To pass the genome from one generation to the next, eukaryotic cells first replicate their DNA, then biorient chromosomes on the mitotic spindle, and finally separate their sister chromatids, thus enabling division of one cell into two genetically identical daughter cells. We are interested in understanding these processes at the molecular level.
- Figure 1 (Click to view legend)
Throughout interphase, numerous sites in mammalian genomes are bound by ring-shaped 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 DNA damage repair, but how cohesion is established is poorly understood. We discovered previously that the establishment of cohesion coincides with particularly stable binding of cohesin to DNA, and that this binding mode depends on a cohesin-associated protein known as sororin. The establishment of cohesion is also known to require acetylation of cohesin. We are therefore trying to understand how sororin and cohesin acetylation convert cohesin into a “cohesive” state during DNA replication.
Although cohesin is best known for its role in mediating cohesion, we and others discovered that it also plays a significant role in gene regulation. We suspect that these functions are the reason why cohesin binds to chromatin even before cohesion is established, and why cohesin associates with DNA even in postmitotic 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. Our future aims are to test whether cohesin plays a general role in forming chromatin loops, and to understand the mechanistic basis of this function and its relationship with cohesin’s role in cohesion.
- Figure 2 (Click to view legend)
- Figure 3 (Click to view legend)
Sister chromatid separation in anaphase depends on the removal of cohesin from chromosomes. Several years ago we discovered that this process depends on two mechanisms in vertebrate 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.
In metaphase, when all chromosomes have been bioriented, the anaphase promoting complex/cyclosome (APC/C) is activated. The APC/C is a 1.5 MDa complex which assembles ubiquitin chains on securin and cyclin B. The subsequent destruction of these proteins by the 26S proteasome allows activation of separase, cleavage of centromeric cohesin, and sister chromatid separation. Until chromosome biorientation is complete, the APC/C is inhibited by the spindle assembly checkpoint (SAC). The SAC ensures that sister chromatids are only separated once chromosomes have become attached to both spindle poles. Despite the crucial importance of APC/C, it is poorly understood how this complex is inhibited by the SAC, how the inhibition is relieved in metaphase, and how active APC/C recruits and ubiquitylates its substrates. We are using biochemical assays and electron microscopic analyses of APC/C in different functional states to address these questions.
Although mitosis has been studied for more than a century, our molecular understanding of this complicated process is far from complete. During the past five years, the MitoCheck consortium funded by the 6th framework program of the European Union has developed and applied genomic and proteomic approaches to study mitosis. The consortium has used RNA interference screens to identify proteins required for mitosis in human cells, tagging of genes in bacterial artificial chromosomes (BACs) to enable intracellular localization and affinity purification of these proteins and mass spectrometry to identify protein complexes and mitosis-specific phosphorylation sites on these. This work identified numerous protein complexes, many of which had previously escaped identification or had been poorly characterized. Importantly, the approaches developed by MitoCheck will generally be applicable to high-throughput analyses of other processes in mammalian cells. In the future we will develop quantitative assays for mitosis in a new project named MitoSys funded by the European Union.
