Functional elucidation of the sequence-encoded regulatory activity of enhancers in vivo in the brain

摘要 基因组的非编码区曾经被认为是垃圾，现在已经成为进化的中心组成部分， 发展和疾病。人类基因组中最常见的非编码调控元件是 增强子，确保目标基因在正确的时间在正确的细胞中表达，通过控制其 激活。对增强子功能的扰动被广泛认为是一种主要的，但仍然知之甚少， 人类大脑进化和疾病的组成部分。在以下方面取得了重大和持续的进展 注释增强剂并预测这些元素在细胞和组织中的活性，包括大脑。尽管 在这些进展中，预测增强子的序列编码功能仍然是一个重大挑战。此外， 动态和上下文相关的染色体相互作用、表观遗传修饰和转录 最终决定增强子介导的基因调控的因子活性通常仍然很低 明白了。这是理解增强子的功能和口译的一个重大障碍 增强子序列变异对人脑发育、进化和疾病的影响。因此，在那里 是确定调控DNA的序列和功能之间关系的关键需要，以及 确定大脑中增强子活性和基因调控的决定因素。在最初的早期阶段 研究人员米拉资助，我们建立了一个富有成效的研究计划，专注于阐明增强剂- 哺乳动物大脑中介导的基因调控线路。我们将功能分析与基因和基因分析配对 用基因组方法模拟增强子、转录因子和染色质重塑的功能 蛋白质在正常和致病脑发育中的作用。我们Mira研究计划的首要目标是 目的是：1)扩展和应用定义哺乳动物大脑中序列编码的增强子活性的方法；2) 确定增强子介导的基因调控和转录编程的分子机制 以及3)表征调控序列变异的后果，以了解 哺乳动物大脑发育、进化和紊乱过程中的增强子DNA。在续期期间，我们 将应用遗传学、基因组学和神经科学的综合方法来解决在理解 序列编码的增强子功能，并回答有关基因调控的基本问题 大脑。我们的工作将解决有关充分性和翻译相关的基本问题和 神经发育基因调控的增强剂的必要性，并将推动新兴的领域 基于增强子的工具，用于标记和操纵大脑中的细胞类型。总体而言，我们的贡献将有所帮助 破译转录控制是如何在遗传和表观遗传水平上编码的，并阐明基因 哺乳动物大脑的调节回路。

SUMMARY Once considered junk, non-coding regions of the genome have emerged as central components of evolution, development, and disease. The most common non-coding regulatory elements in the human genome are enhancers, which ensure expression of target genes at the right time in the right cells by controlling their activation. Perturbation to enhancer function is widely accepted as a major, but still poorly understood, component of human brain evolution and disease. There have been major and continuing advances in annotating enhancers and predicting activity of these elements in cells and tissues, including the brain. Despite these advances, predicting the sequence-encoded function of enhancers remains a major challenge. Further, the dynamic and context-dependent chromosomal interactions, epigenetic modifications, and transcription factor activity that ultimately determine enhancer-mediated gene regulation generally remain poorly understood. This represents a significant barrier in understanding the function of enhancers and in interpreting the effect of enhancer sequence variation on human brain development, evolution and disease. As such, there is critical need to determine the relationship between sequence and function for regulatory DNA, and to define the determinants of enhancer activity and gene regulation in the brain. In the initial early stage investigator MIRA funding, we established a productive research program focused elucidating enhancer- mediated gene regulatory wiring in the mammalian brain. We paired functional assays with genetic and genomic approaches to model the function of enhancers, transcription factors, and chromatin remodeling proteins in normal and pathogenic brain development. The overarching goals of our MIRA research program are to: 1) Extend and apply methods to define sequence-encoded enhancer activity in the mammalian brain, 2) Determine the molecular mechanisms of enhancer-mediated gene regulation and transcriptional programming in the brain, and 3) Characterize the consequences of regulatory sequence variation to understand the role of enhancer DNA in the development, evolution, and disorders of the mammalian brain. In the renewal period, we will apply integrative genetic, genomic, and neuroscience methods to address key gaps in the understanding of sequence-encoded enhancer function and to answer fundamental questions regarding gene regulation in the brain. Our work will address basic and translationally-relevant questions regarding the sufficiency and necessity of enhancers for neurodevelopmental gene regulation, and will advance the emerging field of enhancer-based tools for labeling and manipulation of cell types in the brain. Overall, our contributions will help to decipher how transcriptional control is encoded at the genetic and epigenetic level and to illuminate the gene regulatory circuitry of the mammalian brain.