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.
Aberrant nuclear organization is a key pathological feature of cancer cells and has been linked to various human diseases such as muscular dystrophies, neurodegenerative disorders and the premature aging disease progeria. Studying the molecular communication between chromatin and the nuclear pore complex has the potential to uncover novel levels of gene regulation and to improve our understanding of nuclear organization in normal and pathological cells. Ultimately, our studies might provide new insights into the onset of Acute Myelogenous Leukemia (AML) and lead to the generation of new tools to interfere with malignant cell proliferation and repair dysfunctional genetic programs in aberrant cells.
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