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Final Report Summary - CCC (Chromosome Condensation and Cohesion)

Accurate cell division relies on the fact that the genetic information encoded in the DNA molecules is equally segregated into the two daughter cells. Proper partitioning of the genome, in turn, depends on two key changes in chromosome organization: 1) chromatin is converted into compact structures with the right mechanical properties (size, flexibility, rigidity) to facilitate their segregation; 2) the two sister DNA molecules remain tightly associated with each other from the moment of DNA replication until the metaphase-anaphase transition of the subsequent mitosis. Although chromosomes were long assumed to play rather a passive role during the cell division process, recent evidence suggests that chromosomes play a much more active role in the process of their own segregation. Understanding the “active chromosome” and how chromosome morphology influences mitosis is pivotal to the understanding of novel routes to mitotic defects and causes for aneuploidy.

This project aimed to investigate how dynamic mitotic chromosomes are assembled and how their morphology contributes to various aspects of mitosis. We adopted a multidisciplinary approach, combining acute protein inactivation, 4D-live cell imaging and biophysical/mathematical approaches to evaluate role of chromosome condensation and sister chromatid cohesion in various aspects of mitotic fidelity. Major findings are summarized below.

One of the main aims of this project was to dissect how are mitotic chromosomes assembled during nuclear division. Our work revealed that maintenance of mitotic chromosome organization is far much more dynamic than previously anticipated. To study the function of key proteins in mitotic chromosome organization (e.g. condensin complexes), we have adopted a “reverse and acute approach” to reveal the requirements for these proteins in the maintenance of chromosome structure, with unprecedented temporal resolution. We have successfully developed a TEV protease- cleavage system to inactivate condensin I complexes in the living fly in a fast and temporally controlled manner. Using a series of microinjections in Drosophila embryos, and combining acute protein inactivation with live cell imaging, we have shown that, unexpectedly, condensin I inactivation leads to over-condensation of chromosome arms. Our analysis revealed that this over-condensation results from the re-intertwining of previously separated sister chromatids. Upon inactivation of condensin I in metaphase-arrested embryos, in which sister chromatid resolution is mostly completed, sister chromatids re-catenate and completely fail to segregate. We thus propose that metaphase chromosome organization is actively maintained by condensin I-directed DNA (de)catenation (Piskadlo et al, under review).

In addition to their compacted and individualized state, mitotic chromosomes are also known to acquire a characteristic X-shape with the two sister chromatids linked by cohesins at the centromeric region. Chromosome rearrangements that disrupt this normal architecture are often found in many cancers, but whether or not they influence mitotic fidelity was unknown. Our recent work, performed in collaboration with William Sullivan (University of California, Santa Cruz, USA), revealed that chromosomal rearrangements that misplace long stretches of pericentric heterochromatin within chromosome arms, distal to the centromere, compromise chromosome segregation (Oliveira et al, Plos Biol 2014). Using engineered chromosomes in Drosophila melanogaster carrying chromosomes with chromosomal fusions, inversions or translocations, we demonstrate that the presence of ectopic heterochromatin leads to ectopic cohesion at these sites. This is mediated by an enrichment of cohesin in these regions, driven by the preferential accumulation of the cohesin-loading factor at dense heterochromatic regions. Ectopic cohesion sites, placed away from the centromere, disjoin abnormally during anaphase and chromosomes exhibit significant stretching. Chromosome rearrangements involving long stretches of heterochromatin were previously believed to be problematic due to altered gene expression of the neighbouring genes. Our work now uncovers that in addition to these problems, mitotic errors may account for the adverse effect of these chromosome rearrangements. It thus provides a clear illustration of the “snow ball” effect that may potentiate cancer development by illustrating how pre-existing errors can drive the accumulation of additional abnormalities.

Lastly, we aimed to dissect how chromosome structural errors are detected by the cell cycle sensors that govern mitotic progression. Problems in sister chromatid cohesion and/or chromosome condensation are likely to change the ability to generate (or sense) tension across sister chromatids. Therefore, under such perturbations, the Spindle Assembly Checkpoint (SAC) should halt mitotic progression and prevent the generation of anomalous cells. Strikingly, problems in cohesion and condensation are invariably associated with aneuploid cells indicating that abnormal mitotic exit from a previous mitosis has taken place. This conundrum raises the possibility that the SAC has sub-optimal efficiency and is unable to sustain a stable arrest when in the presence of abnormal chromosome cohesion/condensation. We have recently provided a quantitative analysis of SAC response upon total loss of sister chromatid cohesion (Mirkovic et al Cell Reports 2015). Quantitative live cell imaging, combined with mathematical modelling performed in collaboration with Prof. Bela Novak (University of Oxford, UK), revealed that weak SAC activation upon cohesion loss is caused by weak signal generation. This is further attenuated by several feedback loops across the mitotic signalling network. We propose that multiple feedback loops involving Cyclin-dependent kinase 1 (Cdk1) gradually impair error-correction efficiency and accelerate mitotic exit upon premature loss of cohesion. Our findings explain how cohesion defects may escape SAC surveillance and give rise to aneuploid cells.

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