A Mass Spectrometry Approach to the Genetic and Epigenetic Mechanisms Controlling Neuronal Identity

控制神经元身份的遗传和表观遗传机制的质谱方法

• 批准号: 10339433
• 负责人: Nadia Dahmane
• 金额: $ 51.61万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10339433
• 关键词: ATAC-seq; Acetylation; Address; Adult; Affect; Binding; Brain; Brain Diseases; Brain region; Catalytic Domain; Cell Differentiation process; Cell Lineage; Cells; ChIP-seq; Chromatin; Chromatin Remodeling Factor; Co-Immunoprecipitations; Cognitive; Collaborations; Complex; Corpus Callosum; Coupled; DNA Binding; Data; Defect; Development; Developmental Biology; Diagnosis; Embryo; Epigenetic Process; Epilepsy; Future; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Transcription; Global Change; Grant; Growth; Histones; Human; Immunoprecipitation; In Vitro; Intellectual functioning disability; Lead; Life; Link; Maintenance; Malignant neoplasm of brain; Maps; Mass Spectrum Analysis; Medicine; Mental Retardation; Methylation; Microcephaly; Mitotic; Molecular Analysis; Motor; Mus; Mutation; Neocortex; Neurodegenerative Disorders; Neurologic; Neuronal Differentiation; Neurons; Nucleosomes; POZ-zinc; Pennsylvania; Play; Polycomb; Post-Translational Protein Processing; Process; Proliferating; Proteins; Proteomics; Regulation; Role; SWI/SNF Family Complex; Schizophrenia; Testing; Therapeutic Intervention; Tissues; Universities; Western Blotting; Zinc Fingers; brain malformation; cell type; developmental neurobiology; disability; epigenetic regulation; experimental study; in vivo; insight; life-long learning; loss of function; member; mouse genetics; nervous system disorder; neurogenesis; postnatal; progenitor; programs; transcription factor; transcriptome

Abstract Deciphering the mechanisms controlling cell fate choice and maintenance in the brain is a critical step in understanding devastating neurological disorders such as microcephaly, schizophrenia and brain cancer that result from defects in these processes. An unresolved question is how the mammalian neuronal identity is maintained and protected, particularly at the transcriptional level, during development and adult life. The BTB/POZ and Zinc finger transcription factor RP58 (aka ZBTB18), is required for brain development and neuronal differentiation both in vivo and in vitro; Mutations in RP58 are linked to human microcephaly and corpus callosum agenesis. We have analyzed the embryonic cortical post mitotic neurons’ transcriptome and showed that the expression of gene markers of other cell lineages, such as myogenic lineage, is increased following Rp58 deletion suggesting that RP58 protects the neuronal identity by repressing genes of other lineages. Our overall hypothesis is that RP58 is required to establish and maintain the neuronal identity and that the loss of its transcriptional function may lead to microcephaly and to neurodegenerative diseases in the adult brain. To decipher how RP58 controls cell differentiation in the brain and to identify RP58 protein partners that may be involved in its function, we have performed RP58 immunoprecipitation coupled to mass spectrometry (IP-MS) experiments on mouse embryonic and postnatal cortices. Our data show that RP58 binds to members of both the Polycomb Repressive Complex 2 (PRC2) and the SWI/SNF complex, two critical chromatin remodeling complexes involved in brain development. In addition, using quantitative histone proteomics and western blot analyses, we show that deletion of Rp58 in the embryonic brain leads to global changes in histones post- translational modifications (PTMs) including to decreased H3K27 methylation (H3K27me) and increased H3K9/14 acetylation. These results raise the hypothesis that RP58 controls the identity of developing and adult neurons and thus their transcriptional program by modulating the PRC2 and SWI/SNF complexes function and the chromatin landscape. The following aims will address these hypotheses. Aim 1: Decipher how RP58 and chromatin remodeling complexes interact to establish and maintain neuronal identity. Aim 2: Decipher if and how RP58 is required for histone PTMs in neural cells. Aim 3: Determine the cell and developmental stage-specific RP58 protein-protein interacting network in neural cells. This grant is a collaboration between the Dahmane lab (Weill Cornell Medicine) with expertise in developmental neurobiology and mouse genetics and the Garcia lab (University of Pennsylvania) with expertise in quantitative mass spectrometry as it relates to epigenetic mechanisms and chromatin regulation. Completion of these proposed studies will lead to the elucidation of the cell type and stage specific RP58 regulatory mechanisms controlling neuronal identity and to key insights into the genetic and epigenetic regulation of brain growth and of their misregulation in brain disorders such as microcephaly.

抽象的 破译大脑中控制细胞命运选择和维持的机制是关键一步 了解破坏性神经系统疾病，例如小头畸形、精神分裂症和脑癌 这些过程中的缺陷造成的。一个未解决的问题是哺乳动物的神经元身份是如何的 在发育和成年期间维持和保护，特别是在转录水平上。这 BTB/POZ 和锌指转录因子 RP58（又名 ZBTB18）是大脑发育和发育所必需的 体内和体外神经元分化； RP58 突变与人类小头畸形和语料库有关 胼胝体发育不全。我们分析了胚胎皮质有丝分裂后神经元的转录组并显示 其他细胞谱系（例如肌源性谱系）的基因标记的表达在以下情况下增加 Rp58 缺失表明 RP58 通过抑制其他谱系的基因来保护神经元身份。我们的 总体假设是 RP58 是建立和维持神经元身份所必需的，并且其丧失 转录功能可能导致成人大脑中的小头畸形和神经退行性疾病。到 破译 RP58 如何控制大脑中的细胞分化，并识别可能的 RP58 蛋白伴侣 为了了解其功能，我们进行了 RP58 免疫沉淀偶联质谱 (IP-MS) 小鼠胚胎和出生后皮质的实验。我们的数据显示 RP58 与两者的成员结合 Polycomb 抑制复合体 2 (PRC2) 和 SWI/SNF 复合体，两个关键的染色质重塑 参与大脑发育的复合体。此外，使用定量组蛋白蛋白质组学和蛋白质印迹 分析表明，胚胎大脑中 Rp58 的缺失会导致组蛋白的整体变化。 翻译修饰 (PTM) 包括降低 H3K27 甲基化 (H3K27me) 和增加 H3K9/14 乙酰化。这些结果提出了这样的假设：RP58 控制发育中和成人的身份 神经元及其转录程序通过调节 PRC2 和 SWI/SNF 复合物的功能和 染色质景观。 以下目标将解决这些假设。目标 1：破译 RP58 和染色质重塑的过程 复合物相互作用以建立和维持神经元身份。目标 2：破译 RP58 是否需要以及如何需要 神经细胞中的组蛋白 PTM。目标 3：确定细胞和发育阶段特异性 RP58 蛋白-蛋白 神经细胞中的相互作用网络。这笔赠款是 Dahmane 实验室（威尔康奈尔大学）之间的合作 医学）拥有发育神经生物学和小鼠遗传学方面的专业知识，加西亚实验室（University of 宾夕法尼亚州）拥有定量质谱专业知识，因为它与表观遗传机制有关 染色质调节。完成这些拟议的研究将有助于阐明细胞类型和阶段 控制神经元身份的特定 RP58 调节机制以及对遗传和遗传的关键见解 大脑生长的表观遗传调控及其在小头畸形等大脑疾病中的失调。