Molecular Oncology Programme

Genomic Instability Group

Research highlights

Forward genetic screening (FGS) represents one of the most powerful methods for the discovery of new pathways and/or mechanisms of disease. Haploid organisms, such as yeast, have long been the ideal platform for FGS, since mutations in one allele can suffice to reveal a phenotype. The relevance of this approach is exemplified by the high number of Nobel Prizes that have been awarded in the recent decades to investigations that started with yeast studies. While many biological questions are intrinsic to mammals and cannot be approached through yeast, the presence of a diploid genome (with two copies per gene) has significantly limited FGS approaches in mammalian cells. The recent development of CRISPR-Cas9 gene editing technologies, together with the isolation of mammalian haploid cell lines, has recently changed this landscape. During 2017, our laboratory contributed to this field by finding ways to stabilise the haploid state in mammalian cells, as well as by developing a new method for the identification of genetrap insertions by RNA sequencing that can be coupled to FGS approaches.

TrapSeq: A new method for genetrap-based genetic screenings in mammals

Genetraps are one of the most widely used methods to conduct genetic screenings in mammals. In these studies, identifying the genetrap insertion site was an essential step, which was accomplished via inverse PCR-based methods that (a) are prone to biases and artefacts, and (b) while they are able to identify the insertion site, they do not provide information as to how the insertion affects the expression of the targeted gene. To overcome these limitations, we have now developed an RNA sequencing-based method (TrapSeq) that provides a fast, direct and cost-effective pipeline for the identification of a gene-trap insertion mutation, and which also reveals the impact of the genetrap on the expression of the mutated gene (FIGURE 1). We have now used TrapSeq to conduct several genetic screenings in haploid mammalian cells, including the discovery of mutations that increase the resistance to ATR inhibitors. This screening confirmed the key role of the mitosis-promoting phosphatase CDC25A in the response to ATR inhibition that we had previously identified in CRISPR-based screenings (Ruiz et al., Mol Cell 2016), as well as identified new determinants of the sensitivity to these chemicals such as the oncogene Epithelial Cell Transforming 2 (ECT2). Studies aiming to understand the mechanisms by which the identified mutations alter the response to ATR inhibition are currently underway.

Identification of a ‘Haploidy Checkpoint’

One important limitation of mammalian haploid cell lines is the rapid loss of the haploid state, resulting in cultures becoming rapidly enriched in diploid cells. This phenomenon has been previously assumed to be due to the ‘diploidization’ of the haploid genomes, although how this occurs has remained poorly understood. We have now revealed that the so-called ‘diploidization’ is a consequence of a growing disadvantage of haploid cells, which are outcompeted by the few diploids that are present in these cultures. In support of this, single-cell sorting can significantly stabilise the haploid state. We have also discovered that the reduced fitness of mammalian haploid cells arises as a consequence of problems during chromosome segregation in mitosis, which subsequently lead to the activation of a cytotoxic p53-dependent response. Consequently, p53deletion can increase the stability of haploid cultures in human HAP1 cells or mouse embryonic stem cells. Due to the similarities between our findings and those previously reported in aneuploid or polyploid cells,we propose the existence of a unified p53-dependent ‘ploidy’ checkpoint, which is activated as a consequence of the difficulties in segregating a suboptimal chromosomal content during mitosis.