Development of tools for site-directed analysis of gene function

组蛋白伴侣Rbbp4是多种染色质重塑复合体的组成部分 参与细胞特性、可塑性和重新编程(Cheroufi和Hochedlinger，2017； Clemot等人，2018年；Conway等人，2015年)。Rbbp4在细胞周期控制中也发挥着重要作用。 作为MuvB复合体的一个组成部分，调节细胞周期基因的表达。与.一起 转录因子B-Myb和FOXM1，MuvB复合体控制基因的时序 在G1/S(生长和DNA合成)和G2/M(有丝分裂)中的表达允许进展 通过细胞周期(Fischer和Muller，2017)。我们之前在一条斑马鱼身上展示了 Rb1缺陷性胚胎脑瘤模型，Rbbp4升高10倍以上，我们 结果表明，在rbbp4突变胚胎中，神经前体细胞经历了tp53依赖的细胞凋亡。 (Schultz等人，2018年)。这些结果表明，抑制Rbbp4可能阻止了细胞的增殖和 TP53程序化细胞激活细胞周期停滞对脑癌细胞存活的影响 《死亡之路》(England，2018)。然而，尽管大多数恶性人类脑癌表现出 Rbbp4表达升高(Schultz，Kool，McGrail未发表的结果)，许多人也 使TP53中的突变失活。更深入地了解Rbbp4的作用机制 促进神经前体细胞周期进程可能导致新的靶点，即在 与Rbbp4抑制剂结合，导致脑瘤细胞死亡。为了解决这个问题，我们建议 应用我们创新的CRISPR/CAS9有针对性的整合策略来生成新的语言 细胞周期报告基因和FOXM1条件基因失活斑马鱼。这些将使我们能够 研究Rbbp4和FOXM1在神经前体细胞周期中的特异性合作 在大脑发育过程中的进展。这一结果将对神经前体细胞有新的认识 细胞周期控制及其失调如何导致脑瘤细胞的不受调控的增殖。

The histone chaperone Rbbp4 is a component of multiple chromatin remodeling complexes involved in cellular identity, plasticity, and reprogramming (Cheloufi and Hochedlinger, 2017; Clemot et al., 2018; Conway et al., 2015). Rbbp4 also plays a significant role in cell cycle control as a component of the MuvB complex that regulates cell cycle gene expression. Together with transcription factors B-Myb and FoxM1, the MuvB complex controls the timing of gene expression in G1/S (growth and DNA synthesis) and G2/M (mitosis) that allows progression through the cell cycle (Fischer and Muller, 2017). We previously demonstrated in a zebrafish model of rb1 defective embryonal brain tumors that rbbp4 is elevated more than 10-fold, and we showed that in rbbp4 mutant embryos neural progenitors undergo tp53-dependent apoptosis (Schultz et al., 2018). These results indicate inhibition of Rbbp4 may block the proliferation and survival of brain cancer cells by activating cell cycle arrest through the Tp53 programmed cell death pathway (Engeland, 2018). However, while most malignant human brain cancers show elevated Rbbp4 expression (Schultz, Kool, McGrail unpublished results), many also harbor inactivating mutations in Tp53. A deeper understanding of the mechanism by which Rbbp4 contributes to neural progenitor cell cycle progression may lead to new targets, that in combination with Rbbp4 inhibitors, lead to brain tumor cell death. To address this, we propose to apply our innovative CRISPR/Cas9 targeted integration strategy to generate novel proneural cell cycle reporter and foxm1 conditional gene inactivation zebrafish lines. These will allow us to examine how Rbbp4 and FoxM1 cooperate specifically in neural progenitor cell cycle progression during brain development. The results will yield new insight into neural progenitor cell cycle control and how its dysregulation drives unregulated proliferation of brain tumor cells.