Investigating chromatin architecture with super-resolution microscopy

Guirao Ortiz, Miguel (2026): Investigating chromatin architecture with super-resolution microscopy. Dissertation, LMU München: Faculty of Biology

Abstract

Chromatin, which is composed of DNA and proteins, serves as the backbone of all life. Despite its seemingly disordered appearance, chromatin exhibits a highly organized architecture, where intricate interactions between DNA and proteins precisely regulate gene expression. Packing over 2 meters of DNA together with numerous structural and regulatory proteins into a 10-micrometer nucleus poses fundamental questions about how chromatin is arranged and how its organization shapes gene expression and cellular activity. This work presents the development of methods relevant to chromatin research and investigates the spatial organization of chromatin. Investigating chromatin architecture and its dynamics often requires complex microscopy and staining techniques. A novel oligonucleotide synthesis strategy for fluorescence in situ hybridization (FISH) was developed, enabling flexible incorporation of labeled nucleotides and allowing nanoscale detection of genomic loci. In addition, commercially available fluorophores conjugated to a linker molecule were characterized, enabling adjustment of fluorophore properties within complex environments. Other parts of this thesis focus on the actual investigation of chromatin organization. Despite the depletion of cohesin, a perturbation that disrupts loop extrusion, overall chromatin architecture remains largely preserved. This highlights the robustness of the nuclear organization that is maintained through multiple overlapping mechanisms, even in the absence of an important structural component. In another study, the dynamic nature of chromatin was examined by probing genomic loci at the nanoscale using FISH combined with super-resolution imaging and computational simulations. The resulting distance distributions for active and inactive regulatory regions display substantial variability across individual cells, reflecting underlying differences in chromatin compaction driven by nucleosome occupancy. The primary focus of this thesis is the investigation of promoter-enhancer interactions, which represent a central mechanism of gene regulation. These interactions are commonly studied using chromosome conformation capture techniques; however, such methods rely on large cell populations and cross-linked chromatin, providing only population-averaged insights. Given the dynamic nature of these interactions at the single-cell level, a pipeline was established to precisely measure absolute distances between genomic loci in individual cells using FISH combined with automated STED microscopy. To investigate these interactions, an established enhancer hijacking model was employed that mimics a naturally occurring chromosomal translocation observed in a subset of acute myeloid leukemia patients. The data in this work revealed a significant shift of the hijacked enhancer toward the promoter compared to an equidistant control region, while individual enhancer–promoter distances exhibit a broad distribution.

• Item Type: Theses (Dissertation, LMU Munich)
• Keywords: chromatin, microscopy, STED, FISH
• Subjects: 500 Natural sciences and mathematics; 500 Natural sciences and mathematics > 570 Life sciences
• Faculties: Faculty of Biology
• Language: English
• Date of oral examination: 9. June 2026
• 1. Referee: Leonhardt, Heinrich
• Signature of the printed copy: 0001/UMC 32070