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Super-resolution Imaging of Chromatin and Functional Nuclear Architecture https://doi.org/10.17863/CAM.96447 Ball, Madeleine White, Robert O'Holleran, Kevin The regulation of transcription is well understood to be linked to the three-dimensional organisation of the genome within the nucleus, however, the mechanisms through which the different levels of organisation regulate genes are poorly understood. Newly developing super-resolution imaging techniques offer an important new way to investigate structural features of the genome, however, imaging chromatin within the nucleus is traditionally very difficult due to the dense packaging with a small nuclear volume. The nuclei of Drosophila melanogaster primary spermatocytes have unique properties that can circumvent these limitations, with comparatively large nuclei with well-separated chromosome masses, and transcriptionally active Y loops that expand into the nuclear interior as clearly observable individual fibres. Therefore, during this thesis I took advantage of this model system to image both the chromatin of the Y loops and chromosome masses. A single molecule localisation microscopy technique was optimised for imaging within the nucleus, with both 3D and dual-colour capability. Then, the Y loops were imaged at super-resolution, and a specialised clustering protocol was developed for quantification. This showed that the Y loops are organised as a chain of clusters, with an average width of approximately 50 nm, and an average distance apart from each other of roughly 100 nm. The relationship between actively elongating transcription, as well as different phosphorylation states of RNA polymerase II, and the Y loop chromatin was investigated and quantified, revealing that polymerase appears adjacent to the Y loop fibres, attached via a smaller chromatin loop emanating from the clusters. The role of transcription in chromatin organisation was assessed on the Y loops through the use of transcription inhibition, and on the autosomal chromosomes through mutant fly lines, using empty-space statistics to quantify their organisation. This indicated that the Y loop chains of clusters structure was unlikely to be determined by transcription. Active and inactive histone modifications were labelled along the Y loops, which were quantified for comparative analysis, with implications for the link between chromatin state and function. The work conducted during this thesis identified novel architectures of transcriptionallyactive chromatin and so provides a foundation for understanding chromatin organisation within the nucleus, and the relationship between transcription and chromatin state. University of Cambridge 2022-09-29 Thesis Doctoral Doctor of Philosophy (PhD) eng UKRI - BBSRC-DTP Department of PDN - G. H. Lewes scholarship https://www.repository.cam.ac.uk/handle/1810/349169 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/b773c31c-fd84-4dfb-a755-f834332d22a8/download 2e5ac0418120a0c22c3017cc8c91646d https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/9fcd63af-eb1f-4614-8639-affcd9b7c77f/download 87eda9de84448d1f82354d60eee3eb5f https://www.rioxx.net/licenses/all-rights-reserved/ Chromatin Development Genome architecture Super-resolution Transcription