A Novel Approach to Decoding Vertebrate Gene Regulation

描述(申请人提供)：尽管我们基因组中编码蛋白质的2%已经被成功地注释和表征，但破译嵌入其余98%的调控密码仍然是一个挑战。提高我们对调控代码的理解对于确定管理器官发生并导致疾病的特定细胞类型的调控电路至关重要。海胆的发育调控网络已被全面描述。然而，在脊椎动物中，大多数关于调控变异的研究采取了自上而下的方法，通过计算预测调控元件或通过实验表征单个转录因子的结合位点。尽管许多已知的调节区都很大且复杂，但初步的体内功能研究表明，短至6个碱基对(BP)的DNA序列推动了报告基因在特定组织中的精确表达，并且两个核苷酸的替代导致了表达结构域的变化。这项拟议的项目将开创一种新的自下而上的方法，通过表征所有6碱基对(BP)序列的调控潜力来破译脊椎动物的调控密码。这些短小的模体将通过在发育过程中转基因斑马鱼来测试其增强活性。该项目的第一个具体目标是通过计算设计一组报告结构，紧凑地覆盖所有6-MERS，并能够有效地表征6-MERs增强剂的功能。这是一个具有挑战性的计算问题，在此阶段设计和实现的算法将为未来增强剂实验和许多其他生物测试的低聚物的有效设计提供强大的资源。第二个目标是计算和分析实验验证的增强子的基因组分布，并使用结果来解释表达数据。最后，第三个目标是开发和测试多个监管6-MERs相互作用的模型。将特别注意增强剂组合的相加效应和潜在消音剂的确定。通过从头开始构建和功能翻译监管语言，该项目补充了理解监管代码的自上而下的努力。该项目将对从发育和进化生物学到基因组注释的众多生物学领域产生巨大影响。各种临床应用也将从该项目中受益，例如基于干细胞的再生疗法的重新编程策略。此外，它还将通过对调节元件进行基因工程，在不同的时间点将化合物驱动到特定的组织，为基因治疗铺平道路。 与公共卫生相关：发育通过在空间和时间上精细的基因调控模式进行，这些模式影响从器官形成到人类多样性和疾病的广泛生物过程。这个项目的目标是有效地研究所有短DNA序列作为脊椎动物发育过程中基因表达调控的潜力。这项工作将极大地促进我们对人类基因组和发育基因调控的了解，在合成生物学和治疗学的发展中具有重要的应用。

DESCRIPTION (provided by applicant): While the 2% of our genome that encodes proteins has been successfully annotated and characterized, deciphering the regulatory code embedded in the remaining 98% remains a challenge. Improving our understanding of the regulatory code is essential for determining the cell-type-specific regulatory circuits that govern organogenesis and contribute to disease. Developmental regulatory networks in the sea urchin have been comprehensively described. In vertebrates, however, most studies of regulatory variation have taken a top-down approach, computationally predicting regulatory elements or experimentally characterizing binding sites of individual transcription factors. Although many known regulatory regions are large and complex, preliminary in vivo functional studies indicate that DNA sequences as short as six base pairs (bp) drive precise expression of a reporter gene to specific tissues, and that a two-nucleotide substitution leads to a change in the domain of the expression. The proposed project will pioneer a novel bottom-up approach to decipher the vertebrate regulatory code by characterizing the regulatory potential of all 6-base pair (bp) sequences. These short motifs will be tested for enhancer activity using zebra fish transgenesis during development. The first specific aim of the project is to computationally design a collection of reporter constructs that covers all 6-mers compactly and enables efficient functional characterization of 6-mer enhancers. This is a challenging computational problem, and the algorithms designed and implemented at this stage will be a powerful resource for the efficient design of oligomers for future enhancer experiments and many other biological assays. The second aim is to compute and analyze the genomic distributions of experimentally validated enhancers and use the results to interpret expression data. Finally, the third aim is to develop and test models for the interaction of multiple regulatory 6-mers. Particular attention will be devoted to additive effects of combinations of enhancers and the identification of potential silencers. By building and functionally translating a regulatory language from scratch, this project complements top-down efforts to understand the regulatory code. This project will have an enormous impact on numerous biological fields, from developmental and evolutionary biology to genome annotation. Various clinical applications will also benefit from this project, such as reprogramming strategies for stem-cell-based regenerative therapies. In addition, it will pave the way for gene therapy by genetically engineering regulatory elements that drive compounds to specific tissues at different time points. PUBLIC HEALTH RELEVANCE: Development proceeds via spatially and temporally exquisite patterns of gene regulation, which influence a wide range of biological processes from organ formation to human diversity and disease. The goal of this project is to efficiently study all short DNA sequences for their potential as regulators of gene expression during vertebrate development. This work will significantly advance our knowledge about the human genome and developmental gene regulation, with important applications in synthetic biology and the development of therapeutics.