Predictive understanding of the temporal control of transcription in Drosophila development

对果蝇发育中转录时间控制的预测性理解

• 批准号: 10608142
• 负责人: Hernan Gustavo Garcia
• 金额: $ 30万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10608142
• 关键词: Acetylation; Affect; Binding; Binding Sites; Biochemical; Biological Assay; Biomedical Engineering; Case Study; Cell Cycle; Cell Proliferation; Cells; Chromatin; Cytoplasm; DNA; DNA Sequence; Data; Decision Making; Defect; Development; Developmental Biology; Disease; Dissection; Drosophila genus; Drosophila melanogaster; Electronics; Embryo; Embryonic Development; Engineering; Future; Gene Expression; Genes; Genetic Transcription; Histone Acetylation; Histone Deacetylase; Histones; Human Genome; Investigation; Knock-out; Libraries; Malignant Neoplasms; Measurement; Measures; Messenger RNA; Microscope; Modeling; Molecular; Mutation; Nature; Nucleic Acid Regulatory Sequences; Organism; Output; Pathway interactions; Pattern; Physicians; Play; Precision therapeutics; Process; Production; Proteins; Regulation; Reporter; Role; Scientist; Series; Shapes; Structure; Technology; Testing; Theoretical model; Time; Transcription Initiation; Transcriptional Regulation; Work; chromatin modification; empowerment; experimental study; fly; genome-wide; genome-wide analysis; histone acetyltransferase; in vivo; knock-down; mutant; new technology; optogenetics; predictive modeling; programs; promoter; repaired; single molecule; small hairpin RNA; spatiotemporal; synthetic biology; temporal measurement; theories; tissue fixing; tool; transcription factor

Project Summary/Abstract The correct implementation of developmental programs depends on information encoded in an organism's DNA. Despite decades of work in dissecting the spatial control of gene expression in embryonic development, we know relatively little about the temporal control of these programs—largely due to reliance on dead, fixed tissues. Recently, our lab established new technologies for real-time measurements of input transcription factor concentration dynamics and output transcriptional activity in single cells of the early embryo of the fruit fly Drosophila melanogaster. Our measurements have revealed that the timing of transcription in development is under precise control. Here, we propose a dialogue between theory and experiment to dissect this largely unexplored layer of transcriptional control using the regulation of the hunchback gene by the activator Bicoid and the pioneer-like transcription factor Zelda as a case study. Recent work from our lab has suggested that hunchback transcription ensues as a result of the transition of its promoter through multiple transcriptionally silent states. In this model, Bicoid and Zelda catalyze the transitions between silent states, presumably by triggering the acetylation of nearby histones. In order to systematically test this model of transcriptional onset and reveal the molecular identities of the pathways involved in the dictating transcriptional onset, we will (i) exploit optogenetics to determine whether the timing Bicoid- and Zelda-driven transcriptional onset is independent of the control of mRNA production rates once transcription has already ensued, (ii) harness measurements of the distribution of transcriptional onset times in single cells to uncover the structure of the biochemical cascade leading to transcriptional onset and how this cascade is modulated by Bicoid and Zelda binding, and (iii) identify the histone acetylases and deacetylases invoked by Bicoid and Zelda to dictate transcriptional onset, a process that takes place at time scales that are not accessible by commonplace genome-wide approaches to dissect the chromatin landscape. Overall, our proposed work will establish a clear workflow for the dissection of this new layer of regulatory control given by the timing of transcription in development and for uncovering the underlying molecular pathways. This approach will be amenable to be implemented in other relevant genes in the fruit fly as well as in other workhorses of developmental biology. Further, we envision that our quantitative and predictive approach to dissecting developmental programs will empower future synthetic applications as well as reengineering of multicellular organisms, for example to fix developmental defects or to halt states of unchecked cellular proliferation.

项目摘要/摘要 发展程序的正确实施取决于有机体中编码的信息 脱氧核糖核酸。尽管在剖析了胚胎发育中基因表达的空间控制方面的数十年工作，但 我们对这些程序的临时控制相对较少了解 - 很大程度上是由于死亡，固定的退休 组织。最近，我们的实验室建立了用于实时测量输入转录因子的新技术 果蝇早期胚胎的单细胞中的浓度动力学和输出转录活性 果蝇Melanogaster。我们的测量结果表明，开发中转录的时机是 在精确控制下。 在这里，我们提出了理论与实验之间的对话，以剖析这个很大程度上出乎意料的层 使用激活剂双子和先驱样的转录控制使用驼背基因调节 转录因子Zelda作为案例研究。我们实验室的最新工作表明Hunchback 转录是由于其启动子通过多个转录沉默状态的过渡而导致的。 在此模型中，双子和Zelda催化了无声状态之间的过渡，大概是通过触发 附近组蛋白的乙酰化。为了系统地测试此转录发作模型并揭示 指示转录发作所涉及的途径的分子身份，我们将（i）利用 光遗传学以确定定时双子和ZELDA驱动的转录发作是否独立于 一旦确保了转录，对mRNA生产率的控制，（ii）线索测量 单个细胞中转录发作时间的分布，以发现生化级联的结构 导致转录发作，以及该级联反应如何通过双聚体和Zelda结合调节，以及（iii） 鉴定由双聚体和Zelda引用的组蛋白乙酰酶和脱乙酰基酶决定转录发作，A 在时间尺度上进行的过程无法通过常见的全基因组方法访问 解剖染色质景观。 总体而言，我们提出的工作将建立清晰的工作流程，以解剖这一新的监管层 由发育中转录的时间和揭示基础分子的控制 途径。这种方法将可以在果蝇中的其他相关基因以及 在其他发展生物学的工作中。此外，我们设想我们的定量和预测性 剖析发展计划的方法将增强未来的合成应用以及 重新设计多细胞生物，例如修复发育缺陷或停止的状态 未检查的细胞增殖。