Role of Ubp-M and H2A deubiquitination in chromatin and cellular function

描述(申请人提供)：组蛋白翻译后修饰在调节染色质结构和功能中起着重要作用。组蛋白修饰的一个例子是泛素化，它主要发生在H_2A和H_2B上。虽然最近的研究揭示了H_2A泛素化在HOX基因沉默和X失活中的关键作用，但H_2A泛素化的功能尚不清楚，催化H_2A脱泛素化的酶也尚未确定。在这项提案中提出的初步结果中，我们确定UBP-M是 组蛋白H_2A。进一步的研究表明，UBP-M介导的H_2A去泛素化在细胞周期进程和基因表达中起着重要作用。在这项提案中，我们建议彻底表征UBP-M，长期目标是确定UBP-M及其介导的H2A去泛素化在染色质和细胞调节中的作用和机制。我们的假设是，UBP-M调节细胞周期进程和基因表达，部分是通过过氧化氢A去泛素化。为了解决这一假设，我们确立了三个具体目标： 1.通过定义UBP-M中ITS所必需的区域来表征UBP-M的功能结构域 核小体和H_2A特异性的体内外去泛素化及其正常 亚细胞定位。 2.通过定位磷酸化来确定磷酸化在调节UBP-M功能中的作用 并研究这些位点的突变如何在体外和体内调节UBP-M的功能。 此外，磷酸化在调节UBP-M的核定位信号(NLS)和 将对核出口信号(NES)进行调查。 3.通过首先确认UBP-M与UBP-M的相互作用来鉴定和鉴定UBP-M相互作用的蛋白质 绘制蛋白质-蛋白质相互作用的位置图，然后确定这些相互作用的方式 蛋白质在体外和体内调节UBP-M的细胞功能。 除了这三个具体目标外，我们还概述了生成完整和有条件的UBP-M的计划 并描述我们的实验进展。我们的初步研究表明，UBP-M 非洲爪哇调节早期胚胎发育，并在卵巢癌和乳腺癌中高表达 细胞，强调了确定UBP-M在体内功能的重要性。然而，由于该应用程序中的工作量和这些实验的不确定结果，我们没有列出这些 实验作为一个特定的目标。

DESCRIPTION (provided by applicant): Posttranslational histone modifications play important roles in regulating chromatin structure and function. One example of histone modification is ubiquitination, which occurs predominately on H2A and H2B. Although recent studies have revealed a critical role of H2A ubiquitination in Hox gene silencing and X inactivation, the function of H2A deubiquitination is unknown and the enzymes that catalyze H2A deubiquitination have not been identified. In the preliminary results presented in this proposal, we identified Ubp-M as the deubiquitinase for histone H2A. Further characterization revealed that Ubp-M-mediated H2A deubiquitination plays important roles in cell cycle progression and gene expression. In this proposal we propose to thoroughly characterize Ubp-M, with the long-range goal of determining the roles and elucidating the mechanisms of Ubp-M and its mediated H2A deubiquitination in chromatin and cellular regulation. Our hypothesis is that Ubp-M regulates cell cycle progression and gene expression, in part, through H2A deubiquitination. To address this hypothesis, we have established three Specific Aims to: 1. Characterize the functional domains of Ubp-M by defining regions in Ubp-M that are necessary for its nucleosomal- and H2A-specific deubiquitination in vitro and in vivo as well as for its normal subcellular localization. 2. Determine the role of phosphorylation in regulating Ubp-M's function by mapping the phosphorylation sites and investigating how mutations of these sites regulate Ubp-M's function in vitro and in vivo. Furthermore, the roles of phosphorylation in regulating Ubp-M's nuclear localization signal (NLS) and nuclear export signal (NES) will be investigated. 3. Identify and characterize Ubp-M interacting proteins by first confirming their interactions with Ubp-M and mapping the sites of protein-protein interaction, and then determining how these interacting proteins regulate Ubp-M's cellular functions in vitro and in vivo. In addition to these three Specific Aims, we outline plans to generate Ubp-M complete and conditional knockout mice and describe our experimental progress. Our preliminary studies indicate that Ubp-M regulates early embryogenesis in Xenopus laevis and is highly expressed in ovarian and breast cancer cells, underscoring the importance to determine the function of Ubp-M in vivo. However, due to the amount of work in this application and the uncertain outcome of these experiments, we did not list these experiments as a Specific Aim.