Mechanisms controlling stochastic gene expression during eye development

 描述（由申请方提供）：在开发期间，细胞命运质量标准取决于可重现和随机调节机制。人们对在单个细胞中随机开启或关闭基因表达以使命运多样化的随机机制知之甚少。随机细胞命运选择对于视觉和嗅觉受体的多样化、运动神经元亚型的特化、神经元中的树突状自我回避、免疫细胞命运的确定以及干细胞的分化是重要的。研究随机基因表达将增强我们对视觉障碍、嗅觉丧失、自闭症、免疫缺陷和淋巴瘤的理解。我们的工作的主要目标是解决如何在一个随机的开/关的方式在发展过程中使用果蝇的彩色光感受器规格为模型的基因表达进行调节。 果蝇的色觉系统是由转录因子Spineless的随机开/关表达决定的两种感光器亚型的随机镶嵌。我们最近发现，在暴露于相同反式作用因子环境的单个细胞核内，无脊椎基因的每个拷贝都做出内在的、随机的表达决定。我们的研究表明，随机无刺的表达需要在R7光感受器，绝缘体DNA元件介导的DNA循环和Polycomb响应元件/三胸响应元件（PRE/TREs），调节表达的特定转录因子的调节。 我们假设，一个独特的组合，在所有R7的转录因子表达所需的随机ss表达（目的1）。我们提出，随机开/关表达是由SS基因座随机假设绝缘体介导的两个DNA环配置之一（目的2）。这些构型决定了阻遏性PRE/TRE与ss启动子的接近程度。在on细胞中，抑制性PRE/TRE保持远离启动子以允许激活;在off细胞中，抑制性PRE/TRE接近启动子以抑制表达（Aim 3）。我们将通过评估转录因子结合和组合性来测试这些假设，通过使用CRISPR/Cas9产生缺失并进行转基因测定来确定绝缘子和PRE/TRE DNA元件的功能，并使用最近开发的cgChIP技术检查无脊椎细胞和无脊椎细胞中的DNA循环条件和组蛋白修饰。实现这些目标的特点是如何调控反式因子，绝缘介导的DNA循环，染色质状态控制随机基因表达。我们的发现将对我们理解细胞核的3D结构和表观遗传修饰如何控制基因表达产生深远的影响。

 DESCRIPTION (provided by applicant): During development, cell fate specification depends on both reproducible and stochastic regulatory mechanisms. Very little is known about stochastic mechanisms that turn gene expression on or off randomly in individual cells to diversify fates. Random cell fate choices are important for the diversification of visual and olfactory receptors, specification of motor neuron subtypes, dendritic self-avoidance in neurons, determination of immune cell fates, and differentiation of stem cells. Studying stochastic gene expression will enhance our understanding of vision disorders, anosmia, autism, immunodeficiencies, and lymphoma. The main goal of our work is to address how gene expression is regulated in a stochastic on/off manner during development using specification of Drosophila color photoreceptors as a model. The color vision system of the fly is a random mosaic of two photoreceptor subtypes determined by the stochastic on/off expression of the transcription factor Spineless. We recently discovered that each copy of the spineless gene makes an intrinsic, random expression decision within a single nucleus exposed to the same milieu of trans-acting factors. Our studies suggest that stochastic spineless expression requires regulation by specific transcription factors in R7 photoreceptors, insulator DNA elements that mediate DNA looping and Polycomb Response Elements/Trithorax Response Elements (PREs/TREs) that regulate expression. We hypothesize that a unique combination of transcription factors expressed in all R7s is required for stochastic ss expression (Aim 1). We propose that stochastic on/off expression is dictated by the ss locus randomly assuming one of two DNA looping configurations mediated by insulators (Aim 2). These configurations determine the proximity of a repressive PRE/TRE to the ss promoter. In on cells, the repressive PRE/TRE remains distant from the promoter to allow activation; in off cells, the repressive PRE/TRE is proximal to the promoter to repress expression (Aim 3). We will test these hypotheses by assessing transcription factor binding and combinatoriality, determining functionality of insulators and PRE/TRE DNA elements by generating deletions using CRISPR/Cas9 and conducting transgene assays, and examining the DNA looping conditions and histone modifications in the Spineless on and Spineless off cells using the recently developed cgChIP technology. Achieving these aims will characterize how regulation by trans factors, insulator-mediated DNA looping, and chromatin state control stochastic gene expression. Our findings will have a profound impact on our understanding of how the 3D architecture of the nucleus and epigenetic modifications control gene expression.