The role of enhancer RNAs in neuronal plasticity and neurogenesis

神经元或神经元祖细胞转录程序的变化分别是神经元可塑性和神经发生的核心。调节这些过程的基因受增强子元件的控制，这些增强子元件在神经元刺激和大脑发育期间受到差异调节。增强子是有时由多个转录因子结合的DNA序列。在组合活化过程中，认为所有结合因子的组合活性决定靶基因活化的程度。此外，神经元增强子最近被证明是积极转录，产生非编码增强子RNA（eRNA）。eRNA显著地促进小鼠神经元中大多数立即早期基因的激活。此外，eRNA表达水平比转录因子结合数据作为增强子活性的更好指标。然而，增强子RNA如何能够在体内指导大脑发育和神经元可塑性尚不清楚。特别是，在这些过程中，增强子转录对增强子功能有多大贡献是未知的。我们追求的假设，增强子RNA直接影响RNA聚合酶II（Pol II）的转录机制，减少启动子近端暂停的重要神经元基因。为了验证这一假设，我们将结合联合收割机体外生物化学，下一代测序和斑马鱼神经发生的体内研究。具体而言，我们将解决以下问题：1.神经元增强子RNA能在体外消除启动子近端停顿吗？2.神经元增强子RNA在斑马鱼和小鼠之间是保守的吗？3.增强子RNA在斑马鱼神经系统发育中的作用是什么？斑马鱼是一个非常适合在器官水平上研究eRNA功能的模型系统。它结合了能够轻松成像神经发生的便利性以及使用反义技术和CRISPRi操纵eRNA功能的能力。尽管大多数新兴的非编码RNA在转录水平上影响基因表达，但迄今为止对潜在机制的了解很少。该建议通过直接整合Pol II转录延伸的机制研究与斑马鱼体内神经发生的功能分析来弥合这一差距。

Changes in the transcriptional programs of neurons or neuronal progenitors lie at the heart of neuronal plasticity and neurogenesis, respectively. Genes that regulate these processes are under the control of enhancer elements that are differentially regulated during neuronal stimulation and brain development. Enhancers are DNA sequences bound by at times multiple transcription factors. In a combinatorial activation process the combined activity of all bound factors is believed to determine the extent of target gene activation. In addition, neuronal enhancers were recently shown to be actively transcribed, giving rise to non-coding enhancer RNAs (eRNAs). eRNAs significantly contribute to the activation of most immediate early genes in mouse neurons. Moreover, eRNA expression levels serve as a better indicator of enhancer activity than transcription factor binding data. However, how enhancer RNAs are able to direct brain development and neuronal plasticity in vivo is unknown. In particular, it is unknown how much enhancer transcription contributes to enhancer function during these processes. We pursue the hypothesis that enhancer RNAs directly influence the RNA polymerase II (Pol II) transcription machinery by diminishing promoter-proximal pausing of important neuronal genes. To examine this hypothesis, we will combine in vitro biochemistry, next-generation sequencing and in vivo studies of zebrafish neurogenesis. Specifically, we will address the following questions: 1. Are neuronal enhancer RNAs able to abrogate promoter-proximal pausing in vitro? 2. Are neuronal enhancer RNAs conserved between zebrafish and mouse? 3. What is the role of enhancer RNAs in zebrafish nervous system development?Zebrafish is a uniquely suited model system to study eRNA function on an organismic level. It combines the ease of being easily able to image neurogenesis with the ability to manipulate eRNA functionality using both antisense techniques and CRISPRi. Despite the fact that most emerging classes of non-coding RNA influence gene expression on the transcriptional level there is very little insight to date on the underlying mechanisms. This proposal bridges this gap by directly integrating a mechanistic study on Pol II transcription elongation with functional analyses of zebrafish neurogenesis in vivo.