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

项目总结 在人类细胞中对基因表达的适当调节是通过从不同的 组蛋白修饰发生在染色质结构中。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转录异常引起的疾病 监管。VprBP是一种核蛋白，最初是根据其与 HIV-1病毒蛋白R。虽然VprBP的研究主要与CUL4 E3泛素连接酶有关。 活性，我们最近发现VprBP是一种P53肿瘤抑制因子的跨优势抑制物， 中和P53的反式激活、细胞凋亡和生长抑制功能。的高水平表达 VprBP在广泛的人类肿瘤样本和癌细胞系中表达，但低至检测不到表达 提示VprBP具有致癌作用。我们的发现 VprBP耗尽的癌细胞生长非常缓慢，不会产生肿瘤移植，这也支持了 VprBP促进肿瘤发生的观点。我们的研究表明VprBP与组蛋白H3尾巴相互作用 这种相互作用促进了VprBP的募集和随后的基因 癌细胞中的沉默。出乎意料的是，我们实验室最近的工作发现了 VprBP中组蛋白H2A的苏氨酸120(T120)所特有的激酶活性。我们的功能研究 证明H_2A-T120磷酸化(H_2A-T_(120p))是VprBP驱动的基因沉默所必需的 癌细胞。根据现有证据，VprBP是唯一与H2A-T120p有关的激酶 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来操纵 致癌VprBP激酶活性为更详细地分析VprBP的功能奠定了基础 癌细胞中的异常基因沉默。 这项拟议研究的长期目标是了解VprBP控制的生物过程 以及它作为肿瘤发生介质的分子基础。总体目标是 确定VprBP介导的细胞增殖调控基因失活的机制 开发一套用于控制VprBP靶基因座H_2A-T120P大小的分子工具 严谨的态度。我们的假设是，VprBP建立并维持关键增长的沉默状态 通过两步机制调控基因，其中它通过与基因相互作用被招募到靶向基因。 特定的转录因子，并使H2A-T120磷酸化，作为招募其他 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，在该系统中，我们可以在特定的基因座上操纵H2A-T120p，并识别与 是由VprBP介导的H2AT-120p直接调节的，对VprBP促进的致癌事件起关键作用。在……里面 第二个目标，我们将研究H_2A-T120P作用的分子机制。 通过识别和表征选择性地使靶基因保持沉默状态的因子 识别H_2A-T120P。在第三个目标中，我们将开发效力更高的双底物类似物抑制剂。 VprBP作为控制H_2A-T120p和阻断肿瘤生长增殖的新分子工具 细胞。VprBP介导的H2A-T120p显然是一种非常重要的新致癌机制 开发，这是我们的实验室发现的，我们开发的工具和专业知识使我们处于最好的 推动对这一重要课题的研究。

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.