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: 10443816
• 负责人: Robert John Johnston
• 金额: $ 38.98万
• 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 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10443816
• 关键词: ATAC-seq; Affect; Anosmia; Automobile Driving; Biological Process; Cells; ChIP-seq; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Color; Complement; DNA; Data; Development; Disease; Drosophila genus; Elements; Enhancers; Exhibits; Eye; Eye Development; Fluorescent in Situ Hybridization; Frequencies; Gene Expression; Genes; Genetic Transcription; Genomics; Goals; Human; Image; Immune; Immunologic Deficiency Syndromes; Individual; Investigation; Knock-out; Link; Lymphoma; Modeling; Molecular; Monitor; Mosaicism; Motor Neurons; Mutation; Neurons; Noise; Pattern; Photoreceptors; Physiologic pulse; RNA; Regulation; Regulator Genes; Reporter; Resolution; Retina; Rhodopsin; Role; Signal Transduction; Source; System; Testing; Time; Transcript; Transcription Initiation; Transcriptional Regulation; Undifferentiated; Vision Disorders; Visual impairment; Visualization; autism spectrum disorder; base; cell fate specification; cell type; chromatin remodeling; experimental study; fly; histone modification; imaging approach; mutant; olfactory receptor; olfactory sensory neurons; promoter; retinal neuron; stem; stem cells; transcription factor; visual photoreceptor

Project Summary Cell fate specification is driven by lineage, signaling, and stochastic regulatory inputs. The mechanisms controlling stochastic fate specification, in which a cell randomly chooses between two or more fates, are poorly understood. Stochastic fate specification is critical for diversifying retinal neurons, olfactory sensory neurons, motor neurons, immune cells, and stem cells. Breakdowns in these mechanisms cause debilitating human disorders, including vision impairments, anosmia, autism, immunodeficiencies, and lymphoma. The main goal of this project is to determine how chromatin state and transcriptional variability control stochastic fate specification, using the random patterning of photoreceptor subtypes in the fly retina as a paradigm. The fly eye contains a random mosaic of two color-detecting R7 photoreceptor subtypes, defined by expression of Rhodopsin 4 (Rh4) or Rhodopsin 3 (Rh3). This fate decision is controlled by the transcription factor Spineless (Ss), which is expressed in a random subset of mature R7s. SsON R7s express Rh4, while SsOFF R7s express Rh3. Our data support a two-step mechanism regulating ssON/OFF expression in mature R7s. In step 1, the early enhancer drives an early pulse of ss transcription in R7 precursors that opens the chromatin at the ss locus. In step 2, the transcriptional pulse ceases and chromatin variably closes, defining the accessibility of the late enhancer. Depending on the degree of chromatin compaction, the late enhancer either turns on (open chromatin) or remains off (closed chromatin) for the lifetime of the mature R7. How regulation of transcription and chromatin compaction is integrated to turn genes randomly on or off during development is poorly understood. We will use DNA FISH, genomics, CRISPR, and lacO/LacI-based live imaging approaches to assess the role of chromatin dynamics and the temporality of regulatory inputs in the two-step mechanism (Aim 1). Identification of the source of variability driving stochastic fate specification in metazoans has not been achieved. Our data suggest that variability in transcription (initiation, elongation, frequency, and duration) in individual cells influences terminal R7 fate specification. To test this hypothesis, we will use nascent multi-color RNA FISH and MS2/MCP-based live imaging to assess transcriptional parameters and relate them to R7 subtype fates (Aim 2). Successful completion of these experiments will identify a source of variability that drives a cell fate decision and inform how molecular noise is utilized to diversify cell types during metazoan development.

项目摘要 细胞命运规范是由谱系，信号和随机调控输入驱动的。的 控制随机命运规范的机制，其中细胞随机选择 两个或两个以上的命运，知之甚少。随机命运规范对于多元化至关重要 视网膜神经元、嗅觉神经元、运动神经元、免疫细胞和干细胞。 这些机制的崩溃会导致包括视力在内的人类疾病 损伤、嗅觉丧失、自闭症、免疫缺陷和淋巴瘤。这个项目的主要目标 是确定染色质状态和转录变异性如何控制随机命运 规范，使用果蝇视网膜中感光细胞亚型的随机图案作为一种 范例 蝇眼包含两种颜色检测R7感光器亚型的随机镶嵌， 由视紫红质4（Rh 4）或视紫红质3（Rh 3）的表达定义。这个命运决定是由 转录因子Spineless（Ss），在成熟R7的随机子集中表达。 SsON R7表达Rh 4，而SsOFF R7表达Rh 3。我们的数据支持两步机制 调节成熟R7中ssON/OFF的表达。在步骤1中，早期增强器驱动早期脉冲 在R7前体中ss转录的启动子，打开ss基因座的染色质。在步骤2中， 转录脉冲停止，染色质折叠关闭，定义了晚期细胞的可及性。 增强剂根据染色质致密化的程度，晚期增强子要么开启， (open染色质）或在成熟R7的寿命期间保持关闭（闭合的染色质）。如何 转录和染色质压缩的调节被整合以随机打开基因或 在发展过程中，我们知之甚少。我们将使用DNA FISH，基因组学，CRISPR， 基于lacO/LacI的实时成像方法，用于评估染色质动力学的作用和 两步机制中监管投入的时间性（目标1）。 后生动物中驱动随机命运特化的变异性来源的鉴定 还没有实现。我们的数据表明，转录的变异性（起始，延伸， 频率和持续时间）影响终末R7命运特化。为了验证这一 假设，我们将使用新生多色RNA FISH和基于MS 2/MCP的活体成像， 评估转录参数并将其与R7亚型命运相关（Aim 2）。成功 这些实验的完成将确定驱动细胞命运决定的可变性来源 并告知如何利用分子噪音在后生动物发育过程中使细胞类型多样化。