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Chromosome Architecture and the Fidelity of Mitosis during Development

Periodic Reporting for period 3 - ChromoCellDev (Chromosome Architecture and the Fidelity of Mitosis during Development)

Reporting period: 2018-10-01 to 2020-03-31

Genome stability relies on accurate partition of the genome during nuclear division. Proper mitosis, in turn, depends on changes in chromosome organization, such as chromosome condensation and sister chromatid cohesion. Despite the importance of these structural changes, chromatin itself has been long assumed to play a rather passive role during mitosis and chromosomes are usually compared to a “corpse at a funeral: they provide the reason for the proceedings but do not take an active part in them.” (Mazia, 1961). Recent evidence, however, suggests that chromosomes play a more active role in the process of their own segregation. The present proposal tests the “active chromosome” hypothesis by investigating how chromosome morphology influences the fidelity of mitosis. are using innovative methods for acute protein inactivation, to evaluate the role of two key protein complexes involved in mitotic chromosome architecture - Condensins and Cohesins. Using a multidisciplinary approach, combining acute protein inactivation, 3D-live cell imaging and quantitative methods, we investigate the role of mitotic chromosomes in the fidelity of mitosis at three different levels. The first one uses novel approaches to uncover the process of mitotic chromosome assembly, which is still largely unknown. The second explores how mitotic chromosomes take an active part in mitosis by examining how chromosome condensation and cohesion influence chromosome movement and the signalling of the surveillance mechanisms that control nuclear division. Lastly we evaluate how mitotic errors arising from abnormal chromosome structure impact on development. We aim to evaluate, at the cellular and organism level, how the cell perceives such errors and how (indeed if) they tolerate mitotic abnormalities. By conceptually challenging the passive chromosome view this project has the potential to redefine the role of chromatin during mitosis and further understand how chromosomal abnormalities may underlay several human pathologies, such as cancer, infertility and developmental disorders.
During the reporting period (30 months) major advances have been made in the three major project aims.
Major achievements include the development of TEV protease-mediated inactivation systems for condensin I and II in the living fly and the dissection of condensin I role in the maintenance of chromosome organization (Piskadlo et al eLife 2017). This study provided a paradigm-changing view on chromosome organisation emphasising how controlled resolution of chromosome entanglements is required throughout mitosis (paper video: https://goo.gl/bKBfGJ). These findings contrast with classical views on mitotic chromosome organisation and provide a novel topology-centric model for mitotic chromosome structure (PISKADLO and OLIVEIRA INT J MOL SCI 2017).
We provided further advances in the study of the role of cohesin in chromosome cohesion in a highly quantitative level (CARVALHAL et al, under review at JCB). This study reveals that sister chromatid cohesion is highly resistant to cohesin decay and uncovers previously unrecognised defects associated with cohesin loss.
We also report the discovery that mitotic defects associated with sister chromatid cohesion loss can be recovered by inactivation of the mitotic checkpoint (SILVA et al, under review at Curr Biol) preprint: https://doi.org/10.1101/262204. Long-term consequences of chromosome cohesion defects were also studied in the context of the developing organism, uncovering that neuronal stem cells restrict organism development when challenged with high levels of aneuploidy (Mirkovic et al, manuscript in preparation)
Mitotic chromosome assembly remains a big mystery in biology. Condensin complexes are pivotal for chromosome architecture yet how they shape mitotic chromatin remains unknown. We uncovered that chromosome structure is linked to the state of sister chromatid resolution. We found that the enzyme responsible for resolving DNA entanglements, Topoisomerase II, is capable of re-intertwining previously separated DNA molecules. Correct separation of DNA molecules relies on condensin I activity, continuously required to counteract this erroneous activity. These findings challenge current views on chromosome resolution maintenance and highlight that this is a highly dynamic bidirectional process. Our future work aims to understand the mechanisms underlying chromosome assembly, focusing on the regulation of DNA entanglements, and ultimately, how errors in this process impact mitotic fidelity and organism development. Although errors in mitosis are well studied at the cellular level, how these errors impact on organism development is still largely unexplored. This holistic approach is crucial to elucidate how abnormal chromosomal organization can ultimately dictate disease outcomes. For example, changes to condensin I are common in certain cancers and can also lead to disrupted brain development (e.g. microcephaly).