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Abstract

The cell nucleus is organized into functional domains that form around chromatin, which serves as a scaffold composed of DNA, proteins, and associated RNAs. On the 0.1-1 µm mesoscale these domains can form spatially defined compartments with distinct composition and properties that enrich specific genomic activities like transcription, chromatin modification or DNA repair. In addition, extrachromosomal DNA elements and RNAs can separate from the chromatin template and assemble with proteins into nuclear bodies. The resulting accumulations of proteins and nucleic acids in the nucleus modulate chromatin-templated processes and their organization. The assembly of these compartments occurs in a self-organizing manner via direct and indirect binding of proteins to DNA and/or RNA. Recently, it has been proposed that multivalent interactions drive compartmentalization by inducing phase separation with a non-stoichiometric accumulation of factors into biomolecular condensates. Despite the importance of compartments for genome regulation, insights into their structure and material properties and how these affect their function is limited. To address this issue, it is important to devise approaches that can perturb nuclear compartments in a targeted manner, while also measuring changes in genome activities within the same cell. In this thesis, the methodology to reveal the underlying structure-function relationships of nuclear compartments has been advanced and applied to compartments involved in activation and silencing of chromatin, and telomere maintenance in cancer cells. I first established a toolbox of chromatin effector constructs to probe and perturb properties of nuclear compartments in living cells that comprised different combinations of DNA binding, transcription activation and light-dependent interaction domains. In addition, I developed workflows to quantitatively assess relevant compartment features by fluorescence microscopy. These methods were employed to study the compaction mechanism of mouse pericentric heterochromatin (PCH) foci and to investigate the interplay between transcriptional co-activators, phase separation and transcription at an inducible reporter gene cluster. It revealed determinants of PCH compaction and identified differential co-activator usage and multivalent interactions as contributors to transcription factor (TF) strength. The results furthermore challenged the model of TF phase separation as a general positive driver of gene transcription. In the second part, I focused on exploiting the detection of compartments for measuring activity of the alternative lengthening of telomeres (ALT) pathway used by cancer cells to extend their telomeres in absence of telomerase. I developed ALT-FISH, a scalable and quantitative imaging assay that detects ALT pathway-specific compartments containing large amounts of single-stranded telomeric nucleic acids. I applied the method to cell line models from different cancer entities and to tumor tissue from leiomyosarcoma and neuroblastoma patients. By devising automated ALT-FISH data acquisition and analysis IV workflows, I implemented an approach, which enabled ALT activity measurements in hundreds of thousands of single cells. These technological advancements provided a quantitative description of ALT activity at single cell resolution and were used to characterize the spatial distribution of ALT activity in relation to other biological features and in response to perturbations. Finally, a novel approach for studying the regulation of ALT in tumors could be established by integrating the method with the spatially resolved detection of single cell transcriptomes. In summary, this thesis introduced and utilized several methods to establish connections between nuclear compartment organization, chromatin features, transcription regulation, and telomere maintenance. These perturbation and imaging techniques are versatile and may be applied to dissect nuclear activities related to other compartments and biological model systems. Furthermore, the detection of ALT activity has demonstrated that compartments can offer valuable biological insights into how phenotypic cellular heterogeneity is encoded and linked to diseases such as cancer.