The role of CHD5 in chromatin-mediated regulation of neural stem cells and glioma

描述（由申请人提供）： 该项目的目标是定义调节组织稳态和癌症之间的精细平衡的遗传/表观遗传机制。神经干细胞（Neural stem cells，NSCs）分化为大脑的多个谱系，在这一过程中的扰动可导致胶质瘤，这是一种众所周知的难以治疗的脑癌。CHD 5被发现为在该染色体区域内编码的肿瘤抑制因子，并且CHD 5高表达预测胶质瘤治疗后患者存活率更高。该提案旨在通过确定CHD 5调节染色质以影响控制NSC分化/命运的全局基因表达级联的机制来建立这些初步发现，并确定这些CHD 5介导的途径的扰动如何影响胶质瘤的发展。目的1是确定Chd 5调节神经干细胞染色质标记和基因表达级联的机制。这将通过以下方式实现：A）使用免疫沉淀和质谱法鉴定Chd 5相互作用蛋白，B）通过在来自Chd 5受损小鼠的NSC中进行RNA测序和ChIP测序来定义全局基因表达和共价组蛋白修饰模式，以及C）鉴定Chd 5与其它蛋白相互作用以及引起对其靶基因的转录调节至关重要的共价组蛋白修饰所需的Chd 5的功能基序，以及评估肿瘤来源的CHD 5突变如何阻碍这些能力。目的2的目标是确定Chd 5在调节NSC分化/命运中的作用。这将通过以下方式实现：A）使用流式细胞术确定Chd 5缺乏如何影响干细胞群体、细胞周期动力学和分化，B）使用定量PCR、蛋白质印迹和免疫荧光定义Chd 5相互作用蛋白和靶基因在分化过程中来自Chd 5-lacZ报告小鼠的NSC中的表达和亚细胞定位的时间模式，和C）评估基序特异性和肿瘤来源的突变如何影响Chd 5调节NSC分化/命运的能力。目的3的目标是评估扰乱Chd 5的后果及其对体内胶质瘤形成的调节途径。这将通过以下方式实现：A）使用原位移植确定Chd 5缺陷和Chd 5调节途径中的损伤如何影响胶质瘤发生，B）通过比较Chd 5受损的NSC与患者胶质瘤样品的组蛋白标记和基因表达的总体模式来评估小鼠平行人胶质瘤中Chd 5介导的改变的密切程度，和C）定义基序特异性和肿瘤来源的CHD 5突变对胶质瘤发生的影响。该项目将使用独特的Chd 5模型，以进一步确定Chd 5介导的染色质动力学在神经发生和胶质瘤发展中的生物学作用。此外，这项工作将阐明影响神经胶质瘤的遗传/表观遗传过程，从而揭示癌症特异性的脆弱性，可能为治疗这种目前无法治愈的恶性肿瘤提供药物干预。

DESCRIPTION (provided by applicant): The goal of this project is to define genetic/epigenetic mechanisms that regulate the fine balance between tissue homeostasis and cancer. Neural stem cells (NSCs) differentiate into multiple lineages of the brain, and perturbations in this process can lead to glioma, a brain cancer that is notoriously difficult to treat. A frequent genetic lesion in glioma is deletion of 1p36. CHD5 was discovered as a tumor suppressor encoded within this chromosomal region, and high CHD5 expression predicts better patient survival following glioma treatment. This proposal seeks to build upon these initial discoveries by determining the mechanism whereby CHD5 regulates chromatin to affect global gene expression cascades that control NSC differentiation/fate and to define how perturbation of these CHD5-mediated pathways affects glioma development. The goal of Aim 1 is to determine the mechanism whereby Chd5 regulates chromatin marks and gene expression cascades in NSCs. This will be accomplished by: A) identifying Chd5-interacting proteins using immunoprecipitation and mass spectrometry, B) defining global gene expression and covalent histone modification patterns by performing RNA-sequencing and ChIP-sequencing in NSCs from Chd5-compromised mice, and C) identifying functional motifs of Chd5 required for its interactions with other proteins and for evoking covalent histone modifications critical for transcriptional regulation of its target genes, and by assessing how tumor-derived CHD5 mutations obstruct these capabilities. The goal of Aim 2 is to define the role of Chd5 in regulating NSC differentiation/fate. This will be accomplished by: A) determining how Chd5 deficiency affects stem cell populations, cell cycle dynamics, and differentiation using flow cytometry, B) defining the temporal pattern of expression and subcellular localization of Chd5-interacting proteins and target genes using quantitative PCR, western blotting, and immunofluorescence in NSCs from Chd5-lacZ reporter mice during the differentiation process, and C) assessing how motif-specific and tumor-derived mutations affect Chd5's ability to regulate NSC differentiation/fate. The goal of Aim 3 is to assess the consequence of perturbing Chd5 and the pathways it regulates on glioma formation in vivo. This will be accomplished by: A) determining how Chd5 deficiency and lesions in Chd5-modulated pathways affect gliomagenesis using orthotopic transplantation, B) assessing how closely Chd5-mediated alterations in the mouse parallel human glioma by comparing global patterns of histone marks and gene expression of Chd5-compromised NSCs to those of patient glioma samples, and C) defining the effect that motif-specific and tumor-derived CHD5 mutations have on gliomagenesis. This project will use unique models for Chd5 to further define the biological role of Chd5- mediated chromatin dynamics in neurogenesis and in glioma development. In addition, this work will elucidate genetic/epigenetic processes that impact glioma, thereby revealing cancer-specific vulnerabilities that may offer pharmacological interventions for treating this currently incurable malignancy.