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

基本信息

批准号：
10561685
负责人：
Nadia Dahmane
金额：
$ 51.54万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
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/10561685
关键词：
ATAC-seq Acetylation Adult Affect Binding Brain Brain Diseases Brain region Catalytic Domain Cell Differentiation process Cell Fate Control Cell Lineage Cell Separation Cells Cerebellum 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 Molecular Analysis Motor Mus Mutation Neocortex Neurodegenerative Disorders Neurologic Neuronal Differentiation Neurons Nucleosomes Pennsylvania Polycomb Post-Translational Protein Processing Process Proliferating Proteins Proteomics Regulation 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 gene repression in vivo insight life-long learning loss of function member mouse genetics neocortical nervous system disorder neurogenesis postmitotic postnatal progenitor programs protein protein interaction transcription factor transcriptome

项目摘要

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导致的一个未解决的问题是哺乳动物的神经身份是如何的。在发育和成年期间维持和保护，特别是在转录水平上。 BTB/POZ 和锌指转录因子 RP58（又名 ZBTB18）是大脑发育和体内和体外的神经元分化；RP58 突变与人类小头畸形和小体畸形有关；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。总体假设是 RP58 是建立和维持神经同一性所必需的，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。破译 RP58 如何控制大脑中的细胞分化，并确定可能的 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) 我们的数据表明，RP58 与小鼠胚胎和出生后皮质的成员结合。 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) 和增加这些结果提出了 RP58 控制发育和成人身份的假设。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 和染色质重塑的方式。复合物相互作用以建立和维持神经同一性目标 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。神经细胞中的相互作用网络这项资助是达赫曼实验室（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）拥有定量质谱方面的专业知识，因为它与表观遗传机制有关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。