Peters Group Research
Mitosis and chromosome biology
To pass the genome from one generation to the next, eukaryotic cells first replicate their DNA, then bi-orient chromosomes on the mitotic spindle, and finally separate their sister chromatids, thus permitting division of one cell into two genetically identical daughter cells. We wish to understand these processes at the molecular level.
How is sister chromatid cohesion established and maintained?
- Figure 1 (Click to view legend)
Numerous sites in the genome are bound by cohesin complexes. During DNA replication these complexes establish physical connections between the newly synthesized sister chromatids. It is well established that the resulting cohesion is essential for chromosome segregation and repair of DNA damage, but how cohesion is established and maintained for many hours, or in the case of mammalian oocytes even for years, is poorly understood. We discovered recently that cohesin is converted into a ‘cohesive’ form that binds to DNA very stably by the protein Sororin, which associates with cohesin during DNA replication. Our data indicate that Sororin stabilizes cohesin on DNA by antagonizing a protein that can dissociate cohesin from DNA, known as Wapl.
How does cohesin control chromatin structure and gene regulation?
- Figure 2 (Click to view legend)
Although cohesin is best known for its role in mediating cohesion, we and others have discovered that cohesin plays an important role in gene regulation. We suspect that these functions are the reason why cohesin binds to chromatin before cohesion is established, and why cohesin associates with DNA even in post-mitotic cells, which will never establish cohesion. We found that cohesin co-localizes in mammalian genomes with the transcriptional insulator protein CTCF and showed that cohesin is required for gene regulation at the imprinted H19-IGF2 locus. Gene expression at this locus is believed to be controlled by the formation of a chromatin loop which forms between CTCF sites specifically on the maternal allele. Our recent work indicates that cohesin is required for this chromatin interaction. We intend to test whether cohesin plays a general role in forming chromatin loops and understand the mechanistic basis of this function.
How is sister chromatid cohesion dissolved during mitosis?
- Figure 3 (Click to view legend)
Sister chromatid separation in anaphase depends on the removal of cohesin from chromosomes. We discovered a number of years ago that this process depends on two mechanisms in ver tebrate cells: the dissociation of cohesin from chromosome arms in prophase and the proteolytic cleavage of cohesin at centromeres in metaphase. The prophase pathway depends on the cohesin-associated protein Wapl, whereas the metaphase pathway is mediated by the protease separase. Although the prophase pathway was identified several years ago, its function and importance for chromosome segregation are still unknown. We therefore generated a conditional Wapl “knockout” mouse to study the role of the prophase pathway in vivo.
How does the APC/C initiate anaphase?
In metaphase, when all chromosomes have been bi-oriented, the anaphase promoting complex/cyclosome (AP C/C) is activated. The AP C/C is a 1.5 MDa complex which assembles ubiquitin chains on securin and cyclin B. Subsequent destruction of these proteins by the 26S proteasome allows activation of separase, cleavage of centromeric cohesin, and sister chromatid separation. Until chromosome bi-orientation is complete, AP C/C is inhibited by the spindle assembly checkpoint (SAC). The SAC ensures that sister chromatids are only separated once chromosomes have been at tached to both spindle poles. Despite the crucial importance of the AP C/C, many of its aspects are poorly understood: how this complex is inhibited by the SAC, how the inhibition is relieved in metaphase, and how active AP C/C recruits and ubiquitylates its substrates. We are using biochemical assays and electron microscopic analyses of the AP C/C in different functional states to address these questions.
MitoCheck / MitoSys
|
Although mitosis has been studied for more than a century, our molecular understanding of this complicated process is far from complete. During the last five years the MitoCheck consortium funded by the European Union has developed and used genomic and proteomic approaches to study mitosis. The consor tium employed RNA inter ference screens to identify proteins required for mitosis in human cells, tagging of genes in bacterial artificial chromosomes (BACs) for intracellular localization and affinity purification of these proteins, and mass spectrometry to identify protein complexes and mitosis-specific phosphorylation sites on these. This work has identified about 100 human protein complexes, many of which had previously not or only incompletely been characterized. Importantly, the approaches developed by MitoCheck can generally be used for high-throughput analysis of other processes in mammalian cells. In the future we will develop quantitative assays for mitosis in a new project funded by the European Union, known by the name of MitoSys.
