(Daniel Gerlich, IMBA Group Leader): Indeed, through prior work, the role of condensin in folding the chromatin fiber into loops around a central axis was known. But it had remained unclear why chromosomes appear as dense bodies with a sharp surface rather than a loose structure resembling a bottlebrush. Prior literature hints at the role of a specific chemical modification in the chromatin fiber – histone acetylation – in regulating the level of compaction. However, its interplay with condensin and its functional relevance remained unclear. With our work, we are now able to conceptually disentangle the two mechanisms.
The first thing we did was to remove the condensin complex to study the effect on chromosome organization. We saw that chromosomes completely lost their regular shape in condensin-depleted cells, yet chromatin compacted to the same level as in unperturbed cells. We then tested the effect of increasing histone acetylation and observed a complete suppression of compaction in the condensin-depleted mitotic cells. Importantly, we noticed that elevated acetylation of mitotic chromosomes also resulted in frequent microtubule perforation into chromatin regions and a strong perturbation of chromosome movements to the spindle poles, indicating an important mechanical role of histone acetylation.
Based on our observations and the known functions of condensin and acetylation, we propose that chromatin is organized as a swollen gel throughout most of the cell cycle. During cell division, however, this gel compacts to an insoluble form. To test this model, we investigated the effect of cutting mitotic chromosomes into small pieces by injection of a restriction enzyme. We found that chromosome fragmentation resulted in droplets of liquid chromatin that display rapid internal mobility of the chromatin fiber. These findings support a phase separation process in mitotic chromosome assembly. Interestingly, elevated acetylation on chromatin caused a complete dissolution of chromatin fragments throughout the cell. Hence, mitotic chromatin is immiscible in mitotic cells, and this property depends on low acetylation levels. As a result, we propose that the phase boundary of immiscible chromatin provides mechanical resistance to microtubules.
In collaboration with the Rosen lab at the University of Texas Southwestern Medical Center, we generated liquid droplets from in vitro-assembled chromatin fibers. When we mixed the chromatin droplets with tubulin, we observed efficient exclusion of microtubules. Thus, the intrinsic phase separation properties of chromatin are sufficient to establish a surface that is resistant to microtubule perforation.
Much of our concepts have grown over the years through our multi-disciplinary approaches to studying chromosomes here at IMBA, with support from many fantastic colleagues and core facilities. Our collaboration with Shotaro Otsuka from the neighboring Max Perutz Labs has been key to showing by electron tomography how microtubules interact with chromosome surfaces. Furthermore, this study has been inspired by our interactions with Mike Rosen and his group of chromatin biochemists, whom we met during a research visit to the Marine Biology Laboratory at Woods Hole, Massachusetts, USA. Rosen and his postdoc Bryan Gibson had discovered acetylation-regulated phase transitions in synthetic chromatin, and we could show how this phenomenon contributes to chromosome mechanics inside cells. Our current study has already led to several new ideas that we aim to investigate in the future. These include a better understanding of how electrical charge contributes to chromosome compaction, and how chromosome compaction is regulated in other processes, like programmed cell death.
Schneider M.W.G., et al. “A mitotic chromatin phase transition prevents perforation by microtubules”. Nature, 2022. DOI: 10.1038/s41586-022-05027-y