Molecular Oncology Programme

Chromosome Dynamics Group

Group Leader:  Ana Losada
Research highlights
Cohesin-SA1 and cohesin-SA2 have distinct roles in 3D genome organisation

Cohesin consists of four core subunits, SMC1, SMC3, RAD21 and SA. There are two versions of the SA subunit in vertebrate somatic cells, SA1 and SA2. Loss of function mutations in the STAG2 gene encoding SA2 have been identified in bladder cancer, Ewing sarcoma and other tumour types. Cells lacking cohesin-SA2 can proliferate because cohesin-SA1 performs the essential function of cohesin in cohesion. This explains why STAG2 mutant tumours are not aneuploid. However, it is likely that cohesin-SA1 cannot accomplish the other functions of cohesin-SA2 related with chromatin organisation and gene regulation, thereby providing some advantage to the tumour. Importantly, lack of cohesin-SA2 may also generate vulnerabilities that could be exploited in cancer therapy. To characterise the specific functions of the two variant complexes in chromatin architecture and gene regulation we are pursuing two strategies. The first one is the characterisation of cells derived from mouse embryos deficient for SA1 or SA2. A STAG1 knockout allele was obtained a few years ago (Remeseiro et al., 2012), while a conditional STAG2 knockout allele has been generated more recently in collaboration with Francisco X. Real (CNIO Epithelial Carcinogenesis Group). The second strategy makes use of non-transformed human cell lines before and after downregulation of SA1 or SA2. In these cells, we have analysed the genome-wide distribution of the two cohesin complexes as well as their transcriptomes. Moreover, in collaboration with M. A. Martí-Renom (CRG-CNAG), we have interrogated the genome architecture by Hi-C. From these studies, and in line with our previous work, we conclude that cohesin-SA1 collaborates with CTCF in the demarcation of domain boundaries (FIGURE 1). In contrast, a more dynamic cohesin-SA2 complex promotes cell type-specific interactions between enhancers and promoters within contact domains (or TADs, for Topologically Associated Domains) independently of CTCF. Loss of SA2 rewires local chromatin contacts and alters gene expression. We are currently exploring the molecular mechanisms underlying these functional specificities of cohesin-SA1 and cohesin-SA2.

Pds5 proteins regulate cohesin distribution and dynamics

Two factors associate with chromatin-bound cohesin, Pds5 and Wapl. Wapl promotes cohesin unloading, and in its absence there is an excess of cohesin on chromatin and chromosome organisation is altered, both in interphase and mitosis. The role of Pds5 is less clear. Moreover, there are two versions of Pds5 present in vertebrate cells, Pds5A and Pds5B. In order to explore their specific functions we previously generated murine knock out (KO) alleles for these two genes. We showed that both Pds5A and Pds5B contribute to cohesion establishment during S phase by promoting cohesin acetylation and Sororin binding, with Pds5B being specifically required for cohesion at centromeres (Carretero et al., 2013). Now we have analysed how these proteins regulate cohesin distribution and dynamics. We have found that the presence of Pds5A or Pds5B does not specify cohesin localisation, since either one can be found at most cohesin binding sites. However, genome wide distribution of cohesin clearly becomes restricted in the absence of both (FIGURE 2). Under this condition, the dynamic association of cohesin to chromatin, measured in Fluorescence Recovery After Photobleaching (FRAP) experiments, is significantly decreased. Much milder effects are observed in cells lacking only Pds5A or Pds5B. Aberrant accumulation of cohesin in axial structures known as vermicelli, previously described in Wapl depleted cells (Tedeschi et al., 2013), can be observed only in the absence of the two Pds5 proteins, although overall accumulation of the complex on chromatin is not as dramatic. From these studies we conclude that Wapl and Pds5 work together in cohesin unloading, but that Pds5 has additional functions. Our current hypothesis is that cohesin acetylation, which is carried out by Cohesin Acetyl Transferases that are recruited to cohesin through Pds5, is essential not only for cohesion establishment during S phase but also for proper cohesin dynamics throughout the cell cycle as well as in non-dividing cells.