Cell-type Specific Epigenomics in the Brain

描述（由申请人提供）：表观遗传学，即在不改变潜在DNA序列的情况下修饰基因表达，在调节脑功能（包括记忆、药物成瘾和神经退行性疾病）中起着至关重要的作用。 尽管大脑表观遗传学取得了重要突破，但目前还不存在研究特定细胞亚群表观遗传调控的适当工具。这是由于大脑的复杂异质性，这可能会掩盖发生在特定细胞亚群中的重要信号。为了解决这个问题，我们建议使用tetO调节的HA标记的组蛋白H3.3。组蛋白H3.3结合到DNA复制之外的染色质中，并且优先结合到活跃转录区域中。 四环素反式激活因子（tTA）允许tet调节的转基因的表达，可以使用细胞类型特异性启动子以细胞特异性方式控制。 因此，标记的组蛋白H3.3将是目标细胞中特异性活性染色质的标志物。在本提案中，我们将使用CaMKII â-tTA驱动程序线来 在兴奋性前脑神经元中表达这种标记的组蛋白。 HA标签的ChIP将分离与兴奋性神经元基因组的活性区域结合的核小体，并与Garcia实验室合作，将通过质谱法定量这些核小体上发现的组蛋白修饰。在具体目标1中，我们将表征CaMKII-tTA x tetO-H3. 3-HA小鼠。 将使用免疫染色来确认HA标记的组蛋白仅存在于兴奋性神经元中，并且将测试动物的行为以免受转基因的影响。 将使用HA抗体或内源性H3.3抗体进行ChIP-seq以用于比较，从而从居家和恐惧条件化小鼠中的兴奋性神经元分离含有H3.3-HA的核小体。这将确定H3.3响应兴奋性神经元中的学习而结合的精确基因组区域。在特定目标2中，我们将使用新的组蛋白蛋白质组学策略来量化在家庭饲养和恐惧条件化小鼠中HA免疫沉淀后来自整个海马或分离的核小体的组蛋白修饰。 这将确定在兴奋性神经元的活动区域响应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发生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氏症。 除了解决记忆后组蛋白修饰改变的组合这一重要问题外，该提案还有望提供可供研究人员在与细胞异质性作斗争的所有领域使用的工具。

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.