Deciphering the regulatory principles governing enhancer specificity

摘要 增强子是编码基因何时何地存在的指令的基因组元件 在发展过程中表达。大多数导致疾病的突变被认为存在于 增强剂。然而，我们不知道增强子序列中哪些变化是惰性序列 个体或群体之间的差异，影响基因调控和细胞完整性。 这些基本问题仍然没有解决，因为我们不能将增强子序列与基因联系起来 表达和表型。我们知识上的这种鸿沟正在阻碍我们解释基因组数据的能力 并了解发育、细胞完整性和疾病。增强子序列为 通过识别DNA中的特定签名与转录因子结合。物质上的限制 控制这些蛋白质如何与增强子DNA相互作用可能导致一套语法 可用于理解增强子序列与组织之间关系的限制条件 特定的基因表达。我建议开发一套方法论和方法工具包，以 破译组织特异性增强子活性的语法约束。我将使用高度并行 功能报告分析、高通量基因到表型的研究以及生化 化验、合成生物学和功能丧失策略。这些实验将在 因为它是一个独特的系统，在其中数百万个增强子变体可以 在发育中的胚胎的所有细胞中进行功能测试。在Ciona的工作将得到以下补充 在鸡、鼠和组织培养中直接了解脊椎动物发育和精确定位的实验 突变致病。确定与基因编码序列相关的‘遗传密码’ 对蛋白质的研究提供了对我们基因组的一个主要组成部分的详细洞察。一旦我们有了一个 类似的代码来破译这些基因何时何地表达的指令，我们将拥有 了解基因组如何编码构建和维护指令的强大工具 生活。

Abstract Enhancers are the genomic elements that encode the instructions for when and where genes are expressed during development. The majority of mutations leading to disease are thought to reside in enhancers. However, we do not understand which changes in enhancer sequence are inert sequence variations between individuals or populations and which impact gene regulation and cellular integrity. These fundamental questions remain unsolved, because we cannot relate enhancer sequence to gene expression and phenotype. This gap in our knowledge is stalling our ability to interpret genomic data and understand development, cellular integrity, and diseases. Enhancer sequences provide a scaffold for transcription factors to bind to, by recognizing specific signatures in the DNA. The physical constraints that govern how these proteins interact with enhancer DNA could lead to a set of grammatical constraints that can be used to understand the relationship between enhancer sequence and tissue specific gene expression. I propose the development of a toolkit of methodologies and approaches to decipher the grammatical constraints on tissue specific enhancer activity. I will use highly parallel functional reporter assays, high-throughput genotype to phenotype studies along with biochemical assays, synthetic biology, and loss of function strategies. These experiments will be carried out in the chordate Ciona intestinalis, as it is a unique system in which millions of enhancer variants can be assayed for function in all cells of a developing embryo. Work in Ciona will be complemented with experiments in chick, mouse and tissue culture to directly inform vertebrate development and pinpoint mutations causing disease. Determining the ‘genetic code’ that relates the coding sequences of genes into protein has provided detailed insight into a major component of our genome. Once we have a similar code to decipher the instructions for when and where these genes are expressed, we will have powerful tools to understand how the genome encodes the instructions for building and maintaining life.