The physical and dynamical properties of transcriptional microenvironments during embryonic development

胚胎发育过程中转录微环境的物理和动力学特性

• 批准号: 426035257
• 负责人: Dr. Albert Tsai
• 金额: --
• 依托单位: Europäisches Laboratorium für Molekularbiologie (EMBL)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/426035257?language=en
• 关键词: physical; dynamical; properties; transcriptional; microenvironments

Master developmental transcription factors determine animal body plans through controlling the expression of many downstream target genes. Recent studies have shown that transcription of these genes occur from low affinity binding sites in microenvironments of high local transcription factor concentrations. We have shown in Drosophila melanogaster embryos with the master transcription factor Ultrabithorax (Ubx) that these nuclear microenvironments are highly dynamic. We propose to investigate their physical and dynamical properties inside living Drosophila embryos through the following aims. 1) To track how enhancers interact with microenvironments regardless of their transcriptional states, we plan to directly mark specific loci near Ubx regulated enhancers in Drosophila for imaging in both fixed and live embryos. 2) We have seen that Ubx and AbdA, two factors with indistinguishable consensus binding sequences, spatially segregate in the same nucleus. We aim to fluorescently tag AbdA and additional variants of Ubx with different interaction domains. A toolbox of tagged Ubx and AbdA will allow us to observe how these proteins dynamically sort into their respective microenvironments. 3) The chromatin, which is the backbone for microenvironment, also changes its conformation during development. We plan to generate fly lines with multiple tagged locations on the chromatin. This library will allow us to gauge the positioning and movement dynamics of the chromatin during development to provide a general context within which to place Ubx-driven enhancers. In sum, this proposal seeks to combine rigorous biophysical quantitation with the genetic toolbox of developmental biology to address how the physical organization of the nuclear space can specifically and precisely regulate transcription in both space and time.

发育转录因子通过调控下游靶基因的表达来决定动物的体型。最近的研究表明，这些基因的转录发生在高局部转录因子浓度的微环境中的低亲和力结合位点。我们已经在具有主转录因子Ultrabithorax（Ubx）的果蝇胚胎中表明，这些核微环境是高度动态的。我们建议通过以下目标来研究它们在果蝇胚胎中的物理和动力学特性。1)为了跟踪增强子如何与微环境相互作用，无论其转录状态如何，我们计划直接标记果蝇中Ubx调节增强子附近的特定位点，以在固定和活胚胎中成像。2)我们已经看到，Ubx和AbdA，具有不可区分的共有结合序列的两个因子，在同一个细胞核中空间分离。我们的目标是荧光标记AbdA和其他具有不同相互作用结构域的Ubx变体。标记的Ubx和AbdA工具箱将使我们能够观察这些蛋白质如何动态地分类到各自的微环境中。3)作为微环境骨架的染色质在发育过程中也会改变其构象。我们计划在染色质上生成具有多个标记位置的飞行线。该文库将使我们能够测量染色质在发育过程中的定位和运动动力学，以提供放置Ubx驱动的增强子的一般背景。总而言之，这项建议旨在将联合收割机严格的生物物理定量与发育生物学的遗传工具箱相结合，以解决核空间的物理组织如何在空间和时间上特异性地和精确地调节转录。