Predictive understanding of the temporal control of transcription in Drosophila development

项目摘要/摘要 发育计划的正确实施取决于生物体中编码的信息 DNA尽管对胚胎发育中基因表达的空间控制进行了数十年的研究， 我们对这些程序的时间控制知之甚少--主要是因为依赖于死的、固定的 纸巾。最近，我们实验室建立了实时测量输入转录因子的新技术 果蝇早期胚胎单细胞的浓度动态和输出转录活性 黑腹果蝇。我们的测量显示，在发育过程中转录的时间是 在精确的控制下。 在这里，我们建议在理论和实验之间进行对话，以剖析这一在很大程度上未被探索的层 双核激活因子和类先锋基因调控驼背基因的转录调控 以转录因子塞尔达为例。我们实验室最近的研究表明，驼背 转录是其启动子通过多种转录沉默状态转变的结果。 在这个模型中，双曲面和塞尔达催化沉默状态之间的转换，可能是通过触发 邻近组蛋白的乙酰化。为了系统地测试这一转录起始模型，并揭示 涉及口述转录起始的通路的分子身份，我们将(I)利用 光遗传学来确定双核细胞和塞尔达基因驱动的转录起始时间是否独立于 一旦转录已经发生，就控制信使核糖核酸的产生速率，(Ii)利用对 单细胞转录起始时间的分布揭示生化级联的结构 导致转录的开始，以及这个级联反应是如何被双核和塞尔达结合所调控的，以及(Iii) 确定组蛋白乙酰基酶和去乙酰基酶被BIKID和Zelda激活以决定转录起始，a 发生在时间尺度上的过程，而普通的全基因组方法无法访问 剖析染色质景观。 总体而言，我们提议的工作将为剖析这一新的监管层面建立一个明确的工作流程 由发育中转录的时间和揭示潜在分子的时间所给予的控制 小路。这种方法将易于在果蝇的其他相关基因中实施，以及 在发育生物学的其他主力领域。此外，我们设想我们的量化和预测性 剖析开发计划的方法将使未来的合成应用程序以及 对多细胞生物进行再工程，例如修复发育缺陷或停止 肆无忌惮的细胞增殖。

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.