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

描述（由申请人提供）：神经元基因组的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元细胞命运重要的转录和表观遗传回路。本拨款提案的目的是通过在成体神经干细胞中执行ChIP-Seq和RNA-seq来生成REST/NRSF靶基因的全基因组视图。核心假设是REST/NRSF对干细胞静止至关重要，并且是成人海马神经发生的主要负调节因子。提出这项研究的基本原理是，了解控制神经元谱系程序的发育机制，有可能进一步治疗与神经元丧失或神经元功能异常相关的越来越多的精神障碍。因此，拟议的研究与NIH的使命有关，即获得可能有助于治疗神经系统疾病的基础知识。在强有力的初步数据的指导下，这一假设将通过两个特定目标进行验证：1)成人NSC染色质中REST/NRSF靶点的全基因组鉴定；2)成人NSCs在静止、增殖和神经元分化过程中的转录组分析。目的1将重点研究成体海马神经干细胞REST/NRSF的ChIP-Seq。目的2将集中于在静止、增殖和神经元分化条件下对成人NSCs进行rna测序。Aim 2还将通过比较ChIP-Seq靶点与我们实验室生成的REST/NRSF条件敲除小鼠的基因表达分析，确定哪些靶点依赖于REST/NRSF。概念框架和方法是创新的，因为它是第一个将最先进的深度测序技术应用于生理相关的成体神经干细胞系统，以确定REST/NRSF靶基因的全基因组视图的研究。拟议的R21拨款意义重大，因为它有望推进和扩展我们对调节干细胞静止、增殖和神经元分化的转录网络的基本理解，这可能对恢复脑损伤或疾病后的认知功能特别重要。

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.