Super-resolution imaging of chromatin and nuclear architecture

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.