Insulator-mediated regulation of stochastic gene expression in the eye

项目摘要/摘要 在发育过程中，一些细胞命运的决定是高度刻板的，而另一些则是随机的。随机细胞 命运的规定对于哺乳动物神经系统的发育是必不可少的，包括视觉， 嗅觉和运动系统。尽管随机细胞命运规范在神经科学中的重要性 在发展过程中，这一过程背后的基因调控机制仍然知之甚少。苍蝇 Eyes是一个强大的模型，可以用来解释随机细胞命运指定的潜在机制。在飞行中， 转录因子无刺(SS)在R7感光器中的随机表达建立了一个随机的 两个不同的R7群体的嵌合体。在每只果蝇中，SSON和SSOFR7S的比例约为2：1，但 每个个体R7随机选择是否表达SS。因此，具有相同基因型别的果蝇 SSON：SSOF R7的比例相同，但在每只苍蝇身上，这些光感受器以独特的随机模式分布。 重要的是，SS的每个拷贝的表达都是独立调节的，即使在它们所在的同一细胞核中也是如此 暴露在相同的反式作用因素中，表明导致 SS表达式的决定发生在SS轨迹，而不是SS表达式的上游步骤 路径。本研究的目标是阐明控制随机性的基因调控机制。 SS在R7s中的表达。Ss基因包含几个预测的绝缘子DNA元件。作为绝缘体蛋白 相互作用形成促进DNA环形成的复合体，我们假设 绝缘子介导的DNA环导致sSON和sSON中ss位点随机地呈现不同的构象 SSOF R7s，并且ss的循环状态确定其ON或OFF表达式状态。我们将通过1)测试这一想法 SSON和SSEF R7S中SS轨迹处绝缘子占有率的特征，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)确定和操纵环状状态 SSON和Ssoff R7s中的ss基因座。综上所述，这些实验将提供第一次分析 绝缘子介导的DNA环在随机基因表达调控中的作用 对随机细胞命运规范的机械理解。

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.