<> "The repository administrator has not yet configured an RDF license."^^ . <> . . "Perturbing and imaging nuclear compartments to reveal mechanisms of transcription regulation and telomere maintenance"^^ . "The cell nucleus is organized into functional domains that form around chromatin, which \r\nserves as a scaffold composed of DNA, proteins, and associated RNAs. On the 0.1-1 µm \r\nmesoscale these domains can form spatially defined compartments with distinct composition \r\nand properties that enrich specific genomic activities like transcription, chromatin modification \r\nor DNA repair. In addition, extrachromosomal DNA elements and RNAs can separate from the \r\nchromatin template and assemble with proteins into nuclear bodies. The resulting \r\naccumulations of proteins and nucleic acids in the nucleus modulate chromatin-templated \r\nprocesses 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 \r\nhas been proposed that multivalent interactions drive compartmentalization by inducing phase \r\nseparation with a non-stoichiometric accumulation of factors into biomolecular condensates. \r\nDespite the importance of compartments for genome regulation, insights into their structure \r\nand material properties and how these affect their function is limited. To address this issue, it \r\nis important to devise approaches that can perturb nuclear compartments in a targeted \r\nmanner, while also measuring changes in genome activities within the same cell. In this thesis, \r\nthe methodology to reveal the underlying structure-function relationships of nuclear \r\ncompartments has been advanced and applied to compartments involved in activation and\r\nsilencing of chromatin, and telomere maintenance in cancer cells.\r\nI first established a toolbox of chromatin effector constructs to probe and perturb properties of \r\nnuclear compartments in living cells that comprised different combinations of DNA binding, \r\ntranscription activation and light-dependent interaction domains. In addition, I developed \r\nworkflows to quantitatively assess relevant compartment features by fluorescence \r\nmicroscopy. These methods were employed to study the compaction mechanism of mouse \r\npericentric heterochromatin (PCH) foci and to investigate the interplay between transcriptional \r\nco-activators, phase separation and transcription at an inducible reporter gene cluster. It \r\nrevealed determinants of PCH compaction and identified differential co-activator usage and \r\nmultivalent interactions as contributors to transcription factor (TF) strength. The results \r\nfurthermore challenged the model of TF phase separation as a general positive driver of gene \r\ntranscription. In the second part, I focused on exploiting the detection of compartments for \r\nmeasuring activity of the alternative lengthening of telomeres (ALT) pathway used by cancer \r\ncells to extend their telomeres in absence of telomerase. I developed ALT-FISH, a scalable \r\nand quantitative imaging assay that detects ALT pathway-specific compartments containing \r\nlarge amounts of single-stranded telomeric nucleic acids. I applied the method to cell line \r\nmodels from different cancer entities and to tumor tissue from leiomyosarcoma and \r\nneuroblastoma patients. By devising automated ALT-FISH data acquisition and analysis \r\nIV\r\nworkflows, I implemented an approach, which enabled ALT activity measurements in hundreds \r\nof thousands of single cells. These technological advancements provided a quantitative \r\ndescription of ALT activity at single cell resolution and were used to characterize the spatial \r\ndistribution of ALT activity in relation to other biological features and in response to \r\nperturbations. Finally, a novel approach for studying the regulation of ALT in tumors could be \r\nestablished by integrating the method with the spatially resolved detection of single cell \r\ntranscriptomes.\r\nIn summary, this thesis introduced and utilized several methods to establish connections \r\nbetween nuclear compartment organization, chromatin features, transcription regulation, and \r\ntelomere maintenance. These perturbation and imaging techniques are versatile and may be \r\napplied to dissect nuclear activities related to other compartments and biological model \r\nsystems. Furthermore, the detection of ALT activity has demonstrated that compartments can \r\noffer valuable biological insights into how phenotypic cellular heterogeneity is encoded and \r\nlinked to diseases such as cancer."^^ . "2023" . . . . . . . "Lukas"^^ . "Frank"^^ . "Lukas Frank"^^ . . . . . . "Perturbing and imaging nuclear compartments to reveal mechanisms of transcription regulation and telomere maintenance (PDF)"^^ . . . "Frank_PhD_Thesis_2023.pdf"^^ . . . "Perturbing and imaging nuclear compartments to reveal mechanisms of transcription regulation and telomere maintenance (Other)"^^ . . . . . . "indexcodes.txt"^^ . . . "Perturbing and imaging nuclear compartments to reveal mechanisms of transcription regulation and telomere maintenance (Other)"^^ . . . . . . "lightbox.jpg"^^ . . . "Perturbing and imaging nuclear compartments to reveal mechanisms of transcription regulation and telomere maintenance (Other)"^^ . . . . . . "preview.jpg"^^ . . . "Perturbing and imaging nuclear compartments to reveal mechanisms of transcription regulation and telomere maintenance (Other)"^^ . . . . . . "medium.jpg"^^ . . . "Perturbing and imaging nuclear compartments to reveal mechanisms of transcription regulation and telomere maintenance (Other)"^^ . . . . . . "small.jpg"^^ . . "HTML Summary of #33557 \n\nPerturbing and imaging nuclear compartments to reveal mechanisms of transcription regulation and telomere maintenance\n\n" . "text/html" . . . "500 Naturwissenschaften und Mathematik"@de . "500 Natural sciences and mathematics"@en . . . "570 Biowissenschaften, Biologie"@de . "570 Life sciences"@en . .