Genome-Wide Profiling of REST/NRSF Targets in Adult Neural Stem Cells

描述(申请人提供)：神经元基因组的一个重要守护者是转录抑制因子REST/NRSF。长期目标是更好地了解在生理和病理背景下对神经细胞命运至关重要的转录和表观遗传回路。这项拨款提案的目标是通过在成人神经干细胞中进行CHIP-SEQ和RNA-SEQ来生成REST/NRSF靶基因的全基因组视图。中心假设是REST/NRSF是干细胞静止所必需的，是成年海马神经发生的主要负调控因子。这项拟议研究的基本原理是，了解控制神经元谱系计划的发育机制有可能进一步治疗越来越多与神经元丧失或神经元功能异常有关的精神障碍。因此，这项拟议的研究与NIH使命中与获得基础知识相关的部分相关，这可能有助于治疗神经疾病。在强大的初步数据的指导下，这一假设将通过追求两个特定目标来验证：1)在全基因组范围内鉴定成年NSC染色质中的REST/NRSF靶标；2)对成年NSC在静止、增殖和神经元分化过程中的转录组进行分析。目标1将专注于在成年海马神经干细胞中进行REST/NRSF的芯片序列。目的2将集中于在静止、增殖和神经元分化的条件下进行成年神经干细胞的RNA-SEQ。AIM 2还将通过将CHIP-Seq靶标与我们实验室产生的REST/NRSF条件性基因敲除小鼠的基因表达分析进行比较，确定哪些靶标依赖于REST/NRSF。概念框架和方法是创新的，因为这是第一次将最先进的深度测序技术应用于生理相关的成人神经干细胞系统，以确定REST/NRSF靶基因的全基因组视图。建议的R21拨款意义重大，因为它有望促进和扩大我们对调控干细胞静止、增殖和神经元分化的转录网络的基本理解，这些网络可能对恢复脑损伤或疾病后的认知功能特别重要。 公共卫生相关性：神经系统的疾病、退化或创伤性损伤是美国最大的公共卫生问题之一，通常被认为是无法修复的，往往会对个人的功能能力造成灾难性的损害。然而，现在，定位于大脑特定生发中心和细胞培养中的神经干细胞的特征增加了希望，如果我们学会利用成人神经发生来临床获益，神经组织的功能性再生可能是可行的。这项研究提案将提高对神经干细胞生物学的理解，可能会导致神经系统修复和再生新药的开发。

DESCRIPTION (provided by applicant): An essential guardian of the neuronal genome is the transcriptional repressor REST/NRSF. The long-term goal is to better understand the transcriptional and epigenetic circuitry important for neuronal cell fate, in both physiological and pathological contexts. The objective of this grant proposal is to generate a genome-wide view of REST/NRSF target genes by performing ChIP-Seq and RNA-seq in adult neural stem cells. The central hypothesis is REST/NRSF is essential for stem cell quiescence and is a master negative regulator of adult hippocampal neurogenesis. The rationale for the proposed research is that understanding developmental mechanisms controlling the neuronal lineage program have the potential to further therapeutic approaches to an increasing number of mental disorders linked to neuronal loss or aberrant neuronal function. Thus, the proposed study is relevant to that part of NIH's mission that relates to gaining fundamental knowledge that will potentially help treat neurological disorders. Guided by strong preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Genome-wide identification of REST/NRSF targets in adult NSC chromatin and 2) Transcriptome analysis of adult NSCs during quiescence, proliferation, and neuronal differentiation. Aim 1 will focus on performing ChIP-Seq of REST/NRSF in adult hippocampal neural stem cells. Aim 2 will focus on performing RNA-seq from adult NSCs under conditions of quiescence, proliferation, and neuronal differentiation. Aim 2 will also identify which targets are dependent on REST/NRSF by comparing ChIP-Seq targets to gene expression analysis from REST/NRSF conditional knockout mice generated in our laboratory. The conceptual framework and approach is innovative, because it is the first study to apply state-of-the-art deep sequencing techniques to a physiologically relevant adult neural stem cell system in order to identify a genome-wide view of REST/NRSF target genes. The proposed R21 grant is significant, because it is expected to advance and expand our basic understanding of transcriptional networks regulating stem cell quiescence, proliferation, and neuronal differentiation which may be of particular importance for restoring cognitive function after brain damage or disease. PUBLIC HEALTH RELEVANCE: Disease, degeneration or traumatic injury of the nervous system are among the greatest public health concerns in the United States and are generally considered irreparable, often causing catastrophic damage to the functional capacity of the individual. Now, however, characterization of neural stem cells residing within specific germinal centers of the brain and in cell culture raises hope that functional regeneration of nervous tissue may be feasible, if we learn to exploit adult neurogenesis for clinical benefit. The research proposal will lead to improved understanding of neural stem cell biology, possibly leading to the development of new drugs for repair and regeneration of the nervous system.