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) 是一种小型 DNA 肿瘤病毒，可引起约 80% 的默克尔细胞肿瘤 癌（MCC），一种高度侵袭性的皮肤癌。自 2008 年发现 MCV 以来，已表明 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 细胞中一致表达。仅在 Rat1 细胞中表达 ST 就足以 诱导细胞转化表明 ST 是 MCC 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），并且 ST 突变体无法结合 L-Myc 和 Tip60/p400 复合物 不能转化细胞。 Tip60/p400 复合物是一种大型多亚基复合物，含有赖氨酸 乙酰转移酶活性（Tip60 蛋白）和核小体交换活性（p400 蛋白）。这两个 活性与活跃的基因转录相关，但需要进一步的工作来充分理解 Tip60/p400复合物活性与基因表达之间的关系。我们假设 MCV ST 将 Tip60/p400 复合物重定向至 L-Myc 结合位点，以激活需要的转录程序 它们的赖氨酸乙酰转移酶和组蛋白重塑活性可驱动细胞转化。在目标 1 中， 我们将评估 ST 对 Tip60/p400 复合物结合的影响，并且我们将比较 ST 的直接靶标 Tip60/p400 复合物在对照细胞和表达 ST 的细胞中确定 ST 是否将复合物从 “正常”Tip60/p400 复合物与 SLT 靶基因启动子结合位点以激活 SLT 靶基因 转录。通过比较正常人体细胞中 SLT 复合物直接靶标的基因列表 对于我们之前在病毒阳性 MCC 细胞中发现的基因，我们将生成一个基因列表，这些基因是 始终受 ST 监管。在目标 2 中，我们将评估 Tip60 组蛋白乙酰转移酶 (HAT) 的贡献 和 p400 核小体重塑活性对 ST 介导的基因表达变化和细胞 转变。在目标 3 中，我们将采用 Cas9 进行表观遗传编辑的 Fkbp/Frb 诱导招募（FIRE- Cas9) 方法连接到 Tip60/p400 复合物，然后使用该系统将 Tip60/p400 复合物招募到 SLT 没有 ST 时的靶基因。招募后，我们将测量 ST 靶基因转录的变化 以及 SLT 靶基因启动子处 Tip60/p400 特异性组蛋白标记的水平。获得的见解 研究 ST 与 L-Myc 和 Tip60/p400 复合物的相互作用将提供更深入的了解。 了解这些因素的致癌作用并了解 SLT 的活动 复杂的研究将导致确定改进的靶向疗法来对抗 MCC。