The physical and dynamical properties of transcriptional microenvironments during embryonic development

掌握发育转录因子通过控制许多下游靶基因的表达来决定动物的身体计划。最近的研究表明，这些基因的转录发生在局部转录因子浓度较高的微环境中的低亲和力结合部位。我们已经在含有主要转录因子Ultrabithorax(UBx)的黑腹果蝇胚胎中表明，这些核微环境是高度动态的。我们建议通过以下目的来研究它们在活的果蝇胚胎中的物理和动力学性质。1)为了跟踪增强子与微环境的相互作用，而不考虑它们的转录状态，我们计划直接在果蝇中Ubx调控的增强子附近标记特定的基因座，用于在固定和活胚胎中进行成像。2)我们已经看到Ubx和Abda这两个具有难以区分的共识结合序列的因子在空间上分离在同一核中。我们的目标是用不同的相互作用结构域荧光标记Abda和Ubx的其他变体。一个标记了Ubx和Abda的工具箱将允许我们观察这些蛋白质如何动态地分类到它们各自的微环境中。3)作为微环境中枢的染色质在发育过程中也会发生构象变化。我们计划在染色质上生成带有多个标记位置的飞行线条。这个文库将允许我们在开发过程中测量染色质的位置和运动动态，以提供一个放置UBX驱动的增强子的一般背景。总之，这项建议寻求将严格的生物物理量化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能够在空间和时间上具体和准确地调节转录。

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.