Role of histone kinase VprBP in gene silencing: mechanisms, targets, and regulation

组蛋白激酶 VprBP 在基因沉默中的作用：机制、靶标和调控

• 批准号: 9177706
• 负责人: WOOJIN AN
• 金额: $ 37.74万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-15 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9177706
• 关键词: Abnormal Cell; Address; Affinity; Apoptosis; Architecture; Binding; Biological Process; CRISPR/Cas technology; Cancer Cell Growth; Cancer Etiology; Cancer cell line; Cell Proliferation; Cell physiology; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Coupled; Data; Development; Disease; Event; Failure; Gene Expression; Gene Expression Regulation; Gene Silencing; Gene Targeting; Genes; Genetic Transcription; Goals; Growth; Growth and Development function; Guide RNA; HIV-1; Histone H2A; Histone H3; Human; Laboratories; Light; Link; Malignant Neoplasms; Maps; Mediating; Mediator of activation protein; Modeling; Molecular; Mus; Names; Nuclear; Nuclear Protein; Nucleosomes; Oncogenic; Pathogenesis; Peptides; Phosphorylation; Phosphotransferases; Positioning Attribute; Property; Protamine Kinase; Protein p53; Recruitment Activity; Regulation; Regulator Genes; Research; Resources; Role; Sampling; Secure; Series; Signal Pathway; Signal Transduction; Site; Specificity; Staging; System; TP53 gene; Tail; Threonine; Time; Transactivation; Transcription Repressor/Corepressor; Transcriptional Regulation; Viral Proteins; Work; Xenograft Model; analog; base; cancer cell; cell growth; cell transformation; collaborative environment; epigenome; gene repression; histone modification; inhibitor/antagonist; innovation; insight; novel; programs; research study; tool; transcription factor; tumor; tumor growth; tumor xenograft; tumorigenesis; tumorigenic; ubiquitin-protein ligase

PROJECT SUMMARY The proper regulation of gene expression in human cells is achieved by signals emanating from a distinct histone modification occurring in chromatin architecture. Studies of gene regulation mechanisms by histone modification may thus aid in the understanding and treatment of ailments caused by abnormal transcription regulation. VprBP is a nuclear protein that was originally identified on the basis of its ability to interact with HIV-1 viral protein R. Although VprBP has been studied mainly in connection with Cul4 E3 ubiquitin ligase activity, we recently discovered that VprBP is a transdominant inhibitor of the p53 tumor suppressor and counteracts p53 transactivation, apoptosis, and growth suppression functions. High-level expression of VprBP in a wide range of human tumor samples and cancer cell lines, but low to undetectable expression of VprBP in their normal counterparts, suggests that VprBP possesses oncogenic properties. Our finding that VprBP-depleted cancer cells grow very slow and do not produce tumor xenografts also supports the idea that VprBP facilitates tumorigenesis. Our studies indicated that VprBP interacts with histone H3 tails protruding from nucleosomes and that this interaction facilitates VprBP recruitment and subsequent gene silencing in cancer cells. Unexpectedly, more recent work from our laboratory uncovered the presence of kinase activity specific for threonine 120 (T120) of histone H2A in VprBP. Our functional studies demonstrated that H2A-T120 phosphorylation (H2A-T120p) is essential for VprBP-driven gene silencing in cancer cells. Based on the available evidence, VprBP is the only kinase that is responsible for H2A-T120p occurring in human cancer cells. Importantly, our development of a highly selective inhibitor to manipulate the oncogenic VprBP kinase activity sets the stage for a more detailed analysis of VprBP function in abnormal gene silencing in cancer cells. The long-term goal of the proposed research is to understand the biological processes that VprBP controls and the molecular basis of its action as a mediator of tumorigenesis. The overall objectives are to determine the mechanisms of VprBP-mediated inactivation of the genes that regulate cell proliferation and to develop a set of molecular tools for controlling the magnitude of H2A-T120p at VprBP target loci in a precise manner. Our hypothesis is that VprBP establishes and maintains the silent state of key growth regulatory genes by a two-step mechanism wherein it is recruited to target genes via interaction with gene- specific transcription factors, and phosphorylates H2A-T120 as a mark for the recruitment of additional factors involved in gene repression and cell transformation. In the first Aim, we will employ the RNA-guided CRISPR-Cas9 system in which we can manipulate H2A-T120p at specific loci, and identify the genes that are directly regulated by VprBP-mediated H2AT-120p and critical for VprBP-promoted oncogenic events. In the second Aim, we will investigate the molecular mechanisms underlying the role of H2A-T120p in maintaining the silent state of target genes by identifying and characterizing factors that selectively recognize H2A-T120p. In the third Aim, we will develop bisubstrate analogue inhibitors with higher potency toward VprBP as novel molecular tools to control H2A-T120p and block growth and proliferation of cancer cells. VprBP-mediated H2A-T120p is clearly a very important new causative mechanism for cancer development, which our lab discovered, and we have developed tools and expertise that put us in the best position to advance research on this critical subject.

项目摘要 人类细胞中基因表达的适当调节是通过从一个不同的细胞发出的信号来实现的。 发生在染色质结构中的组蛋白修饰。组蛋白对基因调控机制的研究 因此，修饰可能有助于理解和治疗由异常转录引起的疾病 调控VprBP是一种核蛋白，最初是根据其与蛋白质相互作用的能力鉴定的。 HIV-1病毒蛋白R。尽管VprBP主要与Cul 4 E3泛素连接酶相关地进行了研究， 活性，我们最近发现VprBP是p53肿瘤抑制因子的反式显性抑制剂， 抵消p53反式激活、凋亡和生长抑制功能。的高效表达 VprBP在广泛的人类肿瘤样品和癌细胞系中，但表达低至不可检测 的VprBP在其正常的同行，表明VprBP具有致癌特性。我们的发现 VprBP耗尽的癌细胞生长非常缓慢并且不产生肿瘤异种移植物也支持了 VprBP促进肿瘤发生的观点。我们的研究表明，VprBP与组蛋白H3尾相互作用 这种相互作用促进了VprBP的募集和随后的基因表达。 在癌细胞中沉默。出乎意料的是，我们实验室最近的工作发现了 VprBP中组蛋白H2 A的苏氨酸120（T120）特异性激酶活性。我们的功能研究 证明H2 A-T120磷酸化（H2 A-T120 p）对于VprBP驱动的基因沉默是必需的， 癌细胞根据现有证据，VprBP是唯一负责H2 A-T120 p的激酶 发生在人类癌细胞中。重要的是，我们开发了一种高度选择性的抑制剂， 致癌的VprBP激酶活性为更详细地分析VprBP功能奠定了基础， 癌细胞中异常的基因沉默。 拟议研究的长期目标是了解VprBP控制的生物过程 及其作为肿瘤发生介质的分子基础。总体目标是 确定VprBP介导的调节细胞增殖的基因失活的机制， 开发一套分子工具，用于控制H2 A-T120 p在VprBP靶基因座的大小， 精确的方式。我们的假设是，VprBP建立并维持了沉默的关键增长状态 调节基因通过两步机制，其中它通过与基因的相互作用被募集到靶基因， 特异性转录因子，并磷酸化H2 A-T120作为募集额外的 参与基因抑制和细胞转化的因子。在第一个目标中，我们将使用RNA引导的 CRISPR-Cas9系统，我们可以在特定位点操纵H2 A-T120 p，并鉴定 由VprBP介导的H2 AT-120 p直接调节，对VprBP促进的致癌事件至关重要。在 第二个目的，我们将研究H2 A-T120 p在细胞凋亡中作用的分子机制。 通过识别和表征选择性地抑制靶基因表达的因子， 识别H2 A-T120 p。第三个目标是开发更高效的双底物类似物抑制剂 VprBP作为控制H2 A-T120 p和阻断癌症生长和增殖的新分子工具 细胞VprBP介导的H2 A-T120 p显然是一种非常重要的新的癌症致病机制 开发，这是我们的实验室发现的，我们已经开发了工具和专业知识，使我们在最好的 推动对这一重要课题的研究。