Chromosomal translocations induced at specified loci in human stem cells

Abstract

The precise genetic manipulation of stem and precursor cells offers extraordinary potential for the analysis, prevention, and treatment of human malignancies. Chromosomal translocations are hallmarks of several tumor types where they are thought to have arisen in stem or precursor cells. Although approaches exist to study factors involved in translocation formation in mouse cells, approaches in human cells have been lacking, especially in relevant cell types. The technology of zinc finger nucleases (ZFNs) allows DNA double-strand breaks (DSBs) to be introduced into specified chromosomal loci. We harnessed this technology to induce chromosomal translocations in human cells by generating concurrent DSBs at 2 endogenous loci, the PPP1R12C/p84 gene on chromosome 19 and the IL2R␥ gene on the X chromosome. Translocation breakpoint junctions for t(19;X) were detected with nested quantitative PCR in a high throughput 96-well format using denaturation curves and DNA sequencing in a variety of human cell types, including embryonic stem (hES) cells and hES cell-derived mesenchymal precursor cells. Although readily detected, translocations were less frequent than repair of a single DSB by gene targeting or nonhomologous end-joining, neither of which leads to gross chromosomal rearrangements. While previous studies have relied on laborious genetic modification of cells and extensive growth in culture, the approach described in this report is readily applicable to primary human cells, including mutipotent and pluripotent cells, to uncover both the underlying mechanisms and phenotypic consequences of targeted translocations and other genomic rearrangements.double-strand break repair (DSB repair) ͉ zinc finger nucleases ͉ mesenchymal cells ͉ gene targeting ͉ nonhomologous end-joining (NHEJ) R ecurrent chromosomal translocations are associated with many cancers where they are considered to be the initiating event for tumorigenic transformation. As many as half of hematological malignancies have a specific translocation signature, as do a number of tumors of mesenchymal origin, including Ewing's sarcoma, rhabdomyosarcoma, and synovial sarcoma (1, 2). Recurrent oncogenic chromosomal rearrangements have also recently been identified in some carcinomas, including tumors of the prostate (3) and small cell lung cancer (4), raising the possibility that they have a more widespread contribution to the etiology of solid tumors of epithelial origin than was previously recognized (5).Given the prevalence of chromosomal translocations in human malignancy, understanding how translocations are formed in human cells and the factors involved in their formation could lead to measures to prevent their occurrence. The initiating lesions in most cases are likely to be contemporaneous DNA double-strand breaks (DSBs) on heterologous chromosomes that are misjoined (2, 6). Sequencing of numerous breakpoint junctions from human translocations indicates that a nonhomologous end-joining (NHEJ) pathway of DSB repair gives rise to the misjoining events, sinc...

Targeted chromosomal deletions and inversions in zebrafish

“…Likewise, the generation of DSBs on disparate chromosomes can promote translocations between the cleaved segments albeit at low frequency (Brunet et al 2009;Simsek et al 2011). Targeted genomic deletions have also been generated in invertebrate animals using two SSNs .…”

CRISPR-Cas9–guided oncogenic chromosomal translocations with conditional fusion protein expression in human mesenchymal cells

“…More recently, the development of programmable nucleases has made it possible to cleave endogenous loci without prior modification (14,15), greatly simplifying genome editing approaches, including translocations. For example, nucleases that use an array of DNA binding domains of zinc fingers (ZFN) or TAL effectors (TALEN) fused with FokI endonuclease cleavage domains have been used to generate chromosomal translocations in mammalian cells (11,16,17). More recently, the discovery of RNAguided nucleases in bacterial adaptive immunity (CRISPR-Cas9) has facilitated DSB introduction at desired genomic loci (18,19).…”

“…Not surprisingly then, recovery of clones containing translocations is laborious, relying on sib-selection (16), and can fail with primary cells with limited passage number if translocation fusion protein expression is not sufficient for cellular transformation. Further, fusion gene expression occurs concomitantly with the translocation, which may alter growth properties of cells harboring translocations within the background cell population, complicating the analysis of early steps of oncogenic transformation.…”

“…Compared with repair of a single DSB, repair of two DSBs leading to chromosomal translocation is inefficient, typically occurring at frequencies of ∼10 −3 to 10 −4 , as measured by breakpoint junction analysis in human cells (11,16). Not surprisingly then, recovery of clones containing translocations is laborious, relying on sib-selection (16), and can fail with primary cells with limited passage number if translocation fusion protein expression is not sufficient for cellular transformation.…”

Redesign of extensive protein–DNA interfaces of meganucleases using iterative cycles of in vitro compartmentalization

“…In addition to DSBs at single sites that primarily lead to indels, those generated simultaneously at two chromosomal loci, albeit rare events, can promote even more significant changes of chromosomes including translocation (40). Although further experiments need to be conducted, this study suggests that an engineered meganuclease tethered to a sequence-specific DNA binding domain such as a TAL effector and zinc finger array (which recognizes extremely long target sequences) may be one of platforms that can be exploited for therapeutic applications requiring exceptionally high demands for sequence specificity.…”