The role of the nucleoporin Nup98 in gene regulation

描述(申请人提供)：真核基因的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缩到信使核糖核酸的合成、加工和输出。细胞分化和发育过程中转录程序的精确执行依赖于通过有丝分裂忠实地复制特定的染色质状态。未能通过多个细胞分裂维持这些表观遗传程序通常会导致癌症等病理情况。因此，了解染色质组织是如何建立和传播的，将增强对基因表达中致病错误如何发生和预防的理解。指导这一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发挥重要作用。Nup98将使用生化、遗传学、基因组和细胞生物学方法研究Nup98与三胸蛋白复合体成员之间的潜在相互作用。三胸蛋白复合体是一种众所周知的染色质调节因子和发育主动基因记忆的介体。首先，将建立核内Nup98复合体的分子组成，确定其对染色质的招募机制，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，将研究Nup98在基因组靶点的分子功能。例如，将通过染色质免疫沉淀来分析Nup98基因敲除和过度表达的后果，以调查哪些结合伙伴、组蛋白修饰和RNA聚合酶II磷酸化事件依赖于Nup98。此外，通过RNA荧光原位杂交以及对mRNA加工和核输出因子招募的分析，将确定Nup98对mRNA加工和输出的潜在影响。此外，还将分析Nup98在激活目标基因的发育组织中的招募以及Nup98对已建立的Trx突变表型的遗传影响。最后，将用全基因组技术研究Nup98在基因长程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在转录启动和活性染色质的建立中的作用提供重要的信息。第三, Nup98与哺乳动物的MLL和WDR5之间的相互作用对于理解急性髓系白血病(AML)和这些蛋白的发育相关作用可能被证明是重要的。建议对Nup98在人类细胞系和小鼠造血细胞系中的结合进行全面的全基因组分析。后者已被已知的白血病诱导基因融合Nup98-NSD1转化，并将与正常造血细胞进行比较。这些方法有可能揭示哺乳动物Nup98的基因调控作用，并提供对其导致白血病的潜力的洞察。

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.