Periodic Reporting for period 4 - CHROMDOM (Chromosomal domain formation, compartmentalization and architecture)
Reporting period: 2023-01-01 to 2024-06-30
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).
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.
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.