Genome-Wide Mapping of Enhancer Elements for Neuronal Differentiation Genes

描述(申请人提供)：细胞分化需要基因表达程序的精确编排。染色质调节复合体通过调节基因调节元件的可及性和激活状态来协调这一过程。通过比较细胞类型特定的表观基因组图谱，可以很容易地观察到细胞分化的结束状态。这些分析表明，远端增强子元件的差异调控是基因表达的细胞类型特异性程序的主要决定因素。然而，动态的染色质调节事件塑造了活性增强子的分布，从而推动了任何单一细胞类型的分化，目前仍在很大程度上尚不清楚。这项提议的目标是确定在体内控制神经元分化的染色质调节事件。我们将通过比较从特定神经细胞类型分化的离散阶段获得的染色质的表观基因组图谱，确定控制神经元基因表达同时变化的发育调节的染色质变化。小脑颗粒神经元(CGN)为本研究提供了一个理想的活体模型，因为它们代表了一个基本相同的神经元群体，可以直接从出生后的小鼠脑中获得大量处于不同发育阶段的神经元。利用DNaseI染色质消化和高通量测序(DNase-Seq)技术，我们已经鉴定了在CGN分化过程中可及染色质分布的实质性差异。在这里，我们建议通过对表示这些基因调节元件的性质和激活状态的组蛋白标记进行全基因组染色质免疫沉淀(CHIP)来表征这些神经元中启动子和增强子的发育调节。然后，我们将通过对我们的组合DNase-Seq、ChIP-Seq和RNA-Seq数据集进行隐马尔可夫建模，测试染色质状态与CGN分化过程中基因表达动态变化之间的关系。这项提议的结果将是首次确定一套全面定义的基因调控元件，这些元件控制神经元分化基础上的基因调控的动态变化。

DESCRIPTION (provided by applicant): Cellular differentiation requires the precise orchestration of gene expression programs. Chromatin regulatory complexes coordinate this process by modulating the accessibility and activation state of gene regulatory elements. The end states of cellular differentiation can be readily visualized through the comparison of cell typ specific epigenome maps. Such analyses indicate that the differential regulation of distal enhancer elements is the primary determinant of cell-type specific programs of gene expression. However the dynamic chromatin regulatory events that sculpt the distribution of active enhancers and thus drive the differentiation of any single cell type have remained largely unknown. The goal of this proposal is to identify the chromatin regulatory events that control the differentiation of neurons in vivo. We will identify developmentally regulated chromatin changes that control contemporaneous changes in neuronal gene expression by comparing epigenomic profiles of chromatin harvested from discrete stages in the differentiation of a specific neuronal cell type. Cerebellar granule neurons (CGNs) provide an ideal in vivo model for this study because they represent a largely homogeneous neuronal population that can be obtained in very large numbers at discrete developmental stages directly from the postnatal mouse brain. Using the technique of DNaseI chromatin digestion followed by high-throughput sequencing (DNase-Seq) we have already identified substantial differences in the distribution of accessible chromatin over the course of CGN differentiation. Here we propose to characterize the developmental regulation of promoters and enhancers in these neurons by performing genome-wide chromatin immunoprecipitation (ChIP) for histone marks that denote the nature and activation state of these gene regulatory elements. We will then test the relationship between chromatin states and dynamic changes in gene expression during CGN differentiation through hidden Markov modeling of our combined DNase-Seq, ChIP-Seq, and RNA-Seq datasets. The outcome of this proposal will be the first identification of a comprehensive defined set of gene regulatory elements that control the dynamic changes in gene regulation that underlie neuronal differentiation.