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: 8452084
• 负责人: Martin W Hetzer
• 金额: $ 35.2万
• 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/8452084
• 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）：真核生物的基因表达在细胞核中受到多个水平的调控，从组蛋白修饰和染色质压实到mRNA的合成、加工和输出。在细胞分化和发育过程中，转录程序的精确执行依赖于有丝分裂中特定染色质状态的忠实复制。如果不能通过多次细胞分裂来维持这些表观遗传程序，通常会导致诸如癌症之类的病理状况。因此，了解染色质组织是如何建立和繁殖的，将加强对基因表达中致病错误是如何发生和预防的理解。指导这一建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。生物化学、遗传学、基因组学和细胞生物学方法将用于研究Nup98与三胸蛋白复合物成员之间的潜在相互作用，三胸蛋白复合物是一种众所周知的染色质调节剂和发育活性基因记忆的介质。首先，将建立核内Nup98复合物的分子组成，确定其对染色质的募集机制，从而为Nup98与表观遗传记忆之间的联系提供关键的功能见解。其次，研究Nup98在基因组靶点的分子功能。例如，将通过染色质免疫沉淀分析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磷酸化事件依赖于Nup98。此外，Nup98对mRNA加工和输出的潜在影响将通过RNA荧光原位杂交和mRNA加工和核输出因子的招募分析来确定。此外，将分析Nup98在激活靶基因的发育组织中的募集，以及Nup98对已建立的Trx突变表型的遗传效应。最后，将利用全基因组技术研究Nup98在远程基因相互作用和活性染色质结构域组织中的潜在作用。这些研究将为Nup98在转录起始和活性染色质建立中的作用提供重要信息。第三,