Epigenome Mapping in Cortical Interneurons

描述（由申请人提供）：该项目的两个主要目标是：1)首次为研究界和公共领域提供一个全面的全基因组图谱，该图谱是在小鼠大脑皮层中选定的皮质gaba能中间神经元和其他细胞亚群中组蛋白甲基化景观；2)首次了解gaba能神经元在幼年向成年过渡过程中的染色质重塑机制。先前对人类和小鼠皮层提取的染色质的研究表明，“gaba能基因”启动子的组蛋白甲基化在成熟的延长期间至少在成年早期受到动态调节，从而将染色质重塑机制与发育时钟联系起来。关注皮层中间神经元表观基因组的理由不仅仅是学术上的好奇心。gaba能中间神经元的基因表达失调被认为是分子病理生理学的标志，也是影响精神病或自闭症患者大脑皮层广泛区域的神经网络同步缺陷的主要因素。这些与GABA相关的基因表达缺陷包括不同的细胞类型，如通常由钙结合蛋白Parvalbumin表达识别的快速尖峰中间神经元。因此，在本提案的背景下，我们计划产生4个BAC（细菌人工染色体）转基因小鼠的等基因系，在GABAergic间神经元中表达绿色荧光蛋白（GFP）标记的组蛋白H2B，并在3个特定亚群中表达钙缓冲蛋白Parvalbumin （PARV）或Calbindin （CALB）或Calretinin （CALR）的差异表达。最近的方法进步使我们能够从脑组织中高效分离和分选gfp标记的细胞核，用于染色质免疫沉淀，随后进行大规模平行测序（ChIP-seq）。重点是三甲基和单甲基组蛋白h3 -赖氨酸4 (H3K4me3, H3K4me1)，它们富集于转录起始位点（H3K4me3）和增强子序列，包括从近端启动子进一步移除的序列（H3K4me1），以及与RNA聚合酶II活性和编码区和非编码区转录延伸相关的标记（H3K36me3）。我们预计，在功能和发育历史方面不同的各种中间神经元亚群将在基因组的许多部分显示细胞类型特异性染色质特征。当结合细胞特异性转录组和其他数据集进行分析时，特定中间神经元类型的组蛋白甲基化定位可能为在精神分裂症和相关疾病中起关键作用的细胞的发育历史和（epi）基因组结构提供全新的见解。

DESCRIPTION (provided by applicant): The two major goals of this project is to 1) provide the research community and public domain for the first time with a comprehensive genome-wide atlas of the histone methylation landscape in selected subpopulations of cortical GABAergic interneurons and other cells residing in mouse cerebral cortex; and 2) To gain first insights into chromatin remodeling mechanisms of GABAergic neurons during the transition from juvenile to mature age. Previous work on chromatin extracted from human and mouse cortex indicated that histone methylation at "GABAergic gene" promoters is dynamically regulated during the extended period of maturation at least until early adulthood, thereby linking chromatin remodeling mechanisms to the developmental clock. The rationale to focus on the epigenome of cortical interneurons goes beyond mere academic curiosity. Dysregulated gene expression in GABAergic interneurons is considered a hallmark of the molecular pathophysiology and a major factor for the synchronization deficits in neural networks that affect widespread areas of the cerebral cortex in subjects on the psychosis or autism spectrum. These GABA related gene expression deficits include distinct cell types such as the class of fast spiking interneurons commonly recognized by expression of the calcium binding protein, Parvalbumin. Therefore, in the context of this proposal, we plan to generate 4 isogenic lines of BAC (bacterial artificial chromosome) transgenic mice to express green fluorescent protein (GFP)-tagged histone H2B in GABAergic interneurons overall, and in 3 specific subpopulations defined by differential expression of calcium buffering proteins Parvalbumin (PARV) or Calbindin (CALB) or Calretinin (CALR). Recent methodological advances enable us to separate and sort with high efficiency GFP-tagged nuclei from brain tissue for the purposes of chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq). Focus will be on tri- and mono-methyl-histone H3-lysine 4 (H3K4me3, H3K4me1) which are enriched at transcription start sites (H3K4me3) and enhancer sequences including those further removed from proximal promoters (H3K4me1), and a mark associated with RNA polymerase II activity and transcriptional elongation across coding and non-coding regions (H3K36me3). We expect that the various interneuron subpopulations, which differ in terms of function and developmental history, will show cell type specific chromatin signatures in many portions of the genome. When analyzed in conjunction with cell-specific transcriptomes and other datasets, histone methylation mapping of specific interneuron types is likely to provide radically novel insights into the developmental history and (epi)genomic architecture of cells ascribed a key role in schizophrenia and related disease. PUBLIC HEALTH RELEVANCE: For the majority of patients diagnosed with schizophrenia, no straightforward genetic cause has been identified. One of the important theories about schizophrenia implies that in some brain regions, such as the "prefrontal cortex", a number of genes are not switched on properly during normal development, as they are in healthy subjects. Many of these genes related to a type of cell called the 'GABA neuron' which comprise less than 10% of all cells (nerve cells and others) in the cortex, but are very powerful because in effect they regulate synchronization of large neural networks in the brain. To further understand the role of the GABA neurons in psychiatric disease, and to clarify why gene expression is abnormal in these cells, it will be important to explore their genomes and chromatin architectures at high resolution. This grant proposal is based on extremely innovative techniques that were recently developed in our laboratories. We will be able, for the first time, to selectively isolate chromosomal materials and chromatin from the GABA neurons of the mouse brain for the study of "epigenetic markings" (basically, chemical modifications that regulate gene expression and function without altering the genetic code) on a genome-wide level. We expect that the work resulting from this project will provide a valuable resource /chromatin atlas for the neuroscience research community, and will shed light on some of the developmental mechanisms that govern proper gene expression activity in mature GABA neurons.