Aneuploidy, i.e. missing or supernumerary chromosomes in the cell nucleus, is a hallmark of malignant tumour progression. Many genes that orchestrate faithful chromosome segregation during mitotic cell divisions are oncogenes or tumour suppressors. The aim of the Chromosome Segregation Laboratory is to investigate the function of some of these in safeguarding accurate chromosome segregation. We are investigating the contribution of structural chromosomal proteins to sister chromatid cohesion and chromosome condensation, and the regulation of mitotic progression by the cell division cycle machinery.
Establishment of sister chromatid cohesion during DNA replication
During S-phase of the eukaryotic cell cycle, two identical copies of each chromosome are synthesised by DNA replication. These two copies, the sister chromatids, need to be faithfully distributed between daughter cells during mitosis. To make it possible for the mitotic machinery to recognise pairs of replication products for segregation, these are kept linked to each other by sister chromatid cohesion from their synthesis onwards until they split in anaphase. Sister chromatid cohesion is mediated by the chromosomal cohesin complex, a large ring-shaped multisubunit protein complex that is thought to bind to DNA by topological embrace. Cohesin is loaded onto chromosomes well before S-phase, but it is during S-phase that it establishes physical cohesion between the two newly replicated sister chromatids. One of the interests in our laboratory is to understand the reactions by which cohesin recognises and holds together sister chromatids during DNA replication.
We have in the past analysed what happens to cohesin as the replication fork travels along the DNA during S-phase. Like most other chromosomal proteins, cohesin may have to transiently dissociate from chromosomes as the fork approaches and DNA is replicated. In this case, cohesin needs to be loaded again onto DNA in a cohesive fashion in the wake of the replication fork. However, we found that the protein factors and reactions that load cohesin onto DNA before S-phase are no longer required during DNA replication. This suggests that reactions different from the initial loading reaction take place during S-phase, or that alternatively the replisome might be able to slide through the large cohesin rings to establish sister chromatid cohesion without displacing them from DNA. To distinguish between these two possibilities, and to gain insight into the process of cohesion establishment, we are drawing our attention to a group of proteins known as 'cohesion establishment factors'. In addition to the cohesin complex itself, a number of these additional cohesion establishment factors are required to ensure that cohesin links sister chromatids after DNA replication. Among these factors, the Eco1 acetyl transferase is of particular interest. Besides the subunits of the cohesin complex, Eco1 is the only other protein whose function is essential and therefore indispensable for the establishment of sister chromatid cohesion. We found Eco1 to be part of the replication fork machinery, but how it promotes sister chromatid cohesion and what its acetylation target might be remained unknown.
Eco1-dependent cohesin acetylation during establishment of sister chromatid cohesion
To gain insight into Eco1 function, we isolated spontaneous suppressor mutations in budding yeast that allow cell growth after inactivation of the temperature sensitive eco1-1 allele,conditions that would normally lead to chromosome missegregation and cell death. Genetic analysis of the suppressors indicated that mutations in three different genes were able to restore viability to eco1-1 cells at the restrictive temperature. To identify the suppressor mutations, genomic DNA of the suppressors was hybridised to oligonucleotide tiling microarrays covering the budding yeast genome. Point mutations in unknown genes, should become detectable as reduced hybridisation efficiency due to the mismatch between the mutant genomic DNA and the 25-mer oligonucleotide probes present on the microarray. This strategy allowed us to identify two of the three suppressor mutations (Figure 1a).
Figure 1. Acetylation of the cohesin subunit Smc3 during cohesion establishment. a) Identification of the Smc3 K113N mutation. Genomic
DNA of the eco1-1 parental strain and suppressor a (SUPa) was hybridised to whole genome oligonucleotide tiling arrays. Reduced hybridisation efficiency, as a function of the sequence context, was used to calculate the prediction signal for a base mismatch. The mutation was then confirmed by DNA sequencing. b) Smc3 acetylation at the time of cohesion establishment. G1 cells were released to progress through a synchronous cell cycle. FACS analysis of DNA content is shown, and the percentage of cells in anaphase (binucleates) is indicated. Smc3 was immunopurified from cell extracts at the indicated times via its Pk affinity epitope tag and analysed by Western blotting with α-Pk and α-acetyl lysine antibodies. c) Model for Eco1 function during establishment of sister chromatid cohesion. Wapl destabilises cohesin on chromosomes. To reach stable sister chromatid cohesion, Eco1 acetylates Smc3 as the replication fork passes, making cohesin resistant against Wapl.
One of the suppressors was a lysine to asparagine mutation (K113N) in Smc3, a subunit of the cohesin complex (Figure 1a). Lysines are the targets of acetylation, and an asparagines sidechain shares features with acetylated lysine. In a collaboration with the Protein Analysis at Clare Hall, we discovered that Smc3 is indeed acetylated at both lysine 113 as well as the neighbouring lysine 112 residue. Acetylation occurs in an Eco1-dependent reaction during the establishment of sister chromatid cohesion (Figure 1b). Preventing Smc3 acetylation by replacing lysine 113 with arginine interferes with cohesion establishment. In contrast, the acetylation-mimicking K113N mutation allowed cell growth and establishment of sister chromatid cohesion even in cells lacking the ECO1 gene altogether. This suggests that the essential function of Eco1 during DNA replication is acetylation of the cohesin subunit Smc3.
The significance of Smc3 acetylation became clear from analysis of suppressor mutations in the second complementation group, which inactivated the budding yeast ortholog of the cohesin destabilising protein Wapl. Just like the Smc3K113N mutation, inactivation of Wapl also made Eco1 dispensable for establishment of sister chromatid cohesion. It therefore emerges that an essential aspect of sister chromatid cohesion establishment is cohesin stabilisation against Wapl by acetylation of Smc3 (Figure 1c). These findings explain the essential role of Eco1 in sister chromatid cohesion.
Outlook
In the absence of both Eco1 and Wpl1, the fundamental mechanism for pairing sister chromatids during DNA replication remains intact. This leaves open the important question how cohesin recognises and holds sister chromatids together. Reactions that are innate to the DNA replication process, for example passage of the replication fork through the cohesin ring, may provide the underlying basis for sister chromatid cohesion. We are now interested to learn how additional cohesion establishment factors that are associated with the replisome, including Ctf4 and Ctf18 (Figure 1c), act to promote cohesion establishment.
For a list of refereed research papers, see Publications (in navigation on left).
