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Chromosomal domain formation, compartmentalization and architecture

Periodic Reporting for period 3 - CHROMDOM (Chromosomal domain formation, compartmentalization and architecture)

Reporting period: 2021-07-01 to 2022-12-31

Chromosomes are life's carriers of genetic information. They provide a scaffold for the regulation of gene expression, the transfer of genes to the next generation, and the organization of the genome. In humans, DNA, a molecule that contains the "program" of how proteins are built, is organized on several hierarchical levels. At the fundamental level, DNA is wrapped around nucleosomes, which assemble into chromatin fiber structures. Chromatin fibers themselves interact with one another and form compartments within the cell nucleus. The orderly packing of DNA and chromatin into chromosomes has a direct influence on gene expression and regulation.

Because of the high density of proteins on chromatin and the multitude of interaction partners, the composition of chromosomes is only poorly understood. Moreover, chromosomes change their shapes and functional characteristics during the cell cycle from an open conformation in interphase to their well-known condensed X-shaped form during mitosis. In addition, chromosomes are not uniform but are divided into densely packed heterochromatic regions that coexist alongside with accessible gene- expressing euchromatic regions.

The overall aim of this research proposal is to understand the mechanism that leads to the formation of one of the key features in the organization of interphase chromosomes in higher eukaryotes: topologically associated domains (TADs).
To study the formation of topologically associating domains (TADs) in chromosomes, we aimed to reconstitute the process in a very minimal system and visualize the processes as they are carried out by single molecules. To this end, we built a fluorescence microscopy setup capable of detecting the fluorescence of single molecules. We established a platform of DNA curtains, i.e. microfluidic chips that allow us to build and visualize an array of parallel strands of DNA, together with DNA-interacting proteins.

We recombinantly expressed key components of TAD formation, such as nucleosomes, genomic insulators, SMC complexes and SMC mediators. These proteins were then purified and fluorescently labeled.

In DNA-binding experiments of the fluorescently tagged SMC complex cohesin, we could characterize this interaction and its modulation by ATP and loading mediators. Moreover, we found that cohesin can promote the bridging of two different DNA molecules, or the connection of two different sites of the same DNA molecule. We characterized the requirements for this step and identified a minimal set of factors needed for stable DNA bridging. Recognizing that cohesin-mediated looping of DNA is one of the hallmarks of TAD formation, we tested the mechanical strength of the loops in single molecule pulling experiments. To our surprise, we found two different classes of tethers that differed by their mechanical strength, suggesting that SMC complexes may interact with DNA in more than one way, with possibly different biological functions.
The use of single molecule methods to study interactions of chromosomal proteins has been a successful strategy that also has been recognized in the field. The strategy of single molecule studies, supplemented by bulk biochemistry will therefore be continued in the future.

In continuation of the project, we expect to deepen our understanding of TAD formation in humans by studying the dynamics of TADs in the presence of genomic insulators.