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Modeling of the human alveolar rhabdomyosarcoma Pax3-Foxo1 chromosome translocation in mouse myoblasts using CRISPR-Cas9 nuclease

AutorLagutina, Irina V.; Valentine, Virginia; Picchione, Fabrizio; Harwood, Frank; Valentine, Marcus B.; Villarejo-Balcells, Bárbara; Carvajal, Jaime J. ; Grosveld, Gerard C.
Fecha de publicación6-feb-2015
EditorPublic Library of Science
CitaciónPLoS Genetics 11(2): e1004951 (2015)
ResumenMany recurrent chromosome translocations in cancer result in the generation of fusion genes that are directly implicated in the tumorigenic process. Precise modeling of the effects of cancer fusion genes in mice has been inaccurate, as constructs of fusion genes often completely or partially lack the correct regulatory sequences. The reciprocal t(2;13)(q36.1;q14.1) in human alveolar rhabdomyosarcoma (A-RMS) creates a pathognomonic PAX3-FOXO1 fusion gene. In vivo mimicking of this translocation in mice is complicated by the fact that Pax3 and Foxo1 are in opposite orientation on their respective chromosomes, precluding formation of a functional Pax3-Foxo1 fusion via a simple translocation. To circumvent this problem, we irreversibly inverted the orientation of a 4.9 Mb syntenic fragment on chromosome 3, encompassing Foxo1, by using Cre-mediated recombination of two pairs of unrelated oppositely oriented LoxP sites situated at the borders of the syntenic region. We tested if spatial proximity of the Pax3 and Foxo1 loci in myoblasts of mice homozygous for the inversion facilitated Pax3-Foxo1 fusion gene formation upon induction of targeted CRISPR-Cas9 nuclease-induced DNA double strand breaks in Pax3 and Foxo1. Fluorescent in situ hybridization indicated that fore limb myoblasts show a higher frequency of Pax3/Foxo1 co-localization than hind limb myoblasts. Indeed, more fusion genes were generated in fore limb myoblasts via a reciprocal t(1;3), which expressed correctly spliced Pax3-Foxo1 mRNA encoding Pax3-Foxo1 fusion protein. We conclude that locus proximity facilitates chromosome translocation upon induction of DNA double strand breaks. Given that the Pax3-Foxo1 fusion gene will contain all the regulatory sequences necessary for precise regulation of its expression, we propose that CRISPR-Cas9 provides a novel means to faithfully model human diseases caused by chromosome translocation in mice.
[Author Summary]: Many cancers carry recurrent chromosome translocations, which often result in the formation of fusion genes that are directly involved in the tumorigenic process. Alveolar rhabdomyosarcoma, a muscle tumor in children, is typified by a translocation that fuses the PAX3 gene on chromosome 2 to the FOXO1 gene on chromosome 13. For translocation to occur both genes need to break and the disparate ends need to fuse via a process called non-homologous end joining. We determined that physical proximity of Pax3 and Foxo1 in mouse muscle progenitor cells (myoblasts) facilitates fusion gene formation. Because Pax3 and Foxo1 in the mouse are in an opposite orientation, we used a chromosome engineering strategy to invert the orientation of Foxo1 so that upon translocation a productive Pax3-Foxo1 fusion gene is created. Co-localization of the Pax3 and Foxo1 loci is higher in fore limb than in hind limb myoblasts. Simultaneous induction of a targeted double strand DNA break in each gene by CRISPR-Cas9 nuclease generated more fusion genes in fore limb than in hind limb myoblasts. Thus, gene proximity facilitates fusion gene formation. We propose that CRISPR-Cas9 nuclease can be used for the precise modeling of chromosome translocations of human cancer in mice.
Versión del editorhttp://dx.doi.org/10.1371/journal.pgen.1004951
Identificadoresissn: 1553-7404
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