Mechanisms controlling stochastic gene expression during eye development

眼睛发育过程中控制随机基因表达的机制

• 批准号: 9116841
• 负责人: Robert John Johnston
• 金额: $ 36.45万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9116841
• 关键词: Address; Affect; Anosmia; Architecture; Autistic Disorder; Binding; Biological Assay; Biological Process; Boxing; CRISPR/Cas technology; Cell Nucleus; Cells; Chromatin; Chromatin Loop; Color; Color Visions; DNA; Development; Disease; Distant; Drosophila genus; Elements; Enhancers; Epigenetic Process; Eye; Eye Development; Feedback; Figs - dietary; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Goals; Health; Human; Immune; Immunologic Deficiency Syndromes; Indium; Individual; Light; Lymphoma; Mediating; Modeling; Modification; Molecular; Motor; Motor Neurons; Mutate; Mutation; Neurons; Organism; Photoreceptors; Polycomb; Proteins; Regulation; Regulator Genes; Response Elements; Retinal Cone; Role; Stem cells; System; Technology; Testing; Tissues; Trans-Activators; Transgenes; Variant; Vision; Vision Disorders; Work; cell fate specification; cell type; fly; histone modification; mutant; olfactory receptor; promoter; stem; stem cell differentiation; transcription factor; visual photoreceptor

 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.

 描述（由申请人提供）：在发育过程中，细胞命运规范取决于可重复的和随机的调节机制。对于在个体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。用于视觉和嗅觉受体的多样化、运动神经元亚型的规范、神经元树突自我回避、免疫细胞命运的确定以及干细胞的分化研究。随机基因表达将增强我们对视力障碍、嗅觉丧失、自闭症、免疫缺陷和淋巴瘤的理解。我们工作的主要目标是利用果蝇彩色光感受器的规范来解决在发育过程中如何以随机开/关方式调节基因表达。苍蝇的色觉系统是由转录因子 Spineless 的随机开/关表达决定的两种光感受器亚型的随机镶嵌。无刺基因在暴露于相同反式作用因子环境的单个核内做出内在的、随机的表达决定，我们的研究表明，随机无刺表达需要R7光感受器、介导DNA循环的绝缘体DNA元件中的特定转录因子的调节。调节表达的 Polycomb 响应元件/Trithorax 响应元件 (PRE/TRE) 我们努力发现，随机 ss 表达需要在所有 R7 中表达的转录因子的独特组合。 （目标 1）我们提出，随机的开/关表达是由 ss 基因座决定的，随机假设由绝缘子介导的两种 DNA 环结构之一（目标 2）。在关闭细胞中，抑制性 PRE/TRE 靠近启动子。我们将通过评估转录因子结合和组合性、通过使用 CRISPR/Cas9 生成缺失并进行转基因测定来确定绝缘子和 PRE/TRE DNA 元件的功能以及检查 DNA 循环条件和组蛋白来测试这些假设。使用最近开发的 cgChIP 技术对 Spineless on 和 Spineless off 细胞进行修饰，实现这些目标将表征反式因子、绝缘体介导的 DNA 环和和的调节方式。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。