The Role of Long Non-coding RNAs in Lineage Restriction of Neural Stem Cells

描述（由申请人提供）：转录组分析已经证明70%-90%的基因组被转录，然而令人惊讶的是只有1- 2%编码蛋白质。最近的工作揭示了一类新的非编码转录物，称为长非编码RNA（lncRNA）。这些lncRNA的一个已知的主要功能是特异性地将染色质修饰剂引导到它们的靶基因组范围内。虽然生物信息学的进步已经导致培养细胞中lncRNA的广泛编目和功能分析，但体内数据严重缺乏。小鼠脑的脑室下区（SVZ）是研究lncRNA在体内作用的理想系统。SVZ含有多能神经干细胞（SVZ-NSC），能够产生局部神经胶质细胞以及嗅球（OB）的终末分化神经元。已知干细胞向神经谱系的终末分化细胞的这种命运限制依赖于染色质状态变化和关键转录因子的作用。因此，SVZ中的神经谱系可用于询问染色质状态、转录程序和lncRNA的相互作用。这项工作的主要目标是表征体内神经源性lncRNA的表达和功能。为此，我们对从显微切割的SVZ和OB组织中分离的RNA进行了RNA-seq和转录组重建。这提供了SVZ和OB中表达的所有lncRNA的全面注释，并鉴定了那些特异性富集于神经源性脑区域的转录物。我们将这种RNA-seq分析与ChIP-seq分析相结合，以鉴定在胚胎干细胞、SVZ-NSC和非神经原性成纤维细胞中发生染色质标记变化的lncRNA基因座。这项工作使我们得到了一个转录本，注释为Dlx 1as，一个2.8 kb的非编码RNA转录自Dlx 2基因附近的一个超保守区。随后在培养的SVZ-NSCs中的敲除实验显示，Dlx 1as是这些培养物中有效神经发生所必需的。目的1将评估Dlx 1as是否是促进体内神经发生所必需的。为了测试Dlx 1as是否是必要的，我们将向成年小鼠的脑室注射敲除构建体，并评估SVZ-NSC产生神经元的能力。在目标2中，我们将评估Dlx 1as是否可以指导染色质修饰和修饰。初步数据显示，三胸染色质修饰复合体成员MLL 1在分化过程中定位于Dlx 1/2位点，并且这种定位是RNA依赖性的。Dlx 1as是否负责将MLL 1靶向Dlx 1/2启动子和/或增强子？我们将使用ChIP-qPCR和RNA免疫沉淀（RIP）分析来评估Dlx 1as缺失的SVZ-NSC培养物中Dlx 1/2位点的染色质状态变化。我们将通过RIP-qPCR确定Dlx 1as是否与特异性染色质重塑因子相互作用并可能招募特异性染色质重塑因子。我推测Dlx 1as是体内神经发生所必需的，通过调节Dlx 1/2位点的染色质修饰因子。

DESCRIPTION (provided by applicant): Transcriptome analysis has demonstrated that 70%-90% of the genome is transcribed, yet surprisingly only 1- 2% codes for protein. Recent work has unveiled a new class of non-coding transcript, termed long noncoding RNAs (lncRNAs). A known major function of these lncRNAs is to specifically direct chromatin-modifiers to their targets genome-wide. While bioinformatic advances have lead to extensive cataloguing and functional analysis of lncRNAs in cultured cells, in vivo data is severely lacking. The sub-ventricular zone (SVZ) of the mouse brain represents an ideal system in which to study the role of lncRNAs in vivo. The SVZ contains multipotent neural stem cells (SVZ-NSCs) capable of giving rise to local glial cells as well as terminally differentiated neurons for the olfactory bul (OB). This fate restriction, of stem cells to terminally differentiated cells of the neural lineage is known to be dependent on chromatin-state changes and the actions of key transcription factors. The neural lineage in the SVZ can therefore be used to interrogate the interplay of chromatin state, transcriptional programs, and lncRNAs. The major goal of this work is to characterize the expression and function of a neurogenic lncRNA in vivo. To this end, we have performed RNA-seq and transcriptome reconstruction on RNA isolated from microdissected SVZ and OB tissue. This provided a comprehensive annotation of all lncRNAs expressed in the SVZ and OB, and identified those transcripts specifically enriched in neurogenic brain regions. We combined this RNA-seq analysis with ChIP-seq analysis to identify lncRNA loci that undergo changes in chromatin marks in embryonic stem cells, SVZ-NSCs, and non-neurogenic fibroblasts. This work led us to a transcript annotated as Dlx1as, a 2.8 kb noncoding RNA transcribed from an ultraconserved region adjacent to the Dlx2 gene. Subsequent knockdown experiments in cultured SVZ-NSCs revealed Dlx1as is necessary for efficient neurogenesis in these cultures. Aim 1 will evaluate whether Dlx1as is necessary to promote neurogenesis in vivo. To test if Dlx1as is necessary, we will inject a knockdown construct into the ventricle of adult mice, and assess the ability of the SVZ-NSCs to produce neurons. In Aim 2, we will assess whether Dlx1as can direct chromatin modifiers and modifications. Preliminary data reveals that trithorax chromatin modification complex member MLL1 is localized to the Dlx1/2 locus during differentiation, and this localization is RNA-dependent. Is Dlx1as responsible for targeting MLL1 to the Dlx1/2 promoters and/or enhancers? We will use ChIP-qPCR and RNA immunopreciptation (RIP) analysis to assess the chromatin-state changes at the Dlx1/2 locus in Dlx1as-depleted SVZ-NSC cultures. We will determine whether Dlx1as interacts with and potentially recruits specific chromatin remodeling factors through RIP-qPCR. I hypothesize that Dlx1as is required for neurogenesis in vivo through modulation of the chromatin- modifying factors recruited to the Dlx1/2 locus.