Periodic Reporting for period 5 - CHROMDOM (Chromosomal domain formation, compartmentalization and architecture)
Periodo di rendicontazione: 2024-07-01 al 2025-06-30
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: 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, chromosomal structure is not uniform but divided into densely packed heterochromatic regions that coexist alongside with accessible gene-expressing euchromatic regions.
The overall aim of this research proposal is to shed light on the molecular 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).
Because of the high density of proteins on chromatin and the multitude of interaction partners, the composition of chromosomes is only poorly understood: 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, chromosomal structure is not uniform but divided into densely packed heterochromatic regions that coexist alongside with accessible gene-expressing euchromatic regions.
The overall aim of this research proposal is to shed light on the molecular 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.
TADs in higher eukaryotes are delineated by the zinc finger protein CTCF, which specifically binds DNA motifs at TAD boundaries. We investigated the binding of CTCF to its binding site in high-throughput single molecule experiments. We found that CTCF's zinc fingers mediate the formation of clusters, which enables RNA recruitment. It is possible that these CTCF-RNA clusters create a scaffold for interaction with RNA-binding proteins. Despite its stability, DNA-bound CTCF could be removed from its binding site by transcribing RNA polymerases. This study revealed key aspects of the multifaceted roles of CTCF in genome organization and transcription regulation.
A further member of the SMC family, Smc5/6 has mainly been implicated in processes related to DNA repair, such as helping recovery from stalled replication forks. We performed high-throughput single molecule studies of Smc5/6 binding to DNA substrates and showed that this complex preferentially associates with DNA that bears hallmarks of structural defects. Using single-molecule coupled mechanics and fluorescence measurements, we demonstrated that Smc5/6 exhibits a scanning mode on double-stranded DNA, but stops when it encounters structures which resemble stalled replication forks. Smc5/6 then stabilizes these forks. We speculate that its downstream activation then enables it to promote the recovery pathway.
Inter-nucleosome interactions cause chromatin to self-associate into a densely packed condensed phase. Nucleosome remodelers influence the positioning of nucleosomes and thus have the ability to directly influence the mechanics of condensed chromatin. We studied these effects using a model system of condensed chromatin arrays, and one select remodeler. These experiments revealed that ATP hydrolysis plays a key role in maintaining the mechanical fluidity of chromatin. Failure to hydrolyze ATP drives remodelers into effective chromatin crosslinkers, which solidify chromatin. Our study further revealed a dual role of ATP hydrolysis: It drives the remodeling process itself but also ensures that remodelers remain mobile and can transfer from nucleosome to nucleosome.
The results of our work have been presented at international conferences and published in peer reviewed journals (Science Advances, Nucleic Acids Research, Methods in Enzymology, Cell Reports, Nature Structural and Molecular Biology). Highlights have been disseminated in other formats like an online video or press releases.
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.
TADs in higher eukaryotes are delineated by the zinc finger protein CTCF, which specifically binds DNA motifs at TAD boundaries. We investigated the binding of CTCF to its binding site in high-throughput single molecule experiments. We found that CTCF's zinc fingers mediate the formation of clusters, which enables RNA recruitment. It is possible that these CTCF-RNA clusters create a scaffold for interaction with RNA-binding proteins. Despite its stability, DNA-bound CTCF could be removed from its binding site by transcribing RNA polymerases. This study revealed key aspects of the multifaceted roles of CTCF in genome organization and transcription regulation.
A further member of the SMC family, Smc5/6 has mainly been implicated in processes related to DNA repair, such as helping recovery from stalled replication forks. We performed high-throughput single molecule studies of Smc5/6 binding to DNA substrates and showed that this complex preferentially associates with DNA that bears hallmarks of structural defects. Using single-molecule coupled mechanics and fluorescence measurements, we demonstrated that Smc5/6 exhibits a scanning mode on double-stranded DNA, but stops when it encounters structures which resemble stalled replication forks. Smc5/6 then stabilizes these forks. We speculate that its downstream activation then enables it to promote the recovery pathway.
Inter-nucleosome interactions cause chromatin to self-associate into a densely packed condensed phase. Nucleosome remodelers influence the positioning of nucleosomes and thus have the ability to directly influence the mechanics of condensed chromatin. We studied these effects using a model system of condensed chromatin arrays, and one select remodeler. These experiments revealed that ATP hydrolysis plays a key role in maintaining the mechanical fluidity of chromatin. Failure to hydrolyze ATP drives remodelers into effective chromatin crosslinkers, which solidify chromatin. Our study further revealed a dual role of ATP hydrolysis: It drives the remodeling process itself but also ensures that remodelers remain mobile and can transfer from nucleosome to nucleosome.
The results of our work have been presented at international conferences and published in peer reviewed journals (Science Advances, Nucleic Acids Research, Methods in Enzymology, Cell Reports, Nature Structural and Molecular Biology). Highlights have been disseminated in other formats like an online video or press releases.
This project allowed us to streamline the approach of conducting high-throughput single molecule experiments. As a result, we can now routinely conduct these studies with a wide range of nucleic acid binding complexes. These experiments enable us to collect data with kinetic and molecular detail that surpass the capabilities of conventional bulk biochemical assays.
In addition, we have developed many tools and techniques of analyzing these data, and supplemented these with validation tools such as simulations.
Scientifically, this project has allowed us to uncover important new insights into the molecular mechanisms that maintain chromosome structural integrity
In addition, we have developed many tools and techniques of analyzing these data, and supplemented these with validation tools such as simulations.
Scientifically, this project has allowed us to uncover important new insights into the molecular mechanisms that maintain chromosome structural integrity