Predictive understanding of the temporal control of transcription in Drosophila development

项目总结/摘要 发育程序的正确执行取决于编码在有机体的 DNA.尽管几十年来一直在研究胚胎发育中基因表达的空间控制， 我们对这些程序的时间控制知之甚少，这主要是由于依赖于死的、固定的、 组织中最近，我们的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中的浓度动态和输出转录活性 果蝇我们的测量结果显示，发育过程中转录的时间是 在精确的控制下。 在这里，我们提出了理论和实验之间的对话，以剖析这一基本上未被探索的层面。 转录控制，使用通过激活剂Bicoid和先驱样蛋白调节hunchback基因， 转录因子塞尔达作为一个案例研究。我们实验室最近的工作表明，驼背 转录增强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，Bicoid和Zelda催化了沉默状态之间的转换，大概是通过触发 邻近组蛋白的乙酰化。为了系统地测试这种转录起始模型，并揭示 分子身份的途径参与的支配转录开始，我们将（i）利用 光遗传学，以确定Bicoid和Zelda驱动的转录起始时间是否独立于 一旦转录已经发生，mRNA生产率的控制，（ii）利用测量的 转录起始时间在单细胞中的分布，以揭示生化级联反应的结构 导致转录起始以及该级联如何被Bicoid和Zelda结合调节，以及（iii） 确定Bicoid和Zelda所调用的组蛋白乙酰化酶和去乙酰化酶来决定转录起始， 这一过程发生的时间尺度是普通的全基因组方法无法达到的， 解剖染色质景观。 总的来说，我们提出的工作将建立一个明确的工作流程，以解剖这一新的监管层， 由发育中的转录时间给予的控制，以及用于揭示潜在的分子生物学特征的控制。 途径。这种方法将适用于果蝇中的其他相关基因， 在发育生物学的其他研究中。此外，我们设想，我们的定量和预测 一种剖析发展程序的方法将使未来的综合应用成为可能， 多细胞生物的再工程，例如修复发育缺陷或停止 不受控制的细胞增殖

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.