Super-resolution imaging of chromatin and nuclear architecture

该项目涉及应用超分辨率技术观察果蝇初级精母细胞内的染色质和基因组组织。转录的组织沿着活跃的“Y环”将被解决，RNA聚合酶II的行动有关染色质状态的细节将在野生型精母细胞，以及突变体中超过1000精子发生基因已被敲除进行评估。在这些细胞群中，将研究Y环是如何形成的，转录是如何沿着单个染色质纤维进行的，以及聚合酶沿着染色质纤维的延伸是如何与组蛋白修饰如H3K27 me3相关的。超分辨率的使用提供了能够直接可视化与基因组组织相关的转录机制的独特能力，其中所涉及的因素通常超出衍射极限，因此通常不能使用传统的荧光显微镜进行解析。关于聚合酶的作用，以及如何组织和调节局部转录的“转录工厂”以确保正常的基因功能，仍然存在许多基本问题，而这个项目有能力阐明其中的许多问题。

This project involves the application of super-resolution techniques to observe chromatin and genome organisation within Drosophila melanogaster primary spermatocytes. The organisation of transcription along the active "Y loops" will be resolved, and detail of RNA polymerase II action in relation to chromatin state will be evaluated in wild-type spermatocytes, as well as in mutants where over 1000 spermatogenesis genes have been knocked out. In these groups of cells it will be investigated how the Y loops are formed, how transcription progresses along a single chromatin fibre, and how the elongation of polymerase along a chromatin fibre is related to histone modifications such as H3K27me3. The use of super-resolution offers the unique ability of being able to directly visualise the mechanism of transcription in relation to genome organisation, of which the factors involved usually lie beyond the diffraction limit, and therefore cannot usually be resolved using traditional fluorescence microscopy. Many fundamental questions remain about the action of polymerase, and how "transcription factories" of localised transcription are organised and regulated to ensure normal gene function, and this project has the ability to shed light on many of these.