Insulator-mediated regulation of stochastic gene expression in the eye

绝缘体介导的眼随机基因表达调节

• 批准号: 9544684
• 负责人: Alexandra Neuhaus-Follini
• 金额: $ 5.19万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-12-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9544684
• 关键词: Adopted; Binding; Binding Proteins; Biochemical; Cell Nucleus; Cells; ChIP-seq; Chromatin Loop; Complement; Complex; DNA; Data; Development; Elements; Event; Exposure to; Eye; Fluorescent in Situ Hybridization; Gene Expression; Genes; Genotype; Goals; Individual; Lead; Maintenance; Mediating; Modeling; Molecular Conformation; Mosaicism; Motor; Nervous system structure; Pathway interactions; Pattern; Phenotype; Photoreceptors; Population; Process; Proteins; RNA Interference; Regulation; Regulator Genes; Regulatory Element; Resolution; Role; Site; Stereotyping; System; Testing; Tissues; Trans-Activators; Transcriptional Silencer Elements; Vision Disorders; Visual; Work; cell fate specification; cell type; chromosome conformation capture; experimental study; fly; human disease; knock-down; loss of function mutation; mutant; neurodevelopment; recruit; transcription factor

Project Summary/Abstract During development, some cell fate decisions are highly stereotyped, while others are random. Stochastic cell fate specification is essential for the development of the mammalian nervous system, including the visual, olfactory, and motor systems. Despite the importance of stochastic cell fate specification in neural development, the gene regulatory mechanisms that underlie this process remain poorly understood. The fly eye is a powerful model for elucidating mechanisms underlying random cell fate specification. In the fly, stochastic expression of the transcription factor Spineless (Ss) in R7 photoreceptors establishes a random mosaic of two different populations of R7s. The ratio of Sson:Ssoff R7s is approximately 2:1 in every fly, but each individual R7 makes a random choice to express Ss or not. Thus, flies of the same genotype have the same ratio of Sson:Ssoff R7s, but in every fly, these photoreceptors are distributed in a unique, random pattern. Importantly, expression of each copy of ss is independently regulated, even in the same nucleus where they are exposed to the same complement of trans-acting factors, indicating that the stochastic event that leads to the Ss expression decision occurs at the ss locus, rather than at an upstream step in the Ss expression pathway. The goal of the proposed work is to elucidate the gene regulatory mechanism controlling stochastic ss expression in R7s. The ss locus contains several predicted insulator DNA elements. As insulator proteins interact with each other to form complexes that facilitate the formation of DNA loops, we hypothesize that insulator-mediated DNA looping causes the ss locus to randomly assume distinct conformations in Sson and Ssoff R7s and that the looping state of ss determines its on or off expression state. We will test this idea by 1) characterizing insulator occupancy at the ss locus in Sson and Ssoff R7s, 2) determining the roles of insulator DNA elements and proteins in stochastic ss expression, and 3) determining and manipulating the looping state of the ss locus in Sson and Ssoff R7s. Together, these experiments will provide the first analysis of the role of insulator-mediated DNA looping in the regulation of stochastic gene expression and will enhance our mechanistic understanding of stochastic cell fate specification.

项目总结/摘要 在发育过程中，一些细胞的命运决定是高度定型的，而另一些则是随机的。随机细胞 命运特化对于哺乳动物神经系统，包括视觉， 嗅觉和运动系统。尽管随机细胞命运规范在神经系统中的重要性， 尽管基因调控机制在人类的发育过程中发挥着重要作用，但对这一过程的基因调控机制仍然知之甚少。苍蝇 眼是阐明随机细胞命运特化机制的有力模型。在飞行中， 转录因子Spineless（Ss）在R7光感受器中的随机表达建立了一个随机的 两个不同R7种群的嵌合体在每只果蝇中，Sson：Ssoff R7s的比例约为2：1，但 每个R7都随机选择是否表示Ss。因此，相同基因型的果蝇具有 Sson：Ssoff R7的比例相同，但在每只苍蝇中，这些光感受器以独特的随机模式分布。 重要的是，每个ss拷贝的表达都是独立调节的，即使在同一个细胞核中， 都暴露在相同的反式作用因子中，这表明导致 Ss表达的决定发生在ss基因座，而不是在Ss表达的上游步骤 通路本研究的目的是阐明基因调控机制控制随机 R7s中的ss表达。ss基因座含有几个预测的绝缘子DNA元件。作为绝缘蛋白 相互作用形成复合物，促进DNA环的形成，我们假设， 绝缘子介导的DNA成环导致ss基因座在Sson中随机呈现不同的构象， Ssoff R7s，ss的循环状态决定了它的on或off表达式状态。我们将通过以下方式来验证这个想法：1） 表征Sson和Ssoff R7s中ss基因座处的绝缘子占据，2）确定绝缘子的作用 随机ss表达中的DNA元件和蛋白质，以及3）确定和操纵成环状态 Sson和Ssoff R7中的ss基因座。总之，这些实验将提供第一个分析的作用， 绝缘子介导的DNA环在随机基因表达的调节，并将提高我们的 随机细胞命运规范的机械理解。