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: 9753448
• 负责人: Camille Cushman
• 金额: $ 3.82万
• 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/9753448
• 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： Merkel细胞多病毒（MCV）是一种小的DNA肿瘤病毒，可引起约80％的默克尔细胞 癌（MCC），一种高度侵略性的皮肤癌。自2008年发现MCV以来，已显示 MCV肿瘤抗原小T抗原（ST）和大T抗原（LT）是唯一的病毒蛋白 在病毒阳性MCC细胞中始终表达。单独的ST在Rat1细胞中的表达足以 诱导细胞转化，表明ST是MCC中细胞转化的主要驱动力 发展。我们的实验室确定ST与L-MYC/MAX和TIP60/P400形成特定的复合物 复合物（SLT复合物）和ST突变体与L-MYC和TIP60/P400复合物的结合能力 无法转化细胞。 TIP60/P400综合体是一个具有歌词的大型多工厂综合体 乙酰转移酶活性（TIP60蛋白）和核小体交换活性（P400蛋白）。这两个 活动与主动基因转录有关，但需要进一步的工作才能充分理解 TIP60/P400复合活性与基因表达之间的关系。我们假设MCV ST 将TIP60/P400复合物重定向到L-MYC结合位点，以激活需要转录程序 他们的赖氨酸乙酰转移酶和Hisstone重塑活性以驱动细胞转化。在AIM 1中， 我们将评估ST对TIP60/P400复合物结合的影响，并将比较 对照细胞中的TIP60/P400复合物与表达ST的细胞以确定ST是否从 与SLT靶基因启动子激活SLT靶基因的“正常” TIP60/P400复合结合位点 转录。通过比较正常人细胞中SLT复合物的直接靶标的基因列表 对于我们先前在病毒阳性MCC细胞中发现的人，我们将生成一个基因列表 始终受st的调节。在AIM 2中，我们将评估TIP60组蛋白乙酰转移酶（HAT）的贡献 和P400核小体重塑活性至ST介导的基因表达变化和细胞 转型。在AIM 3中，我们将适应FKBP/FRB诱导招聘的表观遗传编辑（Fire-- CAS9）tip60/p400复合物的方法，然后使用此系统募集TIP60/P400复合物到SLT 靶基因在没有st的情况下。招募后，我们将测量对ST靶基因转录的变化 以及SLT靶基因启动子的TIP60/P400特异性Hisstone标记的水平。从中获得的见解 研究与L-MYC和TIP60/P400复合物的ST相互作用将提供更深的 了解这些因素的致癌作用并了解SLT的活动 复合物将导致对靶向疗法的改进来对抗MCC。