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（由申请人提供）：EB 病毒 (EBV) 在 HIV 感染和免疫抑制的人群中引起淋巴瘤和淋巴增殖性疾病。这些肿瘤细胞在体外经常表达潜伏 III EBV 核抗原 (EBNA) 和潜伏膜蛋白 (LMP)。 , EBV 通过表达以下物质将静息 B 淋巴细胞 (RBL) 转化为持续增殖的淋巴细胞系 (LCL)因此，EBV 将 RBL 转化为 LCL 是一个相关模型，可通过基因操作来研究 EBV 在生长转化中的作用，该模型取决于 EBV 转录因子 (TF) EBNA2、EBNALP、EBNA3A、EBNA3C 和 LMP1 激活的 NF-。最近，我们发现所有必需的 EBNA 和 LMP1 激活的 NF-kB 亚基都聚集到少数增强子上。在这些增强子中，187 个具有明显更高和更宽的组蛋白 H3K27ac 信号，这是超级增强子的特征，被指定为“超级增强子 (SE)”，控制细胞转录、发育、表型和表达。我们发现 ESE 相关基因（包括 MYC 和 BCL2）富含 B 细胞 TF 基序，并且具有较高的 STAT5A 和 STAT5A 水平。 NFAT 共占用基因比其他 LCL 基因表达更高。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 (eRNA)。我们将使用 BioTAP-XL 交联、亲和纯化和质谱来表征 ESE 蛋白质组成分。深度测序 (GRO-seq) 识别受 EBV 超级增强子影响的增强子 RNA，并使用短发夹 RNA (shRNA)、短干扰 RNA (siRNA) 或锁定核酸反义寡核苷酸 (LNA) 敲低以确定其重要性。这里的实验使用综合方法来阐明 ESE 激活关键致癌因素的分子机制。这些研究将确定治疗干预的机会。