Super-Resolution Imaging of Nanoscale Chromatin Dynamics in $\textit{Arabidopsis thaliana}$

Chromatin organisation has long been known to influence gene regulation. How exactly the chromatin structure and function influence one another remains a significant question in the field of genomics. Chromatin consists of DNA wrapped around proteins called nucleosomes and can broadly be divided into two states within the nucleus. Euchromatin describes decondensed, accessible chromatin associated with active gene expression, whereas heterochromatin describes compact, inaccessible chromatin associated with inactive or silenced genes. Spatial organisation of these domains is not random, but highly structured in 3D space. Dynamic changes in the spatial organisation of chromatin are associated to changes in cell fate and cellular responses to stress. In Arabidopsis thaliana, a model organism for plant studies, massive changes in chromatin organisation occur during the dedifferentiation of cells. The digestion of the cell wall of plant cells leads to the release of protoplasts, dedifferentiated cells that can acquire pluripotency and regenerate into whole new plants. The leaf-to-protoplast transition can thus be considered as a paradigm for dedifferentiation and what changes in chromatin organisation occur can offer important insights into the plasticity of plant cells. Global decondensation of heterochromatin during dedifferentiation is accompanied by dramatic deregulation of gene expression and a loss of RNA Pol II. However, how euchromatin is affected is unknown. We asked what changes occur in euchromatin organisation during dedifferentiation and whether active transcription plays a role. To determine this, we used a super-resolution imaging method called STED to quantify changes in occupancy, density and distribution of chromatin as well as the distribution of active and inactive chromatin domains and RNA Pol II via immunostaining. To assess the role of transcription in dedifferentiation we repeated our analysis in a mutant with lower transcriptional activity. We found that transcription partially drives the concurrent shift of actively transcribed permissive chromatin domains towards an expanded interchromatin compartment in protoplasts. In addition to a dynamic chromatin structure, Arabidopsis posses a plant specific 3D chromatin structure called the KNOT. The KNOT consists of several genomic loci called KNOT Engaged Elements (KEEs) across multiple chromosomes that form long distance contacts in the nucleus. Recruitment of invasive DNA, such as transgenes, to the KNOT has been shown to silence expression of these sequences. How the KNOT is organised in 3D space and whether this organisation changes between tissues, or under certain stresses, is unknown. To investigate the question, we used branching oligoFISH to label specific KEEs and visualise their 3D localisation and engagement in isolated leaf nuclei. We found that oligoFISH was effective at labelling KEEs, but the lower than expected co-localisation measures suggested further troubleshooting is required. KEEs do not show similarities in chromatin state or sequence, apart from sharing fragments of certain transposable elements. Further exploration of genomic data could reveal further similarities and shed light on the functional mechanism of KLS. To assist in the exploration of the Arabidopsis genome we developed an RShiny based tool that allowed for 1) browsing of the genome, 2) design of oligo primers (for example for use in FISH) and 3) exploration of chromatin state data. Additionally, to address specific questions, this work also focused on the development and optimisation of workflows for sample preparation, imaging and image quantification for the study of chromatin organisation at the nanoscale. For this we 1) established a method for the isolation, embedding, staining, and 3D STED imaging of Arabidopsis thaliana leaf nuclei, 2) developed a program that allows for automated segmentation of the nucleus and nuclear subcompartments.