Mechanisms controlling stochastic gene expression during eye development

 描述(由申请人提供)：在发育过程中，细胞命运的指定取决于可复制和随机调节机制。对于在单个细胞中随机开启或关闭基因表达以实现命运多样化的随机机制，人们知之甚少。随机的细胞命运选择对于视觉和嗅觉受体的多样化、运动神经元亚型的指定、神经元中的树突自我回避、免疫细胞命运的确定和干细胞的分化都是重要的。研究随机基因表达将增强我们对视力障碍、嗅觉障碍、自闭症、免疫缺陷和淋巴瘤的理解。我们工作的主要目标是以果蝇颜色光感受器的规范为模型，研究发育过程中基因表达是如何以随机开/关的方式进行调节的。苍蝇的色觉系统是由无刺转录因子的随机开/关表达决定的两个光感受器亚型的随机镶嵌。我们最近发现，在暴露于相同环境中的反式作用因子的单个核内，无刺基因的每个拷贝都做出了内在的、随机的表达决定。我们的研究表明，随机的无刺表达需要R7感光细胞中特定转录因子的调节，绝缘体DNA元件介导DNA环，以及多梳反应元件/三胸反应元件(PreS/Tres)调节表达。我们假设，在所有R7中表达的转录因子的唯一组合是随机SS表达所必需的(目标1)。我们认为，随机开/关的表达是由SS轨迹随机决定的，假设绝缘子介导的两种DNA环状构型中的一种(目标2)。这些构型决定了抑制性Pre/TrE与ss启动子的接近程度。在On细胞中，抑制性的Pre/Tre与启动子保持较远的距离以允许激活；在On细胞中，抑制性Pre/Tre位于启动子的近端以抑制表达(目标3)。我们将通过评估转录因子的结合和组合性来检验这些假说，通过使用CRISPR/Cas9产生缺失来确定绝缘体和前/tre DNA元件的功能，并进行转基因检测，以及使用最近发展的cgChIP技术来检查无刺ON和无刺ON细胞中的DNA环条件和组蛋白修饰。实现这些目标将表征反式因子、绝缘体介导的DNA环和染色质状态如何调控随机基因表达。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.