Cell-type Specific Epigenomics in the Brain

大脑中细胞类型特异性表观基因组学

基本信息

批准号：
8822927
负责人：
EDWIN TED G. ABEL
金额：
$ 19.32万
依托单位：
UNIVERSITY OF PENNSYLVANIA
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：
2014-03-14 至 2017-01-01
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8822927
关键词：
Acetylation Acids Active Sites Address Affect Aging Alzheimer&apos 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 Genomic Segment Health 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 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 epigenetic regulation epigenomics excitatory neuron histone modification improved inhibitory neuron interest memory consolidation mouse genome nervous system disorder new therapeutic target novel novel therapeutics promoter research study response tool transcription factor

项目摘要

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（由申请人提供）：表观遗传学，即在不改变潜在 DNA 序列的情况下修改基因表达，在调节大脑功能（包括记忆、药物成瘾和神经退行性疾病）方面发挥着至关重要的作用，尽管大脑表观遗传学取得了重要突破，但适当的工具仍然存在。研究特定细胞亚群中的表观遗传调控目前还不存在，这是由于大脑复杂的异质性，这可能会掩盖特定细胞亚群中发生的重要信号。为了解决这个问题，我们建议使用一种方法。 tetO 调节的 HA 标记组蛋白 H3.3。组蛋白 H3.3 掺入 DNA 复制之外的染色质，并优先掺入活跃转录区域。因此，标记的组蛋白 H3.3 将成为特定细胞中活性染色质的标记。在本提案中，我们将使用 CaMKII¿ -tTA 驱动线在兴奋性前脑神经元中表达这种标记的组蛋白，HA 标签将分离出与兴奋性神经元基因组活性区域结合的核小体，并与 Garcia 实验室合作，通过质谱法对这些核小体上发现的组蛋白修饰进行定量。具体目标 1，我们将对 CaMKII-tTA x tetO-H3.3-HA 小鼠进行免疫染色。用于确认 HA 标记的组蛋白仅存在于兴奋性神经元中，并且将使用 HA 或内源性 H3.3 抗体来测试转基因效果的动物行为，以与分离的 H3 进行比较。 .3-HA 含有来自笼养小鼠和恐惧条件小鼠的兴奋性神经元的核小体，这将确定 H3.3 结合的精确基因组区域，以响应特定目标的兴奋性神经元。 2，我们将使用新的组蛋白蛋白质组学策略来量化在家笼和恐惧条件小鼠中进行 HA 免疫沉淀后来自整个海马或分离核小体的组蛋白修饰，这将确定对兴奋性神经元活跃区域的学习做出反应的精确组蛋白修饰。除了解决重要问题之外，记忆巩固过程中发生的组蛋白修饰可能会发现出现认知缺陷的疾病的新治疗靶点，包括精神分裂症和阿尔茨海默病。其中组蛋白修饰的组合在记忆后会发生变化，该提案有望提供可供所有领域的研究人员使用的工具，以应对细胞异质性。