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

描述（申请人提供）：翻译后组蛋白修饰在调节染色质结构和功能中起重要作用。组蛋白修饰的一个例子是泛素化，其主要发生在H2A和H2B上。虽然最近的研究已经揭示了H2A泛素化在Hox基因沉默和X失活中的关键作用，但H2A去泛素化的功能尚不清楚，催化H2A去泛素化的酶尚未被鉴定。在本提案中提出的初步结果中，我们将Ubp-M鉴定为 组蛋白H2A。进一步的研究表明，Ubp-M介导的H2A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，确定Ubp-M及其介导的H2A去泛素化在染色质和细胞调控中的作用和阐明其机制的长期目标。我们的假设是，Ubp-M调节细胞周期进程和基因表达，部分，通过H2A去泛素化。为了解决这一假设，我们制定了三个具体目标： 1.通过定义Ubp-M中对其功能结构域进行必要的区域来表征Ubp-M的功能结构域。 核小体和H2A特异性去泛素化在体外和体内以及其正常 亚细胞定位。 2.通过绘制磷酸化图谱，确定磷酸化在调节Ubp-M功能中的作用 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，还探讨了磷酸化在调节Ubp-M核定位信号（NLS）和 将研究核输出信号（内斯）。 3.通过首先确认它们与Ubp-M的相互作用来鉴定和表征Ubp-M相互作用蛋白 绘制蛋白质相互作用的位点， 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，我们概述了计划，以产生Ubp-M完全和条件 敲除小鼠并描述我们的实验进展。我们的初步研究表明Ubp-M 调节非洲爪蟾的早期胚胎发生，并在卵巢癌和乳腺癌中高度表达 细胞，强调的重要性，以确定在体内的Ubp-M的功能。然而，由于这项应用的工作量和这些实验的不确定结果，我们没有列出这些 实验作为一个具体的目标。

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.