Periodic Reporting for period 4 - ChromoCellDev (Chromosome Architecture and the Fidelity of Mitosis during Development)
Reporting period: 2020-04-01 to 2021-09-30
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. Our work tested the “active chromosome” hypothesis by investigating how chromosome morphology influences the fidelity of mitosis. We used 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 investigated the role of mitotic chromosomes in the fidelity of mitosis at three different levels. We established novel approaches that allowed us to uncover the maintenance of mitotic chromosome organization by inactivating Condensin in a fast and acute manner (TEV cleavage system). This work changed the view on chromosome architecture and emphasizes how controlled resolution of chromosome entanglements is required throughout mitosis (Piskadlo et al, 2017). It further allowed us to study the role of other players in mitotic chromosomes organization, including topoisomerase 2, histone tails and a poorly characterized helicase-like protein. At a cellular level, we also investigated how mitotic chromosomes take an active part in mitosis by examining how chromosome condensation and cohesion influence chromosome movement and the signaling of the surveillance mechanisms that control nuclear division. Our work elucidated how sister chromatid cohesion defects evade the checkpoint mechanisms that control mitosis (Mirkovic et al, 2015). Also, we discovered that mitotic defects associated with sister chromatid cohesion loss can be recovered by inactivation of the mitotic checkpoint (Silva et al, 2018). The execution of this project also allowed us to establish that cohesin loss induces previously unrecognized defects on mitotic fidelity (Carvalhal et al, 2018). Lastly, our work also evaluated the impact of abnormal chromosome structure and number in development and established the developing brain as the most sensitive tissue in response to aneuploidy (Mirkovic et al, 2019). By conceptually challenging the passive chromosome view this project redefined the role of chromatin during mitosis. It further proposed novel mechanisms for how defects in chromosome assembly may underlay several human pathologies, such as cancer, infertility and development disorders.
ChromoCellDev uncovered major advances on how chromosomes are assembled, how their structure influences the mechanics of mitosis and how defects in these processes impact on organisms development. Major results are summarized below:
• A multidisciplinary approach to elucidate of how sister chromatid cohesion defects evade the checkpoint mechanisms that control mitosis (Mirkovic et al Cell Rep 2015)
• A paradigm-changing view of the process of chromosome organization emphasizing how controlled resolution of chromosome entanglements is required throughout mitosis (Piskadlo et al eLife 2017)
• A quantitative analysis of cohesin decay in mitotic fidelity uncovering previously unrecognized defects associated with cohesin loss (Carvalhal et al, JCB 2018)
• The discovery that mitotic defects associated with sister chromatid cohesion loss can be recovered by inactivation of the Spindle Assembly Checkpoint (Silva et al, Curr Biol 2018)
• The finding that the developing brain is the most sensitive tissue in response to acutely-induced aneuploidy (Mirkovic et al, PlosBiol 2019)
Collectively, these findings also raised novel hypothesis for the process of chromosome organization, currently under investigation in the lab. They were also pivotal to understand how chromosomal abnormalities underlay several human pathologies, such as cancer, infertility and developmental disorders.
• A multidisciplinary approach to elucidate of how sister chromatid cohesion defects evade the checkpoint mechanisms that control mitosis (Mirkovic et al Cell Rep 2015)
• A paradigm-changing view of the process of chromosome organization emphasizing how controlled resolution of chromosome entanglements is required throughout mitosis (Piskadlo et al eLife 2017)
• A quantitative analysis of cohesin decay in mitotic fidelity uncovering previously unrecognized defects associated with cohesin loss (Carvalhal et al, JCB 2018)
• The discovery that mitotic defects associated with sister chromatid cohesion loss can be recovered by inactivation of the Spindle Assembly Checkpoint (Silva et al, Curr Biol 2018)
• The finding that the developing brain is the most sensitive tissue in response to acutely-induced aneuploidy (Mirkovic et al, PlosBiol 2019)
Collectively, these findings also raised novel hypothesis for the process of chromosome organization, currently under investigation in the lab. They were also pivotal to understand how chromosomal abnormalities underlay several human pathologies, such as cancer, infertility and developmental disorders.
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 work also provided critical explanation on how alterations on chromosome structure impacts on cell division, either by problems in cohesion or in condensation. We performed a quantitative analysis of how the mitotic checkpoint (Spindle Assembly Checkpoint, SAC) responds to cohesion loss. This work uncovered that weak SAC activation upon cohesion loss is caused by weak signal generation, which is further attenuated by several feedback loops in the mitotic signaling network. Our findings explain how cohesion defects escape SAC surveillance. We additionally demonstrated that mitotic defects associated with sister chromatid cohesion loss can be recovered by SAC inactivation. More recently, we uncovered that Condensin II complexes have a direct role in the efficiency of SAC response in male spermatocytes. In this project, we also investigated, in a quantitative way, the effects of cohesion decay on chromosome morphology and segregation. We uncovered that in the presence of low levels of cohesin, mitotic defects are associated with wrong chromosome attachments rather than sister chromatid disjunction. This approach revealed previously unrecognized defects associated with mild cohesin loss.
Finally, our ultimate goal in this project was to understand how these changes in chromosome translate in terms of organism development. We established a unique assay that allowed us to provoke chromosome random segregation in a time controlled manner. This allowed us to uncover that cell identity determines cell fate when facing unbalanced chromosome number and that the developing brain is the most critical tissue in response to aneuploidy. This holistic approach is crucial to elucidate how abnormal chromosomal organization can ultimately dictate disease outcomes.
Finally, our ultimate goal in this project was to understand how these changes in chromosome translate in terms of organism development. We established a unique assay that allowed us to provoke chromosome random segregation in a time controlled manner. This allowed us to uncover that cell identity determines cell fate when facing unbalanced chromosome number and that the developing brain is the most critical tissue in response to aneuploidy. This holistic approach is crucial to elucidate how abnormal chromosomal organization can ultimately dictate disease outcomes.