Epigenome Mapping in Cortical Interneurons

描述(申请人提供)：这个项目的两个主要目标是：1)首次为研究界和公共领域提供选定的皮质GABA能中间神经元和其他驻留在小鼠大脑皮层的细胞中组蛋白甲基化情况的全面全基因组图谱；2)初步了解GABA能神经元从幼年到成熟转变过程中的染色质重塑机制。以前从人和小鼠大脑皮质提取的染色质的研究表明，在成熟期延长的过程中，组蛋白甲基化至少在成年早期是动态调节的，从而将染色质重塑机制与发育时钟联系起来。关注皮质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能中间神经元基因表达异常被认为是分子病理生理学的标志，也是神经网络同步缺陷的主要因素，神经网络影响精神病或自闭症患者大脑皮层的广泛区域。这些与GABA相关的基因表达缺陷包括不同的细胞类型，如钙结合蛋白小白蛋白的表达通常识别的一类快速尖峰中间神经元。因此，在这一建议的背景下，我们计划建立4个BAC(细菌人工染色体)转基因小鼠的等基因系，以在整个GABA能中间神经元中表达绿色荧光蛋白(GFP)标记的组蛋白H2 B，并在由钙缓冲蛋白ParvalBrain(PARV)或Calbindin(Calb)或Calretinin(CALR)差异表达的3个特定亚群中表达绿色荧光蛋白(GFP)标记的组蛋白H2 B。最新的方法学进展使我们能够高效地从脑组织中分离和分类GFP标记的核，用于染色质免疫沉淀和大规模平行测序(CHIP-SEQ)。焦点将集中在转录起始点(H3K4me3)和增强子序列(包括进一步从近端启动子(H3K4me1)中移除的那些)上的三甲基和单甲基组蛋白H3-赖氨酸4(H3K4me3，H3K4me1)，以及与RNA聚合酶II活性和跨编码区和非编码区的转录延长相关的标记(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定的染色质特征。当结合细胞特异性转录本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图谱可能为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.