MCV ST activates specific gene transcription by redirecting the activity of the Tip60/p400 complex.

MCV ST 通过重定向 Tip60/p400 复合物的活性来激活特定基因转录。

• 批准号: 9896661
• 负责人: Camille Cushman
• 金额: $ 3.87万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9896661
• 关键词: Acetylation; Acetyltransferase; Address; Antigen Targeting; Binding; Binding Sites; Cells; ChIP-seq; Chromatin; Complex; DNA Tumor Viruses; Data; Deposition; Development; Disease; Epigenetic Process; Gene Activation; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Histones; Human; In Vitro; Incidence; Large T Antigen; Lysine; Malignant Epithelial Cell; Measures; Mediating; Merkel Cells; Merkel cell carcinoma; Methods; Nucleosomes; Oncogenic; Polyomavirus; Polyomavirus Transforming Antigens; Proteins; Reporting; Role; Site; Skin Cancer; Small T Antigen; System; TP53 gene; Transcription Coactivator; Transgenic Mice; Tumor Antigens; Variant; Viral Proteins; Virus; Work; antigen binding; cell transformation; combat; histone acetyltransferase; improved; insight; melanoma; metaplastic cell transformation; mortality; mouse model; mutant; programs; promoter; recruit; targeted treatment; transcriptome sequencing; tumor

Project Abstract: Merkel cell polyomavirus (MCV) is a small DNA tumor virus that causes approximately 80% of Merkel cell carcinoma (MCC), a highly aggressive skin cancer. Since the discovery of MCV in 2008, it has been shown that the MCV tumor antigens small T antigen (ST) and large T antigen (LT) are the only viral proteins consistently expressed in virus-positive MCC cells. Expression of ST alone in Rat1 cells was sufficient to induce cellular transformation suggesting that ST is the primary driver of cellular transformation in MCC development. Our lab determined that ST forms a specific complex with L-Myc/MAX and the Tip60/p400 complex (SLT complex) and that a ST mutant incapable of binding to L-Myc and the Tip60/p400 complex cannot transform cells. The Tip60/p400 complex is a large multi-subunit complex that has lysine acetyltransferase activity (Tip60 protein) and nucleosome exchange activity (p400 protein). Both of these activities have been correlated with active gene transcription but further work is necessary to fully understand the relationship between Tip60/p400 complex activity and gene expression. We hypothesize that MCV ST redirects the Tip60/p400 complex to L-Myc binding sites to activate a transcriptional program requiring their lysine acetyltransferase and histone remodeling activity to drive cellular transformation. In Aim 1, we will assess the effect of ST on Tip60/p400 complex binding and we will compare the direct targets of the Tip60/p400 complex in control cells with cells expressing ST to determine if ST redirects the complex from “normal” Tip60/p400 complex binding sites to SLT target gene promoters to activate SLT target gene transcription. By comparing the list of genes that are direct targets of the SLT complex in normal human cells to those we previously identified in virus-positive MCC cells, we will generate a list of genes that are consistently regulated by ST. In Aim 2, we will assess the contribution of Tip60 histone acetyltransferase (HAT) and p400 nucleosome remodeling activities to ST-mediated gene expression changes and cellular transformation. In Aim 3, we will adapt the Fkbp/Frb inducible recruitment for epigenetic editing by Cas9 (FIRE- Cas9) method to the Tip60/p400 complex and then use this system to recruit the Tip60/p400 complex to SLT target genes in the absence of ST. Upon recruitment, we will measure changes to ST target gene transcription and the level of Tip60/p400-specific histone marks at SLT target gene promoters. Insights gained from investigating the ST interaction with L-Myc and the Tip60/p400 complex will provide a deeper understanding of the oncogenic roles of these factors and understanding the activities of the SLT complex will lead to identification of improved targeted therapies to combat MCC.

项目摘要： 默克尔细胞多瘤病毒（MCV）是一种小DNA肿瘤病毒，其引起约80%的默克尔细胞 癌（MCC），一种高度侵袭性的皮肤癌。自2008年发现MCV以来， MCV肿瘤抗原小T抗原（ST）和大T抗原（LT）是仅有的病毒蛋白， 在病毒阳性MCC细胞中一致表达。在Rat 1细胞中单独表达ST足以 诱导细胞转化，表明ST是MCC中细胞转化主要驱动力 发展我们的实验室确定ST与L-Myc/MAX和Tip 60/p400形成特异性复合物 ST突变体不能结合L-Myc和Tip 60/p400复合物， 无法转换细胞。Tip 60/p400复合物是一种大的多亚基复合物，具有赖氨酸 乙酰转移酶活性（Tip 60蛋白）和核小体交换活性（p400蛋白）。这两 活性与活跃的基因转录相关，但进一步的工作是必要的，以充分了解 Tip 60/p400复合物活性与基因表达的关系。我们假设MCV ST 将Tip 60/p400复合物重定向到L-Myc结合位点，以激活转录程序， 它们的赖氨酸乙酰转移酶和组蛋白重塑活性来驱动细胞转化。在目标1中， 我们将评估ST对Tip 60/p400复合物结合的影响，并将比较ST的直接靶点。 用表达ST的细胞在对照细胞中检测Tip 60/p400复合物，以确定ST是否将复合物从 “正常”Tip 60/p400复合物结合位点与靶基因启动子的结合以激活靶基因 转录。通过比较正常人类细胞中作为cDNA 3复合物直接靶点的基因列表， 与我们以前在病毒阳性MCC细胞中鉴定的那些基因相比，我们将生成一个基因列表， 在目标2中，我们将评估Tip 60组蛋白乙酰转移酶（HAT）的贡献。 和p400核小体重塑活动对ST介导的基因表达变化和细胞 转型在目标3中，我们将调整Fkbp/Frb诱导型募集用于通过Cas9（FIRE-Cas9）进行表观遗传编辑。 Cas9）方法，然后使用该系统将Tip 60/p400复合物募集到细胞中， 招募后，我们将测量ST靶基因转录的变化， 和Tip 60/p400特异性组蛋白标记在靶基因启动子处的水平。从以下方面获得的见解： 研究ST与L-Myc和Tip 60/p400复合物的相互作用将提供更深入的研究。 了解这些因素的致癌作用，了解肿瘤细胞的活动， 复杂的将导致确定改进的靶向治疗，以打击MCC。