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)是一种小的DNA肿瘤病毒，约80%的默克尔细胞 癌症(MCC)，一种高度侵袭性的皮肤癌。自2008年发现MCV以来，已经显示出 MCV肿瘤抗原小T抗原(ST)和大T抗原(LT)是仅有的病毒蛋白 在病毒阳性的MCC细胞中持续表达。在Rat1细胞中单独表达ST足以 诱导细胞转化提示ST是MCC细胞转化的主要驱动因素 发展。我们实验室确定ST与L-MYC/MAX和Tip60/P400形成了特异性的复合体 一个不能与L-Myc和Tip60/P400复合体结合的ST突变体 无法转换单元格。Tip60/P400复合体是一种含有赖氨酸的大型多亚单位复合体 乙酰转移酶活性(Tip60蛋白)和核小体交换活性(P400蛋白)。这两个都是 活动与活跃的基因转录相关，但还需要进一步的工作才能充分了解 Tip60/P400复合体活性与基因表达的关系我们假设MCV ST 将Tip60/P400复合体重定向到L-Myc结合位点以激活需要 它们的赖氨酸乙酰转移酶和组蛋白重塑活性驱动细胞转化。在目标1中， 我们将评估ST对Tip60/P400复合体结合的影响，并比较 对照细胞中表达ST的Tip60/P400复合体以确定ST是否将该复合体从 SLT靶基因启动子与Tip60/P400复合结合位点激活SLT靶基因 抄写。通过比较正常人细胞中作为SLT复合体直接靶点的基因清单 对于我们之前在病毒阳性的MCC细胞中发现的那些基因，我们将生成一个基因列表，这些基因是 始终如一地受到ST。在目标2中，我们将评估Tip60组蛋白乙酰转移酶(HAT)的作用 和P400核小体重塑活性对ST介导的基因表达变化和细胞 转型。在目标3中，我们将使FkBP/FRB可诱导招募用于Cas9的表观遗传编辑(FIRE- Cas9)方法克隆到Tip60/P400复合体，然后利用该系统将Tip60/P400复合体招募到SLT 在没有ST的情况下靶基因。招募后，我们将测量ST目标基因转录的变化 SLT靶基因启动子Tip60/P400特异性组蛋白标志物水平。从以下方面获得的见解 研究ST与L-Myc和Tip60/P400复合体的相互作用将提供更深层次的 了解这些因素的致癌作用和了解SLT的活动 复杂性将导致确定更好的靶向疗法来对抗MCC。