Cell-type Specific Epigenomics in the Brain

描述(申请人提供)：表观遗传学，在不改变潜在DNA序列的情况下改变基因表达，在调节大脑功能方面发挥关键作用，包括记忆、药物成瘾和神经退行性疾病。尽管在大脑的表观遗传学方面取得了重大突破，但目前还不存在研究特定细胞亚群中表观遗传学调控的适当工具。这是由于大脑的复杂异质性，它可以掩盖出现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在DNA复制外结合到染色质中，并优先进入活跃转录的区域。四环素反式激活剂(TTA)允许表达tet调节的转基因，可以使用细胞类型特异性启动子以细胞特异性的方式进行控制。因此，标记的组蛋白H3.3将成为活性染色质的标记，特异性地存在于感兴趣的细胞中。在本提案中，我们将使用CaMKII？-TTA驱动程序行来 在兴奋性前脑神经元中表达这种标记的组蛋白。HA标签芯片将分离与兴奋性神经元基因组的活性区域结合的核小体，并与加西亚实验室合作，通过质谱仪对这些核小体上发现的组蛋白修饰进行量化。在特定目标1中，我们将鉴定CaMKII-TTA x Teto-H3.3-HA小鼠。免疫染色将用于确认HA标记的组蛋白仅存在于兴奋性神经元中，并将测试转基因对动物行为的影响。将使用HA抗体或内源性H3.3抗体进行CHIP-SEQ，以与从在家和恐惧条件下小鼠的兴奋性神经元中分离出含有核小体的H3.3-HA进行比较。这将确定H3.3在兴奋性神经元中对学习的反应所结合的精确基因组区域。在特定的目标2中，我们将使用新的组蛋白蛋白质组学策略来量化在家和恐惧条件下小鼠的整个海马区或分离的核小体在HA免疫沉淀后的组蛋白修饰。这将确定在兴奋性神经元的活跃区对学习做出反应的精确的组蛋白修饰。了解记忆巩固过程中发生的组蛋白组合修饰，可能会为包括精神分裂症和阿尔茨海默氏症在内的认知障碍疾病发现新的治疗靶点。除了解决组蛋白修饰的哪些组合在记忆后发生变化这一重要问题，这一提议有望提供工具，供所有与细胞异质性作斗争的领域的研究人员使用。

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.