The role of the nucleoporin Nup98 in gene regulation

核孔蛋白 Nup98 在基因调控中的作用

• 批准号: 8598482
• 负责人: Martin W Hetzer
• 金额: $ 36.48万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-05 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8598482
• 关键词: Acute Myelocytic Leukemia; Binding; Binding Proteins; Binding Sites; Biochemical Genetics; Biological; Cell Differentiation process; Cell Line; Cell Nucleus; Cell Proliferation; Cell division; Cells; Chromatin; Communication; Complex; Development; Developmental Process; Disease; Dissection; Drosophila genome; Drosophila genus; Embryo; Epigenetic Process; Event; Failure; Fluorescent in Situ Hybridization; Gap Junctions; Gene Activation; Gene Expression; Gene Expression Regulation; Gene Fusion; Gene Structure; Gene Targeting; Generations; Genes; Genetic; Genetic Programming; Genetic Transcription; Genetic screening method; Genomics; Goals; Hematopoietic; Homologous Gene; Human; Human Cell Line; Immunoprecipitation; Investigation; Lead; Link; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Membrane Proteins; Memory; Messenger RNA; Methods; Mitosis; Modeling; Molecular; Mus; Muscular Dystrophies; Neurodegenerative Disorders; Nuclear; Nuclear Envelope; Nuclear Export; Nuclear Pore; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Phenotype; Phosphorylation; Play; Premature aging syndrome; Process; Progeria; Proteins; RNA; RNA Polymerase II; Recruitment Activity; Regulation; Regulator Genes; Relative (related person); Reproduction; Role; Site; Techniques; Testing; Tissues; Transcriptional Activation; Transcriptional Regulation; Transgenic Organisms; cancer cell; cell transformation; chromatin immunoprecipitation; design; embryonic stem cell; gene interaction; genome-wide; genome-wide analysis; histone modification; human disease; improved; insight; leukemia; mRNA Export; member; mutant; nerve stem cell; novel; prevent; programs; protein complex; repaired; tool

DESCRIPTION (provided by applicant): Eukaryotic gene expression is regulated at multiple levels in the cell nucleus, from histone modification and chromatin compaction to the synthesis, processing and export of mRNA. The precise execution of transcriptional programs during cell differentiation and development relies on faithful reproduction of a specific chromatin state through mitosis. Failure to maintain these epigenetic programs through multiple cell divisions commonly leads to pathological conditions such as cancer. Thus, understanding how chromatin organization is established and propagated will enhance the understanding of how disease-causing errors in gene expression can occur and be prevented. The central hypothesis guiding this proposal is that the nuclear pore component Nup98 plays an essential role in transcriptional activation of developmentally regulated genes. Biochemical, genetic, genomic and cell biological methods will be used to investigate the potential interaction between Nup98 and the members of the trithorax protein complex, a well-known chromatin regulator and mediator of developmental active gene memory. First, the molecular composition of the intranuclear Nup98 complex will be established, its recruitment mechanism to chromatin identified and thus provide key functional insights into the link between Nup98 and epigenetic memory. Second, the molecular function of Nup98 at genomic target sites will be investigated. For instance, the consequences of Nup98 knockdown and over-expression will be analyzed by chromatin immune- precipitation to investigate which binding partners, histone modifications and RNA polymerase II phosphorylation events depend on Nup98. In addition, a potential effect of Nup98 on mRNA processing and export will be determined by RNA fluorescence in situ hybridization and analysis of recruitment of mRNA processing and nuclear export factors. Furthermore, Nup98 recruitment in developing tissues that activate targets genes as well as genetic effects of Nup98 on established Trx mutant phenotypes will be analyzed. Finally, a potential role of Nup98 in long-range gene interactions and organization of active chromatin domains will be investigated with genome-wide techniques. These studies will provide important information about the role of Nup98 in transcriptional initiation and establishment of active chromatin. Third, the interaction between Nup98 and mammalian MLL and Wdr5 may prove to be important in understanding Acute Myelogenous Leukemia (AML) and development-associated roles of these proteins. Proposed is a comprehensive genome-wide analysis of Nup98 binding in human cell lines and in mouse hematopoietic cell lines. The latter have been transformed by a known leukemia-inducing gene fusion Nup98- NSD1 and will be compared relative to normal hematopoietic cells. These approaches have the potential to reveal the gene regulatory role of mammalian Nup98 and provide insight into its leukemia-causing potential.

描述（由申请人提供）：真核基因表达在细胞核中以多个水平调节，从组蛋白的修饰和染色质压实到mRNA的合成，加工和导出。细胞分化和发育过程中转录程序的精确执行取决于通过有丝分裂对特定染色质状态的忠实繁殖。无法通过多个细胞分裂维持这些表观遗传程序，通常会导致病理状况，例如癌症。因此，了解如何建立染色质组织和传播将增强对基因表达中引起疾病​​的错误的理解，并被预防。指导该提议的中心假设是核孔成分NUP98在发育调节基因的转录激活中起着至关重要的作用。生化，遗传，基因组和细胞生物学方法将用于研究NUP98与Trithorax蛋白复合物的成员之间的潜在相互作用，Trithorax蛋白质复合物是众所周知的染色质调节剂和发育活性基因记忆的介体。首先，将建立核内NUP98复合物的分子组成，其募集机制鉴定为染色质，从而为NUP98与表观遗传记忆之间的联系提供了关键的功能见解。其次，将研究NUP98在基因组靶位点的分子功能。例如，将通过染色质免疫沉淀来分析NUP98敲低和过表达的后果，以研究哪些结合伴侣，组蛋白修饰和RNA聚合酶II磷酸化事件取决于NUP98。此外，NUP98对mRNA加工和输出的潜在影响将由RNA荧光原位杂交和分析mRNA加工和核输出因子的募集分析。此外，将分析NUP98在开发组织中募集基因以及NUP98对已建立的TRX突变表型的遗传作用。最后，将使用全基因组技术研究NUP98在远程基因相互作用和主动染色质结构域的组织中的潜在作用。这些研究将提供有关NUP98在转录启动和活性染色质建立中的作用的重要信息。第三， NUP98与哺乳动物MLL和WDR5之间的相互作用可能对了解这些蛋白质的急性髓质白血病（AML）和与发育相关的作用很重要。提出的是对人类细胞系和小鼠造血细胞系中NUP98结合的全面基因组分析。后者已通过已知的诱导白血病基因融合NUP98-NSD1进行了转化，并将相对于正常的造血细胞进行比较。这些方法有可能揭示哺乳动物NUP98的基因调节作用，并洞悉其引起白血病的潜力。