Analysing the various layers of genome architecture using a high-throughput single-molecule technique

Huber, Jonas (2025): Analysing the various layers of genome architecture using a high-throughput single-molecule technique. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

In eukaryotes, genetic information is stored in mitochondria and the nucleus as long strands of deoxyribonucleic acid (DNA) (1). Nuclear DNA extends for roughly 6.3 million base pairs (bp) with a total length of about 2 meters (1). As the nucleus is only 10 μm in diameter (2), DNA needs to be highly compacted to fit inside, which is a challenging task for eukaryotic cells. DNA compaction and organisation are accomplished by a variety of different DNA-interacting proteins, which additionally regulate genome accessibility and, hence, gene expression in different cells, tissue types and organisms (3–7). To study DNA-protein interactions a single-molecule assay based on total internal fluorescence microscopy called DNA curtains is applied (8). Here hundreds of recombinantly expressed and fluorescently labelled proteins can be visualised on parallelly aligned DNA molecules simultaneously, which will be explained in more detail in Chapter I 2.. Polymerases bind to and translocate on DNA to copy genetic information during DNA replication and transcription, requiring accessible DNA (9). This is influenced by DNA sequence, other proteins and the activity of the polymerase itself, which I present in Chapter II 1. and Chapter II 4.. In the nucleus, DNA is normally wrapped into nucleosomes, composed of 146 bp of DNA wound around an octameric protein complex (10). DNA in this conformation, called chromatin fibre, is compacted and less accessible to regulatory proteins (11). Accessibility depends on the positioning and spacing of nucleosomes and in Chapter II 2. I analyse nucleosome positioning depending on DNA sequence. In addition to chromatin fibre formation, additional compaction is required to fit the whole DNA into the nucleus. In the next compaction step DNA is folded into spatial domains called topologically associating domains (TADs) by a process called loop extrusion (12–15). TADs are characterised by shorter three-dimensional (3D) genomic distances, increasing regulatory interactions within them while decreasing interactions with neighbouring regions (16, 17). They are formed by the ring-shaped cohesin complex, which generates DNA loops and the architectural protein CCCTC-binding factor (CTCF) residing on its genomic binding sites as an anchor point for these loops (18, 19). Cohesin additionally functions in cell division, preventing early separation of sister chromatids (20). Chapter II 3. shows that cohesin can form tethers between two DNAs and stable bridges on single DNA molecules, revealing potential mechanisms for holding sister chromatids together and for loop formation. CTCF’s high stability on its DNA binding site and its ability to recruit secondary binding partners like cohesin’s SA subunit and ribonucleic acid (RNA) are displayed in Chapter II 4.. Chromatin domains further assemble into higher-order chromatin compartments, referred to as A- (transcriptionally active) or B- (transcriptionally inactive) compartments (21). Their formation depends on phase separation, a process in which the interaction of specific nucleic acids and proteins leads to the formation of dynamic phases segregated from the surrounding liquid (22–26). Phase-separating proteins often contain intrinsically disordered regions (IDRs) and nucleic acid-binding domains (27, 28), as is the case for CTCF (29, 30). Chapters II 4. and 5. display that CTCF forms oligomers under physiological conditions, enabling it to form clusters with DNA and capture RNA, which might create an interaction hub for phase-separating proteins. This study reveals mechanistic insights into genome organisation involving transcription, chromatin formation and the different layers of genome architecture by studying protein-DNA interactions and higher-order complex formation on a single-molecule level. Additionally, it sheds light on the mutual interplay of these complex processes.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 4. April 2025
• 1. Bericht­erstatter:in: Stigler, Johannes