The role of chromatin dynamics in stochastic cell fate specification

项目概要：细胞随机选择细胞命运的随机细胞命运规范是关键 在发展过程中。随机机制使视觉和嗅觉受体、运动神经元、免疫 细胞和干细胞。这些机制的破坏导致视力障碍、嗅觉丧失、免疫缺陷， 自闭症和淋巴瘤。我研究发展中的果蝇视觉系统作为一个范例，以了解 控制随机细胞命运规范的机制。在蝇眼内，R7光感受器产生随机的 两种亚型之间的二元命运选择，其特征在于光检测视紫红质（Rh 3或Rh 4）的表达。 Rh4）。这个决定是由转录因子Spineless（Ss）的随机ON/OFF表达控制的。 SsON R7表达Rh 4，SsOFF R7表达Rh 3。每个R7独立地做出这个随机决定， 有67%的机会选择SsON R7命运，33%的机会选择SsOFF R7命运。因此，视网膜具有一致的 亚型比率，但独特的，随机模式。 我们已经确定了一个两步机制控制生长抑素的表达在发展过程中。在第一步中， 早期的表达脉冲打开了前体R7中的ss基因位点。在第二步中，轨迹压缩为变化的 程度，决定了在末端R7亚型特异化后重新启动表达的能力。这最后 然后在动物的一生中维持表达决定。我在研究动态染色质 在ss基因座的压实影响随机R7亚型特化。我假设R7的前体 在表达的早期脉冲后，在ss位点快速重组染色质，以确定ON/OFF 末端R7s中ss表达的状态。 为了验证这一假设，我将使用固定和实时成像技术来跟踪R7期间的ss压缩 子类型规范。为了在固定图像中可视化压实，我开发了一种三色DNA-FISH策略 其标记SS基因座、上游区域和下游区域。测量这些物体之间的3D距离 区域提供了细胞类型特异性定量的压缩在发展中的R7。作为对这种方法的补充， 我开发了一种利用LacO/LacI报告系统的实时成像方法。将LacO重复插入到 ss上游和下游区域，我将测量两个LacI报告点之间的3D距离 在区分R7时，重点关注无法用固定成像识别的过渡点。以促进 定量，我开发了一个自动化的图像分析管道，以评估染色质压实固定和 活组织（Aim 1）。我将通过进行ATAC-seq鉴定染色质来补充这些方法 在正常和突变条件下SS基因座的可接近性。我将特别侧重于 晚期增强子，其活性最终决定ssON或ssOFF R7的命运（目的2）。该项目的结果 将阐明随机过程中染色质动态和表达状态之间的调控关系， 细胞命运规格。

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.