From molecular mechanisms to oncogenesis
- Erika Brunet, CR1 Inserm
- Marion Piganeau, PhD student
- Armêl Millet, PhD student
- Babin Loelia, PhD student
- Khadija Lamribet, IE
- Sonia Dubois, Post-Doc
- Benjamin Renouf, Post-Doc (2013-2015)
- Hind Ghezraoui, PhD student (2011-2015)
- Antoine Marmignon, Post-Doc (2013-2015)
Brief research summary
Recurrent chromosomal alterations are characteristic features of numerous cancers. From a mechanistic point of view, it has been shown that translocations can arise from at least two simultaneous double-strand breaks (DSBs) and that misrepair of those DNA breaks leads to the inter-chromosomal exchange of DNA pieces. This exchange can lead to expression of a new chimeric protein, when two genes are cut to form a new active gene, or the overexpression of preexisting oncogenes, or the ablation of tumor suppressor genes. Yet, despite the wide range of recurrent chromosomal translocations identified in various cancers over the past decades, the origin of translocations and the path from their formation to tumorigenesis is still unclear.
In order to study chromosomal translocation, our strategy relies on engineered nucleases (TALEN and CRISPR/Cas9) to induce concomitant DSBs on two chosen chromosomes. Misrepair of the 2 DSBs can induce chromosomal translocation by exchanging the arms of the two heterologous chromosomes and thus creating two derivative chromosomes. This approach obviates the need for prior genetic manipulation or cloning of cells, significantly expanding the repertoire of human cells that can be interrogated for translocation formation.
Two angles of the chromosomal translocation paradox piqued our interest:
- What are the molecular mechanism(s) that induce chromosomal translocation? Are the canonical DSB repair mechanisms directly implicated in the formation of these translocation events? Is the translocation mechanism just misrepair of DSBs or are those events part of the normal repair process?
- What does a specific translocation do to the cells? What are the cellular consequences of cancer fusion genes expression? What is the path from translocation formation to tumorigenesis?
The group developed a comprehensive knowledge base of all nuclear DNA repair pathways and pioneered approaches to study DNA repair using novel genome engineering methods using Zinc Finger, TALE and CRISPR/ CAS9 nucleases. We were the first to induce cancer translocations in human stem cells using these methods. Thus we deciphered the DNA repair mechanism implicated in translocation formation in human cells and we revealed an unexpected and striking species-specific difference between mouse and human: while cells classic NHEJ suppresses translocation formation in mouse, classic NHEJ promotes translocations in human cells. Our group continues to develop and apply these tools to uncover the intricacies of chromosomal translocations and their role in oncogenesis.
- Renouf, B., Piganeau, M., Ghezraoui, H., Jasin, M., and Brunet, E. (2014). Creating Cancer Translocations in Human Cells Using Cas9 DSBs and nCas9 Paired Nicks. Methods in enzymology 546, 251-271.
- Ghezraoui, H., Piganeau, M., Renouf, B., Renaud, J.B., Sallmyr, A., Ruis, B., Oh, S., Tomkinson, A.E., Hendrickson, E.A., Giovannangeli, C., Jasin, M., and Brunet, E. (2014). Chromosomal translocations in human cells are generated by canonical nonhomologous end-joining. Mol Cell 55, 829-842.
- Piganeau, M., Ghezraoui, H., De Cian, A., Guittat, L., Tomishima, M., Perrouault, L., Rene, O., Katibah, G.E., Zhang, L., Holmes, M.C., Doyon, Y., Concordet, J.P., Giovannangeli, C., Jasin, M., and Brunet, E. (2013). Cancer translocations in human cells induced by zinc finger and TALE nucleases. Genome research 23, 1182-1193.
- Simsek, D., Furda, A., Gao, Y., Artus, J., Brunet, E., Hadjantonakis, A.K., Van Houten, B., Shuman, S., McKinnon, P.J., and Jasin, M. (2011). Crucial role for DNA ligase III in mitochondria but not in Xrcc1-dependent repair. Nature 471, 245-248.
- Simsek, D., Brunet, E., Wong, S.Y., Katyal, S., Gao, Y., McKinnon, P.J., Lou, J., Zhang, L., Li, J., Rebar, E.J., Gregory, P.D., Holmes, M.C., and Jasin, M. (2011). DNA ligase III promotes alternative nonhomologous end-joining during chromosomal translocation formation. PLoS Genet 7, e1002080.
- Nakanishi, K., Cavallo, F., Perrouault, L., Giovannangeli, C., Moynahan, M.E., Barchi, M., Brunet, E., and Jasin, M. (2011). Homology-directed Fanconi anemia pathway cross-link repair is dependent on DNA replication. Nature structural & molecular biology 18, 500-503.
- Nakanishi, K., Cavallo, F., Brunet, E., and Jasin, M. (2011). Homologous recombination assay for interstrand cross-link repair. Methods in molecular biology 745, 283-291.
- Brunet, E., Simsek, D., Tomishima, M., DeKelver, R., Choi, V.M., Gregory, P., Urnov, F., Weinstock, D.M., and Jasin, M. (2009). Chromosomal translocations induced at specified loci in human stem cells. Proc Natl Acad Sci U S A 106, 10620-10625.
- Weinstock, D.M., Brunet, E., and Jasin, M. (2008). Induction of chromosomal translocations in mouse and human cells using site-specific endonucleases. J Natl Cancer Inst Monogr, 20-24.
- Weinstock, D.M., Brunet, E., and Jasin, M. (2007). Formation of NHEJ-derived reciprocal chromosomal translocations does not require Ku70. Nat Cell Biol 9, 978-981.