compared with inhibition of TLR.


Assuntos
Células Endoteliais/imunologia , Células Epiteliais/imunologia , Infecções por Haemophilus/imunologia , Haemophilus parasuis/imunologia , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Suínos/imunologia , Animais , Linhagem Celular , Quimiocina CCL4/metabolismo , Quimiocina CCL5/metabolismo , Células Endoteliais/microbiologia , Células Epiteliais/microbiologia , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD2/genética , RNA Interferente Pequeno/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética , Transdução de Sinais
8.
Cell Host Microbe ; 22(4): 561-573.e4, 2017 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-29024646

RESUMO

Epstein-Barr virus (EBV) transforms B cells to continuously proliferating lymphoblastoid cell lines (LCLs), which represent an experimental model for EBV-associated cancers. EBV nuclear antigens (EBNAs) and LMP1 are EBV transcriptional regulators that are essential for LCL establishment, proliferation, and survival. Starting with the 3D genome organization map of LCL, we constructed a comprehensive EBV regulome encompassing 1,992 viral/cellular genes and enhancers. Approximately 30% of genes essential for LCL growth were linked to EBV enhancers. Deleting EBNA2 sites significantly reduced their target gene expression. Additional EBV super-enhancer (ESE) targets included MCL1, IRF4, and EBF. MYC ESE looping to the transcriptional stat site of MYC was dependent on EBNAs. Deleting MYC ESEs greatly reduced MYC expression and LCL growth. EBNA3A/3C altered CDKN2A/B spatial organization to suppress senescence. EZH2 inhibition decreased the looping at the CDKN2A/B loci and reduced LCL growth. This study provides a comprehensive view of the spatial organization of chromatin during EBV-driven cellular transformation.


Assuntos
Linfócitos B/virologia , Cromatina/virologia , Herpesvirus Humano 4/genética , Interações Hospedeiro-Patógeno , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor de Quinase Dependente de Ciclina p15/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p18/genética , Inibidor de Quinase Dependente de Ciclina p18/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Humanos , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transativadores/genética , Transativadores/metabolismo , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo
9.
Proc Natl Acad Sci U S A ; 114(36): 9683-9688, 2017 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-28831010

RESUMO

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.


Assuntos
Carcinoma/genética , Elementos Facilitadores Genéticos , Neoplasias Nasofaríngeas/genética , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Repressoras/genética , Adolescente , Adulto , Idoso , Animais , Azepinas/farmacologia , Carcinoma/etiologia , Carcinoma/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Elementos Facilitadores Genéticos/efeitos dos fármacos , Epigênese Genética , Infecções por Vírus Epstein-Barr/complicações , Feminino , Genoma Viral , Xenoenxertos , Sequenciamento de Nucleotídeos em Larga Escala , Código das Histonas/genética , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/etiologia , Neoplasias Nasofaríngeas/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Prognóstico , Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Triazóis/farmacologia , Adulto Jovem
10.
J Clin Invest ; 127(6): 2066-2080, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28504647

RESUMO

Primary effusion lymphoma (PEL) is a largely incurable malignancy of B cell origin with plasmacytic differentiation. Here, we report the identification of a highly effective inhibitor of PEL. This compound, 6-ethylthioinosine (6-ETI), is a nucleoside analog with toxicity to PEL in vitro and in vivo, but not to other lymphoma cell lines tested. We developed and performed resistome analysis, an unbiased approach based on RNA sequencing of resistant subclones, to discover the molecular mechanisms of sensitivity. We found different adenosine kinase-inactivating (ADK-inactivating) alterations in all resistant clones and determined that ADK is required to phosphorylate and activate 6-ETI. Further, we observed that 6-ETI induces ATP depletion and cell death accompanied by S phase arrest and DNA damage only in ADK-expressing cells. Immunohistochemistry for ADK served as a biomarker approach to identify 6-ETI-sensitive tumors, which we documented for other lymphoid malignancies with plasmacytic features. Notably, multiple myeloma (MM) expresses high levels of ADK, and 6-ETI was toxic to MM cell lines and primary specimens and had a robust antitumor effect in a disseminated MM mouse model. Several nucleoside analogs are effective in treating leukemias and T cell lymphomas, and 6-ETI may fill this niche for the treatment of PEL, plasmablastic lymphoma, MM, and other ADK-expressing cancers.


Assuntos
Adenosina Quinase/metabolismo , Antineoplásicos/farmacologia , Linfoma de Efusão Primária/tratamento farmacológico , Nucleosídeos de Purina/farmacologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Humanos , Concentração Inibidora 50 , Linfoma de Efusão Primária/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Proc Natl Acad Sci U S A ; 114(18): 4751-4756, 2017 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-28351978

RESUMO

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.


Assuntos
Regulação Neoplásica da Expressão Gênica/imunologia , Regulação Viral da Expressão Gênica/imunologia , Herpesvirus Humano 4/imunologia , Doença de Hodgkin/imunologia , Linfoma de Células B/imunologia , Neoplasias Experimentais/imunologia , Proteínas da Matriz Viral/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/patologia , Centro Germinativo/imunologia , Centro Germinativo/patologia , Herpesvirus Humano 4/genética , Doença de Hodgkin/genética , Doença de Hodgkin/patologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Linfoma de Células B/genética , Linfoma de Células B/patologia , Camundongos , Camundongos Mutantes , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Linfócitos T/imunologia , Linfócitos T/patologia , Proteínas da Matriz Viral/genética
12.
Proc Natl Acad Sci U S A ; 113(49): 14121-14126, 2016 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-27864512

RESUMO

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.


Assuntos
Elementos Facilitadores Genéticos , Herpesvirus Humano 4/fisiologia , Interações Hospedeiro-Patógeno , Transtornos Linfoproliferativos/virologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular , Endonucleases , Humanos , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo
13.
Chest ; 148(6): 1470-1476, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26270005

RESUMO

BACKGROUND: Indwelling arterial catheters (IACs) are used extensively in the ICU for hemodynamic monitoring and for blood gas analysis. IAC use also poses potentially serious risks, including bloodstream infections and vascular complications. The purpose of this study was to assess whether IAC use was associated with mortality in patients who are mechanically ventilated and do not require vasopressor support. METHODS: This study used the Multiparameter Intelligent Monitoring in Intensive Care II database, consisting of > 24,000 patients admitted to the Beth Israel Deaconess Medical Center ICU between 2001 and 2008. Patients requiring mechanical ventilation who did not require vasopressors or have a diagnosis of sepsis were identified, and the primary outcome was 28-day mortality. A model based on patient demographics, comorbidities, vital signs, and laboratory results was developed to estimate the propensity for IAC placement. Patients were then propensity matched, and McNemar test was used to evaluate the association of IAC with 28-day mortality. RESULTS: We identified 1,776 patients who were mechanically ventilated who met inclusion criteria. There were no differences in the covariates included in the final propensity model between the IAC and non-IAC propensity-matched groups. For the matched cohort, there was no difference in 28-day mortality between the IAC group and the non-IAC group (14.7% vs 15.2%; OR, 0.96; 95% CI, 0.62-1.47). CONCLUSIONS: In hemodynamically stable patients who are mechanically ventilated, the presence of an IAC is not associated with a difference in 28-day mortality. Validation in other datasets, as well as further analyses in other subgroups, is warranted.


Assuntos
Cateterismo Periférico , Monitorização Fisiológica/métodos , Insuficiência Respiratória , Adulto , Idoso , Gasometria/métodos , Cateterismo Periférico/efeitos adversos , Cateterismo Periférico/métodos , Cateterismo Periférico/mortalidade , Cateteres de Demora/efeitos adversos , Feminino , Hemodinâmica , Mortalidade Hospitalar , Humanos , Unidades de Terapia Intensiva/estatística & dados numéricos , Tempo de Internação/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Avaliação de Resultados em Cuidados de Saúde , Pontuação de Propensão , Respiração Artificial/métodos , Insuficiência Respiratória/sangue , Insuficiência Respiratória/diagnóstico , Insuficiência Respiratória/fisiopatologia , Insuficiência Respiratória/terapia , Medição de Risco , Estados Unidos
14.
Proc Natl Acad Sci U S A ; 112(37): 11612-7, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26305967

RESUMO

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.


Assuntos
Centro Germinativo/metabolismo , Herpesvirus Humano 4/metabolismo , Proteínas da Matriz Viral/metabolismo , Animais , Autoanticorpos/química , Doenças Autoimunes/metabolismo , Doenças Autoimunes/virologia , Diferenciação Celular , Linhagem da Célula , Cruzamentos Genéticos , Epigênese Genética , Citometria de Fluxo , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Heterozigoto , Imunidade Humoral , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Baço/citologia , Dedos de Zinco
15.
PLoS Pathog ; 11(5): e1004890, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25996949

RESUMO

The Epstein-Barr virus (EBV) encoded oncoprotein Latent Membrane Protein 1 (LMP1) signals through two C-terminal tail domains to drive cell growth, survival and transformation. The LMP1 membrane-proximal TES1/CTAR1 domain recruits TRAFs to activate MAP kinase, non-canonical and canonical NF-kB pathways, and is critical for EBV-mediated B-cell transformation. TRAF1 is amongst the most highly TES1-induced target genes and is abundantly expressed in EBV-associated lymphoproliferative disorders. We found that TRAF1 expression enhanced LMP1 TES1 domain-mediated activation of the p38, JNK, ERK and canonical NF-kB pathways, but not non-canonical NF-kB pathway activity. To gain insights into how TRAF1 amplifies LMP1 TES1 MAP kinase and canonical NF-kB pathways, we performed proteomic analysis of TRAF1 complexes immuno-purified from cells uninduced or induced for LMP1 TES1 signaling. Unexpectedly, we found that LMP1 TES1 domain signaling induced an association between TRAF1 and the linear ubiquitin chain assembly complex (LUBAC), and stimulated linear (M1)-linked polyubiquitin chain attachment to TRAF1 complexes. LMP1 or TRAF1 complexes isolated from EBV-transformed lymphoblastoid B cell lines (LCLs) were highly modified by M1-linked polyubiqutin chains. The M1-ubiquitin binding proteins IKK-gamma/NEMO, A20 and ABIN1 each associate with TRAF1 in cells that express LMP1. TRAF2, but not the cIAP1 or cIAP2 ubiquitin ligases, plays a key role in LUBAC recruitment and M1-chain attachment to TRAF1 complexes, implicating the TRAF1:TRAF2 heterotrimer in LMP1 TES1-dependent LUBAC activation. Depletion of either TRAF1, or the LUBAC ubiquitin E3 ligase subunit HOIP, markedly impaired LCL growth. Likewise, LMP1 or TRAF1 complexes purified from LCLs were decorated by lysine 63 (K63)-linked polyubiqutin chains. LMP1 TES1 signaling induced K63-polyubiquitin chain attachment to TRAF1 complexes, and TRAF2 was identified as K63-Ub chain target. Co-localization of M1- and K63-linked polyubiquitin chains on LMP1 complexes may facilitate downstream canonical NF-kB pathway activation. Our results highlight LUBAC as a novel potential therapeutic target in EBV-associated lymphoproliferative disorders.


Assuntos
Linfócitos B/metabolismo , Transformação Celular Viral , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/metabolismo , Fator 1 Associado a Receptor de TNF/metabolismo , Ubiquitinação , Proteínas da Matriz Viral/metabolismo , Linfócitos B/imunologia , Linfócitos B/virologia , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Células HEK293 , Herpesvirus Humano 4/imunologia , Humanos , Lisina/metabolismo , Mutação , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Fator 1 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/antagonistas & inibidores , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Complexos Ubiquitina-Proteína Ligase/antagonistas & inibidores , Complexos Ubiquitina-Proteína Ligase/genética , Complexos Ubiquitina-Proteína Ligase/metabolismo , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética
16.
Cell Host Microbe ; 17(2): 205-16, 2015 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-25639793

RESUMO

Super-enhancers are clusters of gene-regulatory sites bound by multiple transcription factors that govern cell transcription, development, phenotype, and oncogenesis. By examining Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs), we identified four EBV oncoproteins and five EBV-activated NF-κB subunits co-occupying ∼1,800 enhancer sites. Of these, 187 had markedly higher and broader histone H3K27ac signals, characteristic of super-enhancers, and were designated "EBV super-enhancers." EBV super-enhancer-associated genes included the MYC and BCL2 oncogenes, which enable LCL proliferation and survival. EBV super-enhancers were enriched for B cell transcription factor motifs and had high co-occupancy of STAT5 and NFAT transcription factors (TFs). EBV super-enhancer-associated genes were more highly expressed than other LCL genes. Disrupting EBV super-enhancers by the bromodomain inhibitor JQ1 or conditionally inactivating an EBV oncoprotein or NF-κB decreased MYC or BCL2 expression and arrested LCL growth. These findings provide insight into mechanisms of EBV-induced lymphoproliferation and identify potential therapeutic interventions.


Assuntos
Linfócitos B/virologia , Proliferação de Células , Regulação da Expressão Gênica , Herpesvirus Humano 4/fisiologia , Interações Hospedeiro-Patógeno , Proteínas Oncogênicas/metabolismo , Proteínas Virais/metabolismo , Linfócitos B/fisiologia , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Análise de Sequência de DNA , Transcrição Genética
17.
Proc Natl Acad Sci U S A ; 112(2): 554-9, 2015 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-25540416

RESUMO

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.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , DNA/genética , DNA/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/genética , Genoma Viral , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/patogenicidade , Linfócitos B/metabolismo , Linfócitos B/virologia , Sítios de Ligação/genética , Linhagem Celular , Quimiocina CXCL10/genética , Quimiocina CXCL9/genética , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Ciclina D2/genética , Elementos Facilitadores Genéticos , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Genes bcl-2 , Genes myc , Genes p16 , Genoma Humano , Herpesvirus Humano 4/fisiologia , Interações Hospedeiro-Patógeno/genética , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Fatores Reguladores de Interferon/metabolismo , Regiões Promotoras Genéticas , Proteínas Virais/genética , Proteínas Virais/metabolismo
18.
Cell Rep ; 8(5): 1595-606, 2014 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-25159142

RESUMO

The nuclear factor κB (NF-κΒ) subunits RelA, RelB, cRel, p50, and p52 are each critical for B cell development and function. To systematically characterize their responses to canonical and noncanonical NF-κB pathway activity, we performed chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) analysis in lymphoblastoid B cell lines (LCLs). We found a complex NF-κB-binding landscape, which did not readily reflect the two NF-κB pathway paradigms. Instead, 10 subunit-binding patterns were observed at promoters and 11 at enhancers. Nearly one-third of NF-κB-binding sites lacked κB motifs and were instead enriched for alternative motifs. The oncogenic forkhead box protein FOXM1 co-occupied nearly half of NF-κB-binding sites and was identified in protein complexes with NF-κB on DNA. FOXM1 knockdown decreased NF-κB target gene expression and ultimately induced apoptosis, highlighting FOXM1 as a synthetic lethal target in B cell malignancy. These studies provide a resource for understanding mechanisms that underlie NF-κB nuclear activity and highlight opportunities for selective NF-κB blockade.


Assuntos
Linfócitos B/metabolismo , Elementos Facilitadores Genéticos , Redes Reguladoras de Genes , Genoma Humano , NF-kappa B/metabolismo , Linhagem Celular Tumoral , Proteína Forkhead Box M1 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , NF-kappa B/genética , Regiões Promotoras Genéticas , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ativação Transcricional
19.
PLoS One ; 9(6): e99857, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24914816

RESUMO

BACKGROUND: Epstein-Barr Virus (EBV) is a globally prevalent herpesvirus associated with infectious mononucleosis and many malignancies. The survey on EBV prevalence appears to be important to study EBV-related diseases and determine when to administer prophylactic vaccine. The purpose of this retrospective study was to collect baseline information about the prevalence of EBV infection in Chinese children. METHODOLOGY/PRINCIPAL FINDING: We collected 1778 serum samples from healthy children aged 0 to 10, who were enrolled in conventional health and nutrition examinations without any EBV-related symptom in 2012 and 2013 in North China (n = 973) and South China (n = 805). We detected four EBV-specific antibodies, i.e., anti-VCA-IgG and IgM, anti-EBNA-IgG and anti-EA-IgG, by ELISA, representing all of the phases of EBV infection. The overall EBV seroprevalence in samples from North and South China were 80.78% and 79.38% respectively. The EBV seropositivity rates dropped slightly at age 2, and then increased gradually with age. The seroprevalence became stabilized at over 90% after age 8. In this study, the seroprevalence trends between North and South China showed no difference (P>0.05), and the trends of average antibody concentrations were similar as well (P>0.05). CONCLUSIONS/SIGNIFICANCE: EBV seroprevalence became more than 50% before age 3 in Chinese children, and exceed 90% after age 8. This study can be helpful to study the relationship between EBV and EBV-associated diseases, and supportive to EBV vaccine development and implementation.


Assuntos
Grupo com Ancestrais do Continente Asiático/estatística & dados numéricos , Infecções por Vírus Epstein-Barr/epidemiologia , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/fisiologia , Especificidade de Anticorpos/imunologia , Criança , Pré-Escolar , China/epidemiologia , Demografia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Prevalência , Estudos Retrospectivos , Estudos Soroepidemiológicos
20.
Biol Direct ; 9: 5, 2014 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-24708540

RESUMO

BACKGROUND: H. sapiens-M. tuberculosis H37Rv protein-protein interaction (PPI) data are essential for understanding the infection mechanism of the formidable pathogen M. tuberculosis H37Rv. Computational prediction is an important strategy to fill the gap in experimental H. sapiens-M. tuberculosis H37Rv PPI data. Homology-based prediction is frequently used in predicting both intra-species and inter-species PPIs. However, some limitations are not properly resolved in several published works that predict eukaryote-prokaryote inter-species PPIs using intra-species template PPIs. RESULTS: We develop a stringent homology-based prediction approach by taking into account (i) differences between eukaryotic and prokaryotic proteins and (ii) differences between inter-species and intra-species PPI interfaces. We compare our stringent homology-based approach to a conventional homology-based approach for predicting host-pathogen PPIs, based on cellular compartment distribution analysis, disease gene list enrichment analysis, pathway enrichment analysis and functional category enrichment analysis. These analyses support the validity of our prediction result, and clearly show that our approach has better performance in predicting H. sapiens-M. tuberculosis H37Rv PPIs. Using our stringent homology-based approach, we have predicted a set of highly plausible H. sapiens-M. tuberculosis H37Rv PPIs which might be useful for many of related studies. Based on our analysis of the H. sapiens-M. tuberculosis H37Rv PPI network predicted by our stringent homology-based approach, we have discovered several interesting properties which are reported here for the first time. We find that both host proteins and pathogen proteins involved in the host-pathogen PPIs tend to be hubs in their own intra-species PPI network. Also, both host and pathogen proteins involved in host-pathogen PPIs tend to have longer primary sequence, tend to have more domains, tend to be more hydrophilic, etc. And the protein domains from both host and pathogen proteins involved in host-pathogen PPIs tend to have lower charge, and tend to be more hydrophilic. CONCLUSIONS: Our stringent homology-based prediction approach provides a better strategy in predicting PPIs between eukaryotic hosts and prokaryotic pathogens than a conventional homology-based approach. The properties we have observed from the predicted H. sapiens-M. tuberculosis H37Rv PPI network are useful for understanding inter-species host-pathogen PPI networks and provide novel insights for host-pathogen interaction studies.


Assuntos
Proteínas de Bactérias/genética , Hidrolases/genética , Mycobacterium tuberculosis/genética , Mapeamento de Interação de Proteínas/métodos , Proteínas de Bactérias/metabolismo , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Hidrolases/metabolismo , Mycobacterium tuberculosis/metabolismo , Tuberculose/metabolismo , Tuberculose/microbiologia
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