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 IL2Rgamma 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 multipotent and pluripotent cells, to uncover both the underlying mechanisms and phenotypic consequences of targeted translocations and other genomic rearrangements.

Fig. 1.

Induction of chromosomal translocations in TOS4A cells. (A) Design of the translocation system. DSBs are induced at the I-SceI and ZFN^IL2Rγ sites in the genome of TOS4A cells. After concurrent DSB formation and misjoining, translocations can in principle lead to either monocentric derivative chromosomes, der(6) and der(X), or dicentric and acentric chromosomes. (B) PCR approach to identify translocation breakpoint junctions for der(6) and der(X). Fragment sizes are calculated with the overhangs filled in (black). (C) Recovery of t(6;X) translocation after 2 rounds of PCR enrichment for the der(X) breakpoint junction. FISH using whole chromosome paints to chromosome 6 (green) and the X chromosome (red) verified the translocation. Breakpoint junction sequences are shown. (D) Translocation NHEJ, single-break NHEJ, and HR in TOS4A cells after expression of I-SceI and ZFN^IL2Rγ. Translocation frequency is evaluated with 2 primer sets. Single-break NHEJ is measured with primers that flank the I-SceI site. To differentiate imprecise NHEJ from HR and SSA, PCR fragments are cleaved with I-SceI and/or LweI. Imprecise NHEJ products are resistant to both (*), whereas HR and SSA products are cleaved by LweI but not I-SceI. (See also Fig. S2C.) Z, ZFN^IL2Rγ; S, I-SceI^hi. HR is the percent GFP+ cells. Results from 3 independent experiments are shown with 1 standard deviation from the mean.

Fig. 2.

Induction of chromosomal translocations at endogenous human loci. (A) Design of the translocation system. DSBs are induced at the ZFN^IL2Rγ and ZFN^p84 sites, as indicated. Only der(19) and der(X) monocentric chromosomes are shown. The PCR approach to identify translocation breakpoint junctions for der(6) and der(X) is shown. Fragment sizes are calculated with the overhangs filled in (black). (B) PCR for der(19) and der(X) breakpoint junctions in TOS4A cells after ZFN^p84 and ZFN^IL2Rγ expression. (C) Sequences of der(X) breakpoint junction sequences from hES-MP cells. Microhomologies are underlined. The boxed C indicates that ZFN^p84 cleavage may sometimes result in an alternative overhang. The 231-bp insertion is described in Fig. S3C legend.

Fig. 3.

Comparison of DSB repair pathways in multipotent hES-MP cells. (A) DSB-mediated gene targeting strategy to quantify HR. A promoterless GFP donor (GFP^p84) can target the p84 locus upon ZFN^p84 cleavage and be expressed from the p84 promoter. Homology arms (blue) are each approximately 750 bp. (B) Flow cytometric analysis of hES-MP cells 2 weeks after transfection with GFP^p84 and the indicated ZFNs. Cells transfected with ZFN^p84 (Right) contain a significant GFP+ population which was sorted for further analysis. (C) PCR analysis of genomic DNA from sorted GFP+ cells to verify DSB-mediated gene targeting. PCR primers and fragment sizes are shown in A. (D) Translocation NHEJ, single-break NHEJ, and HR after ZFN^IL2Rγ and ZFN^p84 cleavage. Translocation frequency is quantified for the der(X) breakpoint junction. For single-break NHEJ, imprecise repair products were detected by PCR amplification across the ZFN^p84 site and colony hybridization (Fig. S4). HR is quantified by DSB-mediated gene targeting. Results from 3 independent experiments are shown with 1 standard deviation from the mean.