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Development of tools for site-directed analysis of gene function

基因功能定点分析工具的开发

基本信息

批准号：
10185650
负责人：
KARL J CLARK
金额：
$ 3.12万
依托单位：
IOWA STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-06-01 至 2024-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10185650
关键词：
Address Apoptosis Brain Brain Neoplasms CRISPR/Cas technology Cell Cycle Cell Cycle Arrest Cell Cycle Progression Cell Cycle Regulation Cell Death Chromatin Chromatin Remodeling Factor Cloning Clustered Regularly Interspaced Short Palindromic Repeats Complement Factor B Complex DNA Methylation DNA biosynthesis Data Deletion Mutation Development Development Plans Documentation Embryo FOXM1 gene Future Gene Expression Gene Silencing Genes Genetic Genotype Goals Growth Guide RNA Health Heterochromatin Histones Human Image Knowledge Laboratory Research Lead Light Malignant - descriptor Malignant neoplasm of brain Mentors Methodology Methods Mitosis Modeling Molecular Molecular Biology Molecular Chaperones Mutation Neurons Pathway interactions Play Regulation Reporter Research Research Personnel Research Project Grants Resolution Reverse Transcription Role Scientist Sequence Analysis Site Statistical Data Interpretation Technical Expertise Testing Tissues Training Transgenic Organisms Zebrafish cancer cell career career development cdc Genes chromatin remodeling confocal imaging data acquisition design design and construction experimental study functional genomics gene function graduate student in vivo inhibitor/antagonist innovation insight mutant neoplastic cell nerve stem cell novel prevent promoter responsible research conduct skills sound tool development transcription factor vector

项目摘要

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.

组蛋白伴侣 Rbbp4 是多种染色质重塑复合物的组成部分参与细胞身份、可塑性和重编程（Cheloufi 和 Hochedlinger，2017；克莱莫特等人，2018；康威等人，2015）。 Rbbp4 在细胞周期控制中也发挥着重要作用作为调节细胞周期基因表达的 MuvB 复合体的组成部分。连同转录因子 B-Myb 和 FoxM1，MuvB 复合体控制基因的时序 G1/S（生长和 DNA 合成）和 G2/M（有丝分裂）中的表达，允许进展整个细胞周期（Fischer 和 Muller，2017）。我们之前在斑马鱼中演示过 rb1 缺陷型胚胎脑肿瘤模型中，rbbp4 升高了 10 倍以上，我们研究表明，在 rbbp4 突变胚胎中，神经祖细胞经历 tp53 依赖性细胞凋亡（舒尔茨等人，2018）。这些结果表明抑制 Rbbp4 可能会阻止增殖和通过 Tp53 编程细胞激活细胞周期停滞来维持脑癌细胞的存活死亡途径（英格兰，2018）。然而，虽然大多数恶性人类脑癌显示 Rbbp4 表达升高（Schultz、Kool、McGrail 未发表的结果），许多也含有 Tp53 失活突变。更深入地了解 Rbbp4 的机制有助于神经祖细胞周期进展可能会导致新的目标，即与Rbbp4抑制剂结合，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 靶向整合策略来生成新型原神经细胞周期报告基因和foxm1条件基因失活斑马鱼系。这些将使我们能够检查 Rbbp4 和 FoxM1 在神经祖细胞周期中如何特异性合作大脑发育过程中的进展。这些结果将为神经祖细胞带来新的见解细胞周期控制及其失调如何驱动脑肿瘤细胞不受控制的增殖。