The role of the nucleoporin Nup98 in gene regulation

描述（由申请人提供）：真核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。细胞分化和发育过程中转录程序的精确执行依赖于特定染色质状态通过有丝分裂的忠实再现。未能通过多次细胞分裂维持这些表观遗传程序通常会导致病理状况，如癌症。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。指导这一建议的中心假设是，核孔组件Nup 98在发育调控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被用来研究Nup 98和三胸蛋白复合物，一个众所周知的染色质调节和发育活性基因记忆的介质的成员之间的潜在相互作用。首先，将建立核内Nup 98复合物的分子组成，鉴定其向染色质的募集机制，从而为Nup 98和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Nup 98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Nup 98敲低和过表达的后果，以研究哪些结合配偶体、组蛋白修饰和RNA聚合酶II磷酸化事件依赖于Nup 98。此外，Nup 98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募集分析来确定。此外，将分析Nup 98在发育组织中的募集，其激活靶基因以及Nup 98对已建立的Trx突变表型的遗传效应。最后，Nup 98在长距离基因相互作用和组织活性染色质结构域的潜在作用将与全基因组技术进行研究。这些研究将为Nup 98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、 Nup 98与哺乳动物MLL和Wdr 5之间的相互作用可能被证明在理解急性髓性白血病（AML）和这些蛋白的发育相关作用中是重要的。提出了在人细胞系和小鼠造血细胞系中Nup 98结合的全面基因组分析。后者已被已知的白血病诱导基因融合体Nup 98-NSD 1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Nup 98的基因调控作用，并深入了解其导致白血病的潜力。

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.