Cell-type Specific Epigenomics in the Brain

描述（由申请人提供）：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（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。尽管在大脑表观遗传学方面取得了重大突破，但目前尚不存在研究特定细胞亚群表观遗传学调控的适当工具。这是由于大脑复杂的异质性，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标记组蛋白H3.3。组蛋白H3.3结合到DNA复制外的染色质中，并优先进入活跃转录区域。四环素反激活因子（tTA）允许表达受tet调控的转基因，可以使用细胞类型特异性启动子以细胞特异性方式进行控制。因此，标记的组蛋白H3.3将成为感兴趣细胞中活性染色质的特异性标记。在本提案中，我们将使用CaMKII -tTA驱动线来

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.