Functional Elucidation of the Sequence-Encoded Regulatory Activity of Enhancers in Vivo in the Brain

脑内增强子体内序列编码调控活性的功能阐明 P.I.-亚历克斯·诺德，博士 研究摘要： 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。虽然最近的努力在注释基因组中的增强子方面取得了进展，但表征 它们的序列编码功能仍然是一个重大挑战。这是一个很大的障碍 理解增强剂在复杂生物过程中的重要作用，以及在解释 增强子序列变异对人类进化和疾病的影响。全基因组的快速采用 测序有望放大这一问题，因为将识别出更多潜在的因果突变 而不是使用目前的方法在功能上进行分类。因此，迫切需要有效地 在序列水平上对调控DNA进行功能表征以揭示非编码作用的机制 人类发展的要素。大规模平行的报告分析提供了一个潜在的解决方案 剖析序列编码的增强子活性，实现数百到数千的定量测量 单个实验中的单个候选增强子序列。在活体内应用这种方法可以 阐明增强子在细胞集合中的正常和致病功能，包括 哺乳动物组织。它还可以揭示它们在复合体中介导基因与环境相互作用的作用 条件，如学习和压力。在初步实验中，我们采用了高通量的记者 分析了小鼠额叶皮质的增强子功能，并将单细胞转录组学应用于 定义发育中的小鼠大脑的体内增强子功能。这些新颖的方法开启了令人兴奋的长久以来 术语研究途径，能够在体内测试增强子序列变异和细胞特异性功能 规模和分辨率在以前是不可能的。我们将把我们强大的功能分析与单细胞相结合 基因组学，以解决有关大脑中基因调控线路的重大问题。我们建议：1) 将我们基于功能的方法扩展到研究小鼠脑内增强子的活体活性，以及2)应用 这些方法来表征增强子的功能，我们之前映射了控制阶段特定的 与神经可塑性相关的基因表达，并测试其对体内增强子功能的影响 非编码变异与大脑进化和病理有关。这些科学问题建立在我们之前的 致力于大脑中的增强剂，使用我们的新方法来转变我们可以解决关键问题的范围 对大脑中增强子功能和基因调控的基本问题的理解存在差距。这 这项工作将产生长期影响，为研究增强子功能和 揭示大脑中的基因调控是如何在DNA序列水平上编码的，并着眼于 阐明了人类进化、健康和疾病背后的非编码遗传电路。

Title - Functional elucidation of the sequence-encoded regulatory activity of enhancers in vivo in the brain P.I. – Alex Nord, PhD Research 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. While recent efforts have made headway annotating enhancers in the genome, characterization of their sequence-encoded function remains a major challenge. This represents a significant barrier in understanding the important role of enhancers in complex biological processes, as well as in interpreting the effect of enhancer sequence variation on human evolution and disease. Rapid adoption of whole genome sequencing promises to amplify this issue, since considerably more potential causal mutations will be identified than can be functionally classified using current approaches. As such, there is a critical need for effective mechanisms to functionally characterize regulatory DNA at the sequence level to reveal the role of non-coding elements in human development. Massively parallel reporter assays provide a potential solution toward dissecting sequence-encoded enhancer activity, enabling quantitative measurement of hundreds to thousands of individual candidate enhancer sequences in a single experiment. Applying this approach in vivo could illuminate the normal versus pathogenic functions of enhancers in the assemblage of cells comprising mammalian tissues. It also could reveal their role mediating gene-by-environment interactions in complex conditions, such as learning and stress. In preliminary experiments, we adapted a high-throughput reporter assay to characterize enhancer function in mouse frontal cortex, and we applied single cell transcriptomics to define enhancer function in vivo in the developing mouse brain. These novel approaches open exciting long- term research avenues, enabling testing of enhancer sequence variation and cell-specific function in vivo at a scale and resolution not previously possible. We will couple our powerful functional assay with single cell genomics to address significant questions regarding gene regulatory wiring in the brain. We propose to: 1) Extend our function-based methods for the study of enhancer activity in vivo in mouse brain, and 2) Apply these methods to characterize the function of enhancers we previously mapped that control stage-specific gene expression associated with neuroplasticity, and to test the consequences on in vivo enhancer function of non-coding variation linked to brain evolution and pathology. These scientific questions build on our previous work on enhancers in the brain, using our new methods to transform the scope at which we can address key gaps in the understanding of enhancer function and fundamental questions of gene regulation in the brain. This work will have long-term impact, generating powerful new methods for studying enhancer function and revealing how gene regulation in the brain is encoded at the DNA sequence level, with an eye toward illuminating the non-coding genetic circuitry underlying human evolution, health and disease.