Targeting Epstein-Barr Virus super-enhancers

针对 Epstein-Barr 病毒超级增强剂

• 批准号: 9082368
• 负责人: ELLIOTT D KIEFF
• 金额: $ 44.38万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-01 至 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9082368
• 关键词: Affect; Affinity Chromatography; Antisense Oligonucleotides; B-Lymphocytes; BCL2 gene; Biological Assay; Bromodomain; Burkitt Lymphoma; Cell Line; Cells; ChIP-seq; Characteristics; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; Development; EBV-associated malignancy; Elements; Enhancers; Epstein-Barr Virus Nuclear Antigens; Epstein-Barr pathogenesis; Gene Expression; Genes; Genetic Transcription; Growth; HIV; Health; Histones; Hodgkin Disease; Human; Human Herpesvirus 4; Immune; In Vitro; Indium; Intervention; Lymphoma; Lymphoproliferative Disorders; Mass Spectrum Analysis; Membrane Proteins; Modeling; Molecular; NF-kappa B; Nasopharynx Carcinoma; Oncogenes; Oncogenic; Phenotype; Proliferating; Proteins; Proteomics; RNA; Reporter; Rest; Role; Running; Signal Transduction; Site; Stat5 protein; System; Therapeutic; Therapeutic Intervention; Viral Proteins; base; chromatin remodeling; crosslink; deep sequencing; global run on sequencing; inhibitor/antagonist; insight; knock-down; locked nucleic acid; lymphoblast; lymphoblastoid cell line; malignant stomach neoplasm; neoplastic cell; novel therapeutics; public health relevance; research study; small hairpin RNA; transcription factor; tumorigenesis

 DESCRIPTION (provided by applicant): Epstein-Barr Virus (EBV) causes lymphomas and lymphoproliferative diseases in HIV infected and immune suppressed people. These tumor cells frequently express Latency III EBV Nuclear Antigens (EBNAs) and Latent Membrane Proteins (LMP). In vitro, EBV converts Resting B Lymphocytes (RBLs) to continuously proliferating Lymphoblasts Cell Lines (LCLs) by expressing the same viral proteins. EBV conversion of RBLs to LCLs is therefore a relevant model that can be genetically manipulated to investigate EBV's role in growth transformation. LCL growth depends on EBV transcription factors (TFs) EBNA2, EBNALP, EBNA3A, EBNA3C and LMP1 activated NF-κB. Recently, we found that all the essential EBNAs and LMP1 activated NF-kB subunits converge to a small number of enhancers sites. Of these enhancers, 187 had markedly higher and broader histone H3K27ac signals, characteristic of super-enhancers, and were designated "EBV super-enhancers (ESE)." Super-enhancers (SE) govern cell transcription, development, phenotype, and oncogenesis. We found ESE- associated genes included the MYC and BCL2 oncogenes. ESEs were enriched for B cell TF motifs and had high STAT5A and NFAT co-occupancy. ESE associated genes were more highly expressed than other LCL genes. Disrupting ESEs by the bromodomain inhibitor JQ1 or conditionally inactivating an EBV oncoprotein or NF-kB decreased MYC or BCL2 expression and arrested LCL growth. To further characterize the ESEs, we will examine their (1) DNA elements, (2) protein compositions, and (3) associated enhancer RNAs (eRNAs). We will use reporter assays and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) to identify the DNA elements essential for ESE activity. We will use BioTAP-XL crosslinking, affinity purification followed by mass spectrometry to characterize the ESE proteomic components. We will also use Global Run On followed by deep sequencing (GRO-seq) to identify enhancer RNAs affected by EBV super-enhancers and useshort hairpin RNAs (shRNAs), short interferring RNAs (siRNAs) or Locked Nucleic Acid anti-sense oligonucleotides (LNAs) knock down to determine their significance. The experiments here in use integrative approaches to elucidate the molecular mechanism by which ESEs activate key oncogenic divers. These studies will identify opportunities for therapeutic intervention.

 描述(申请人提供)：爱泼斯坦-巴尔病毒(EBV)会导致艾滋病毒感染者和免疫抑制者的淋巴瘤和淋巴增殖性疾病。这些肿瘤细胞经常表达潜伏期III EBV核抗原(EBNAs)和潜伏膜蛋白(LMP)。在体外，EBV通过表达相同的病毒蛋白将静止的B淋巴细胞(RBL)转化为持续增殖的淋巴母细胞系(LCLS)。因此，从RBL到LCLS的EBV转换是一个相关的模型，可以通过基因操作来研究EBV在生长转化中的作用。LCL的生长依赖于EB病毒转录因子EBNA2、EBNAALP、EBNA3A、EBNA3C和LMP1激活的NF-κB。最近，我们发现所有必需的EBNAs和LMP1激活的NF-kB亚单位都集中在少量的增强子位点。在这些增强子中，187个具有明显更高和更广泛的组蛋白H3K27ac信号，这是超级增强子的特征，被命名为“EBV超级增强子(ESE)”。超级增强子(SE)调控细胞的转录、发育、表型和肿瘤发生。我们发现ESE相关基因包括MYC和BCL2癌基因。ESES富含B细胞TF基序，并具有较高的STAT5A和NFAT共占率。ESE相关基因的表达高于其他LCL基因。用溴域抑制剂JQ1破坏ESES，或有条件地灭活EBV癌蛋白或核因子-kB，会降低MYC或BCL2的表达，并阻止LCL的生长。为了进一步确定ESES的特征，我们将检查它们的(1)DNA元件，(2)蛋白质组成，和(3)相关增强子RNA(ERNAs)。我们将使用报告分析和聚集的规则间隔短回文重复序列(CRISPR)来鉴定ESE活性所必需的DNA元件。我们将使用BioTAP-XL交联法、亲和纯化和质谱法对ESE蛋白质组分进行表征。我们还将使用全局Run on和深度测序(Gro-seq)来识别受EBV超级增强子影响的增强子RNA，并使用短发夹状RNA(ShRNAs)、短干扰RNA(SiRNAs)或锁定核酸反义寡核苷酸(LNAs)敲除来确定它们的意义。这里的实验使用综合的方法来阐明ESES激活关键致癌潜水员的分子机制。这些研究将确定治疗干预的机会。