In the first half of the project we made important progress in three areas as summarized below.
1) In preparation for mitotic cell division, the nuclear DNA of human cells is compacted into individualized, X-shaped metaphase chromosomes. This dramatic metamorphosis is driven mainly by the combined action of condensins and Topoisomerase IIα (TOP2A) and has been observed using microscopy for over a century. Nevertheless, remarkably little is known about the structural organization of a mitotic chromosome. In this first and the most essential project, we developed a workflow to interrogate the organization of human chromosomes based on optical trapping and manipulation. This allows high-resolution force measurements and fluorescence visualization of native chromosomes to be conducted under tightly controlled experimental conditions. We have used this method to extensively characterize chromosome mechanics and structure. Notably, we find that under increasing mechanical load, chromosomes exhibit nonlinear stiffening behavior, distinct from classical polymer models. To explain this anomalous stiffening, we introduce a Hierarchical Worm-like Chain model that describes the chromosome as a heterogeneous assembly of nonlinear worm-like chains. Moreover, through inducible degradation of TOP2A specifically in mitosis, we provide evidence of a role for TOP2A in the preservation of chromosome compaction. Our innovations open the door to a wide array of new investigations on the structure and dynamics of both healthy and disease-associated chromosomes.
2) In anaphase, any unresolved DNA entanglements between the segregating sister chromatids can give rise to chromatin bridges. To prevent genome instability, chromatin bridges must be resolved prior to cytokinesis. The SNF2 protein PICH has been proposed to play a direct role in this process through the remodeling of nucleosomes. However, direct evidence of nucleosome remodeling by PICH has remained elusive. In this second project, we present an in vitro single-molecule assay that mimicks chromatin under tension, as is found in anaphase chromatin bridges. Applying a combination of dual-trap optical tweezers and fluorescence imaging of PICH and histones bound to a nucleosome-array construct, we show that PICH is a tension- and ATP-dependent nucleosome remodeler that facilitates nucleosome unwrapping and then subsequently slides remaining histones along the DNA. This work elucidates the role of PICH in chromatin-bridge dissolution, and might provide molecular insights into the mechanisms of related SNF2 proteins.
3) During mitosis in eukaryotic cells, mechanical forces generated by the mitotic spindle pull the sister chromatids into the nascent daughter cells. In this third project we wondered how do mitotic chromosomes achieve the necessary mechanical stiffness and stability to maintain their integrity under these forces? To address this we use optical tweezers to show that ions involved in physiological chromosome condensation are crucial for chromosomal stability, stiffness and viscous dissipation. We combine these experiments with high-salt histone-depletion and theory to show that chromosomal elasticity originates from the chromatin fiber behaving as a flexible polymer, whereas energy dissipation can be explained by interactions between chromatin loops. Taken together, we show how collective properties of mitotic chromosomes, a biomaterial of incredible complexity, emerge from molecular properties, and how they are controlled by the physico-chemical environment.
