Nup50 control of neurogenesis and cancer stem cell transformation

描述（由申请人提供）：核孔复合物（NPC）是由约30个核孔蛋白（Nups）的多个拷贝组成的大型蛋白质通道。NPC跨越核包膜（NE）并促进所有核运输。在对NPC有贡献的约30个Nups中，有两大类-(1)与NE稳定关联的Nups和(2)穿梭于核内部的动态Nups。Hetzer实验室和其他人最近的研究结果发现，移动nup与npc以外的染色质相互作用。这是首次报道nup直接影响转录。特别是，NUP98和NUP50结合近端活性基因，包括许多发育调节位点。令人惊讶的是，敲低Nups会降低靶基因的表达，这表明它们在复杂的基因网络中发挥着未被重视的调节作用。初步数据表明，NUP98与染色质修饰子结合。考虑到NUP98和NUP50结合基因的重叠（约75%），推测NUP50也参与了染色质重塑。本文提出的实验集中于NUP50将组蛋白甲基化复合物靶向到特定位点从而促进主动转录的假设。初步数据进一步表明Nup50影响神经干细胞的分化。包括Nup98在内的许多Nups与多种癌症相关，尽管它们在肿瘤生长过程中的机制尚不清楚。阐明Nups与正常发育和癌细胞转化相关的机制，将为研究在许多癌症中观察到的一类新的蛋白质提供一个立足点。本研究旨在阐明NUP50调控脊椎动物发育和肿瘤形成的机制。初步实验将在体外神经发生过程中表征NUP50，包括其蛋白伴侣和染色质占用的变化。表征分化过程中NUP50的变化将包括对Erk磷酸化如何调节NUP50与染色质相互作用能力的详细研究。最后，将测定体外和体内神经发生过程中对Nup50的需求。体内分析将利用现有的工具开发，同时将结果转化为哺乳动物系统，以便

DESCRIPTION (provided by applicant): 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.