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

CHD5 在染色质介导的神经干细胞和神经胶质瘤调节中的作用

基本信息

批准号：
8994369
负责人：
Alea A. Mills
金额：
$ 10万
依托单位：
COLD SPRING HARBOR LABORATORY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-16 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8994369
关键词：
1p36 Affect Behavior Control Biological Brain Cancer Etiology Cell Cycle Cells ChIP-seq Chromatin Chromosome Mapping Chromosomes DNA DNA Binding Domain DNA Packaging Defect Development Diffuse Engineering Epigenetic Process Equilibrium Family Flow Cytometry Future Gene Expression Gene Expression Regulation Gene Targeting Genes Genetic Genome Glioma Gliomagenesis Goals Health Histone H3 Histones Homeostasis Human Immunofluorescence Immunologic Immunoprecipitation Intervention LacZ Genes Lead Lesion Malignant Childhood Neoplasm Malignant Neoplasms Malignant neoplasm of brain Mass Spectrum Analysis Mediating Modeling Molecular Mus Mutate Mutation Neurons Pathway interactions Patients Pattern Phenotype Play Pontine structure Prevalence Process Prognostic Marker Proteins RNA Sequences Regulation Reporter Role Sampling Staging Stem cells Tissues Transcriptional Regulation Transplantation Tumor Suppression Tumor Suppressor Genes Tumor Suppressor Proteins Tumor-Derived Western Blotting Work base brain cell cancer gene expression cancer therapy cell behavior chromatin remodeling design effective therapy genetic manipulation helicase histone modification in vivo insight member mouse model nerve stem cell neurogenesis neuroregulation novel prevent programs stem stem cell biology stem cell differentiation stem cell population transcriptome sequencing tumor tumorigenic

项目摘要

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.

描述(由申请人提供)：该项目的目标是确定基因/表观遗传机制，以调节组织内稳态和癌症之间的微妙平衡。神经干细胞(NSCs)分化为多个大脑谱系，这一过程中的干扰可能会导致胶质瘤，这是一种出了名的难以治疗的脑癌。胶质瘤中一种常见的遗传损害是1p36的缺失。CHD5被发现是在该染色体区域编码的一种肿瘤抑制基因，CHD5的高表达预示着胶质瘤治疗后患者更好的生存。这项建议旨在通过确定CHD5调节染色质从而影响控制NSC分化/命运的全球基因表达级联的机制，并确定这些CHD5介导的通路的扰动如何影响胶质瘤的发展，以期建立在这些初步发现的基础上。目标1的目的是确定Chd5调节神经干细胞中染色质标记和基因表达级联的机制。这将通过以下方式完成：A)使用免疫沉淀和质谱分析确定Chd5相互作用的蛋白质；B)通过在Chd5基因受损的小鼠神经干细胞中进行RNA测序和芯片测序来定义全球基因表达和共价组蛋白修饰模式；以及C)确定Chd5与其他蛋白质相互作用所需的功能基序和引发对其目标基因的转录调控至关重要的共价组蛋白修饰，并评估肿瘤来源的CHD5突变如何阻碍这些功能。目标2的目的是确定Chd5在调节NSC分化/命运中的作用。这将通过以下方法完成：A)使用流式细胞仪确定ChD5缺乏如何影响干细胞群、细胞周期动力学和分化；B)通过定量聚合酶链式反应、Western blotting和免疫荧光技术确定ChD5-lacZ报告鼠神经干细胞在分化过程中ChD5相互作用蛋白和靶基因的表达和亚细胞定位的时间模式；以及C)评估基序特异性和肿瘤衍生突变如何影响ChD5的S调节神经干细胞分化/命运的能力。目标3的目的是评估干扰Chd5的后果以及它在体内调节胶质瘤形成的途径。这将通过以下方法完成：A)确定Chd5缺乏和Chd5调节通路中的损伤如何通过原位移植影响胶质瘤形成；B)通过比较Chd5受损神经干细胞和患者胶质瘤样本的组蛋白标记和基因表达的全球模式，评估Chd5介导的小鼠平行人脑胶质瘤中的变化有多接近；以及C)确定基序特异的和肿瘤衍生的CHD5突变对胶质瘤形成的影响。该项目将使用Chd5的独特模型来进一步确定Chd5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胶质瘤的遗传/表观遗传过程，从而揭示癌症特有的脆弱性，可能为治疗这种目前无法治愈的恶性肿瘤提供药理学干预。