Nuclear pore complexes (NPC) are large protein channels composed of multiple copies of ~30 individual nucleoporins (Nups). NPC span the nuclear envelope (NE) and facilitate all nuclear transport. Among the ~30 Nups that contribute to the NPC, there are two primary categories - (1) Nups that stably associate with the NE and (2) dynamic Nups that shuttle into the nuclear interior. Recent results from the Hetzer laboratory and others found that mobile Nups interact with chromatin away from NPCs. These were the first reports that Nups directly influence transcription. In particular, NUP98 and NUP50 bind proximal to active genes, including many developmentally regulated loci. Amazingly, knockdown of Nups reduces target gene expression and suggests that they serve unappreciated regulatory roles in complex gene networks. Preliminary data indicates that NUP98 binds to chromatin modifiers. Given the overlap (~75%) of NUP98 and NUP50 bound genes, it is speculated that NUP50 is also involved in chromatin remodeling. The experiments proposed here focus on the hypothesis that NUP50 targets histone methylation complexes to specific loci thereby promoting active transcription. Preliminary data further suggests that Nup50 influences neural stem cell differentiation. Many Nups, including Nup98, are correlated with a diverse array of cancers although their mechanisms during tumor growth are poorly understood. Elucidating mechanisms relating Nups to normal development and cancer cell transformation would provide a foothold into a new class of proteins observed in many cancers. This proposal aims to clarify how NUP50 regulates vertebrate development and neoplasia. Initial experiments will characterize NUP50 during in vitro neurogenesis, including changes in its protein partners and chromatin occupancy. Characterizing changes in NUP50 during differentiation will include a detailed study of how Erk phosphorylation regulates the ability of NUP50 to interact with chromatin. Lastly, the requirement for Nup50 will be assayed during in vitro and in vivo neurogenesis. In vivo analysis will utilize existing tools developed while translating results into a mammalian system in order to provide physiological relevance and insight into how individual Nups orchestrate mammalian development.
The nuclear pore complex is a ubiquitous macromolecular structure associated with many tissue-specific developmental defects and forms of cancer, yet the causative mechanisms are largely unknown. One individual nucleoporin, Nup50, causes developmental defects in the nervous system, which we propose arise from its interaction with chromatin during differentiation. Misregulated chromatin remodeling during developmental neurogenesis has been linked to aggressive pediatric brain tumors and we propose experiments to clarify the role of Nup50 during neurogenesis and tumor formation.