Development of tools for site-directed analysis of gene function

组蛋白伴侣Rbbp 4是多种染色质重塑复合物的组分 参与细胞身份，可塑性和重新编程（Cheloufi和Hochedlinger，2017; Clemot等人，2018; Conway等人，2015年）。RBBP 4在细胞周期调控中也起着重要作用 作为调节细胞周期基因表达的MuvB复合物的组分。连同 转录因子B-Myb和FoxM 1，MuvB复合物控制基因转录的时间。 在G1/S（生长和DNA合成）和G2/M（有丝分裂）中表达， 通过细胞周期（Fischer和Muller，2017）。我们之前在斑马鱼身上展示了 rb 1缺陷性胚胎性脑肿瘤模型，rbbp 4升高超过10倍，我们 显示在rbbp 4突变胚胎中，神经祖细胞经历tp 53依赖性凋亡， （Schultz等人，2018年）。这些结果表明，抑制Rbbp 4可以阻断增殖， 通过Tp 53程序化细胞激活细胞周期阻滞使脑癌细胞存活 死亡途径（Engeland，2018）。然而，虽然大多数恶性人类脑癌显示， 升高的Rbbp 4表达（Schultz，Kool，McGrail未发表的结果），许多还具有 Tp 53的失活突变。更深入地了解Rbbp 4 有助于神经祖细胞周期的进展可能会导致新的目标，在 与Rbbp 4抑制剂组合，导致脑肿瘤细胞死亡。为了解决这个问题，我们建议 应用我们创新的CRISPR/Cas9靶向整合策略， 细胞周期报告基因和foxm 1条件基因失活斑马鱼系。这些将使我们能够 研究Rbbp 4和FoxM 1如何在神经祖细胞周期中特异性合作 在大脑发育过程中。这些结果将对神经祖细胞产生新的认识。 细胞周期控制及其失调如何驱动脑肿瘤细胞的不受调节的增殖。

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 However, while most malignant human brain cancers show elevated Rbbp4 expression (Schultz, Kool, McGrail unpublished results), many also harbor inactivating mutations in Tp53.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.