Cell-type Specific Epigenomics in the Brain

大脑中细胞类型特异性表观基因组学

• 批准号: 8620437
• 负责人: EDWIN TED G. ABEL
• 金额: $ 23.34万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-03-14 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8620437
• 关键词: Acetylation; Acids; Active Sites; Address; Affect; Aging; Alzheimer' s Disease; Animal Behavior; Antibodies; Area; Astrocytes; Behavior; Behavioral; Binding; Bioinformatics; Brain; Cells; Chromatin; Cognition; Cognitive deficits; Collaborations; Complex; DNA Sequence; DNA biosynthesis; Data; Disease; Drug Addiction; Epigenetic Process; Experimental Designs; Flow Cytometry; Gene Expression; Genes; Genetic Transcription; Genome; Genomics; Heterogeneity; Hippocampus (Brain); Histone Acetylation; Histone H3.3; Histones; Immunoprecipitation; Lead; Learning; Mass Spectrum Analysis; Memory; Mental disorders; Methods; Microglia; Modification; Mus; N-terminal; Neurodegenerative Disorders; Neurons; Nucleosomes; Organ; Play; Population; Post-Translational Protein Processing; Problem Solving; Prosencephalon; Proteins; Proteomics; Protocols documentation; Regulation; Research Personnel; Role; Schizophrenia; Shotguns; Signal Transduction; Sorting - Cell Movement; Tail; Techniques; Testing; Tetanus Helper Peptide; Tetracyclines; Tissue Sample; Trans-Activators; Transgenes; Transgenic Mice; Variant; addiction; base; brain tissue; calmodulin-dependent protein kinase II; cell type; chromatin immunoprecipitation; combinatorial; conditioned fear; epigenomics; excitatory neuron; histone modification; improved; inhibitory neuron; interest; mouse genome; nervous system disorder; new therapeutic target; novel; novel therapeutics; promoter; public health relevance; research study; response; tool; transcription factor

DESCRIPTION (provided by applicant): Epigenetics, the modification of gene expression without altering the underlying DNA sequence plays a crucial role in regulating brain function including memory, drug addiction, and neurodegenerative disease. Despite important breakthroughs in epigenetics in the brain, the proper tools to study epigenetic regulation in specific cellular subpopulations do not currently exist. This is due to the complex heterogeneity of the brain, which can obscure important signals that occur in specific subsets of cells To solve this problem, we propose using a tetO-regulated, HA-tagged histone H3.3. Histone H3.3 incorporates into chromatin outside of DNA replication and preferentially into actively transcribed regions. The tetracycline transactivator (tTA), which allows expression of tet-regulated transgenes, can be controlled in a cell-specific manner using cell-type specifc promoters. Therefore, the tagged histone H3.3 will be a marker of active chromatin specifically in cells of interest. In this proposal, we will use the CaMKII¿-tTA driver line to express this tagged histone in excitatory forebrain neurons. ChIP for the HA tag will isolat nucleosomes bound to active regions of the excitatory neuron genome, and in collaboration with the Garcia lab, the histone modifications found on these nucleosomes will be quantified by mass spectrometry. In Specific Aim 1, we will characterize CaMKII-tTA x tetO-H3.3-HA mice. Immunostaining will be used to confirm the HA-tagged histone is present exclusively in excitatory neurons and behavior of the animals will be tested fr effects of the transgene. ChIP-seq will be performed using either an HA antibody or a endogenous H3.3 antibody for comparison to isolate H3.3-HA containing nucleosomes from excitatory neurons in homecage and fear conditioned mice. This will determine the precise genomic regions bound by H3.3 in response to learning in excitatory neurons. In Specific Aim 2, we will use novel histone proteomics strategies to quantify histone modifications from whole hippocampi or isolated nucleosomes after HA immunoprecipitation in homecage and fear conditioned mice. This will determine the precise histone modifications that respond to learning at active regions in excitatory neurons. Understanding the combinatorial histone modifications that occur during memory consolidation may uncover novel therapeutic targets for diseases in which cognitive deficits occur, including schizophrenia and Alzheimer's. In addition to addressing the important question of which combinations of histone modifications change after memory, this proposal promises to provide tools that can be used by researchers in all fields that struggle with cellular heterogeneity.

描述（应用程序提供）：表观遗传学，基因表达的修饰而不改变潜在的DNA序列在调节脑功能中起着至关重要的作用，包括记忆，药物成瘾和神经退行性疾病。尽管大脑表观遗传学的重要突破，但目前尚不存在研究特定细胞亚群中研究表观遗传调节的合适工具。这是由于大脑的复杂异质性，这可能会掩盖细胞特定子集中发生的重要信号以解决此问题，因此我们建议使用TETO调节的，标有HA的组蛋白H3.3。组蛋白H3.3掺入DNA复制外的染色质中，优选地纳入主动转录区域。可以使用细胞类型的特定启动子以细胞特异性的方式控制TETCRDENS基因的四环素反式激活器（TTA）。因此，标记的组蛋白H3.3将是专门在感兴趣的细胞中的活性染色质的标记。在此提案中，我们将使用camkii¿-tta驱动程序线 在兴奋的前脑神经元中表达这种标记的组蛋白。 HA标签的芯片将隔离到兴奋神经元基因组的活性区域的核产品，并与Garcia实验室合作，这些核产品上发现的组蛋白修饰将通过质谱法量化。在特定的目标1中，我们将表征camkii-tta x teto-h3.3-ha小鼠。免疫染色将用于确认具有HA标记的组蛋白仅存在于兴奋性神经元中，而动物的行为将受到转化的FR效应。将使用HA抗体或内源性H3.3抗体进行CHIP-SEQ，以比较与Homecage中的兴奋性神经元中的含有核小体分离的H3.3-HA，并进行恐惧的小鼠。这将确定响应于兴奋性神经元学习的H3.3束缚的精确基因组区域。在特定的目标2中，我们将使用新型的组蛋白蛋白质组学策略来量化HA免疫沉淀在归宿和恐惧条件小鼠后的整个海马或分离核酶体中的组蛋白修饰。这将确定兴奋性神经元中活跃区域学习的精确组蛋白修饰。了解记忆巩固过程中发生的组合组蛋白修饰可能会发现出现认知缺陷的疾病的新型治疗靶标，精神分裂症和阿尔茨海默氏症。除了解决记忆之后组蛋白修饰的组合发生变化的重要问题外，该提案还有望提供工具，这些工具可以被所有与细胞异质性困难的领域的研究人员一起使用。