The role of chromatin dynamics in stochastic cell fate specification

染色质动力学在随机细胞命运规范中的作用

• 批准号: 10557193
• 负责人: Lukas Voortman
• 金额: $ 2.73万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-16 至 2023-04-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10557193
• 关键词: 3-Dimensional; ATAC-seq; Animals; Anosmia; Binding Sites; Cells; Chromatin; Code; Color; Complement; Computer Analysis; Culture Techniques; DNA; Data; Development; Disease; Drosophila genus; Elements; Enhancers; Eye; Fluorescent in Situ Hybridization; Gene Expression; Generations; Genes; Genetic Complementation Test; Genetic Transcription; Goals; Hour; Human; Image; Imaging Techniques; Immune; Immunologic Deficiency Syndromes; Individual; Knock-out; Label; Lead; Light; Lymphoma; Measures; Methods; Molecular; Molecular Conformation; Motor Neurons; Pattern; Photoreceptors; Physiologic pulse; Reporter; Retina; Rhodopsin; Role; Signal Transduction; Specific qualifier value; System; Testing; Tissues; Undifferentiated; Vision Disorders; Visual System; Visual impairment; Visualization; analysis pipeline; autism spectrum disorder; automated image analysis; cell fate specification; cell type; color detection; experimental study; fly; genomic locus; imaging approach; mosaic; mutant; olfactory receptor; promoter; stem cells; transcription factor

Project Summary: Stochastic cell fate specification, in which cells randomly choose between cell fates, is critical during development. Stochastic mechanisms diversify visual and olfactory receptors, motor neurons, immune cells, and stem cells. Breakdowns in these mechanisms lead to vision disorders, anosmia, immunodeficiency, autism, and lymphomas. I study the developing Drosophila visual system as a paradigm to understand the mechanisms controlling stochastic cell fate specification. Within the fly eye, R7 photoreceptors make a stochastic binary fate choice between two subtypes, characterized by expression of light-detecting rhodopsins (Rh3 or Rh4). This decision is controlled by the stochastic ON/OFF expression of the transcription factor Spineless (Ss). SsON R7s express Rh4 and SsOFF R7s express Rh3. Each R7 independently makes this random decision with a 67% chance of taking the SsON R7 fate and 33% chance of taking the SsOFF R7 fate. Thus, retinas have consistent subtype ratios, but unique, random patterns. We have identified a two-step mechanism controlling ss expression during development. In step one, an early pulse of expression opens the ss gene locus in precursor R7s. In step two, the locus compacts to varying degrees, determining the ability to reinitiate expression upon specification of terminal R7 subtypes. This final expression decision is then maintained for the lifetime of the animal. I am investigating how dynamic chromatin compaction at the ss locus influences stochastic R7 subtype specification. I hypothesize that R7 precursors rapidly reorganize chromatin at the ss locus following the early pulse of expression to determine the ON/OFF state of ss expression in terminal R7s. To test this hypothesis, I will use fixed and live imaging techniques to track ss compaction during R7 subtype specification. To visualize compaction in fixed images, I developed a three-color DNA-FISH strategy that labels the ss locus, upstream region, and downstream region. Measuring the 3D distance between these regions provides cell-type-specific quantification of compaction in developing R7s. Complementing this method, I developed a live imaging approach utilizing the LacO/LacI reporter system. Inserting LacO repeats into the regions upstream and downstream of ss, I will measure the 3D distance between the two LacI reporter punctae in differentiating R7s, focusing on transition points that cannot be identified with fixed imaging. To facilitate quantification, I developed an automated image analysis pipeline to evaluate chromatin compaction in fixed and live tissue (Aim 1). I will complement these approaches by conducting ATAC-seq to identify chromatin accessibility at the ss locus in normal and mutant conditions. I will focus specifically on the accessibility of the late enhancer whose activity ultimately determines the ssON or ssOFF R7 fate (Aim 2). The results of this project will elucidate the regulatory relationship between chromatin dynamics and expression state during stochastic cell fate specification.

项目摘要：随机细胞命运规范，其中细胞在细胞命运之间随机选择是至关重要的 在开发过程中。随机机制使视觉和嗅觉受体多样化，运动神经元，免疫 细胞和干细胞。这些机制的故障导致视力障碍，厌食，免疫缺陷， 自闭症和淋巴瘤。我研究了发展中的果蝇视觉系统，以了解 控制随机细胞命运规范的机制。在苍蝇眼中，R7感光器使随机性 两种亚型之间的二元命运选择，其特征是表达光检测的视紫红蛋白（RH3或 RH4）。该决定由转录因子无脊椎（SS）的随机开/关表达控制。 SSON R7S Express RH4和SSOFF R7S Express RH3。每个R7独立做出随机决定 67％的机会取得SSON R7命运和33％的机会取得SSOFF R7命运的机会。因此，视网膜具有一致的 亚型比率，但独特的随机模式。 我们已经确定了一种控制开发过程中SS表达的两步机制。在第一步，一个 表达的早期脉冲在前体R7中打开SS基因基因座。在第二步中，该基因座压缩为变化 学位，确定在终端R7亚型规范时重新表达表达的能力。决赛 然后，在动物的生命中保持表达决策。我正在研究动态染色质 SS基因座的压实会影响随机R7亚型规范。我假设R7前体 在表达早期脉冲之后，在SS基因座处迅速重组染色质，以确定ON/OFF 终端R7中的SS表达状态。 为了检验该假设，我将使用固定和实时成像技术来跟踪R7期间的SS压实 亚型规范。为了可视化固定图像中的压实，我制定了三色的DNA-fish策略 这标记了SS基因座，上游区域和下游区域。测量这些之间的3D距离 区域提供开发R7中压实的细胞类型定量。补充此方法， 我使用LACO/LACI报告程序系统开发了一种实时成像方法。将LACO重复插入 SS上游和下游的区域，我将测量两个LACI记者点之间的3D距离 在区分R7中，专注于无法用固定成像识别的过渡点。促进 定量，我开发了一个自动图像分析管道，以评估固定和 活组织（目标1）。我将通过进行ATAC-SEQ识别染色质来补充这些方法 在正常和突变条件下在SS基因座的可访问性。我将专门关注 晚期的增强剂的活动最终决定了SSON或SSOFF R7命运（AIM 2）。这个项目的结果 将阐明随机过程中染色质动力学与表达状态之间的调节关系 细胞命运规范。