To assay chromosomal translocations, we use novel reporters that involve targeted DNA double-strand breaks induced by endonucleases, including zinc finger nuclease and CRISPR. Translocation formation between breaks on heterologous chromosomes establishes a functional selection marker, allowing for the isolation and analysis of individual translocation events.
Our preliminary studies using this system determined that the “classic” pathway of nonhomologous end-joining (NHEJ), which is essential for resistance to ionizing radiation and normal lymphocyte ontogeny, is completely dispensable for translocation formation. Thus, alternative NHEJ pathways may play an important role in chromosomal rearrangements, including those found in human cancers.
We have built on these studies to complete agnostic screens for new factors that modulate translocation formation, including mediators of chromatin dynamics and targetable enzymes.