Abstract
Many 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.
Highlights
Rhabdomyosarcoma (RMS) is the third most common soft-tissue sarcoma in children with an annual incidence of five new cases per million
We determined that physical proximity of Pax3 and Foxo1 in mouse muscle progenitor cells 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
Summary
Rhabdomyosarcoma (RMS) is the third most common soft-tissue sarcoma in children with an annual incidence of five new cases per million. It accounts for 5–8% of all pediatric cancer. RMS belongs to the family of small round blue cell tumors of childhood and exhibits histological features of skeletal muscle. Two major histological subtypes of RMS can be distinguished, embryonal (E-RMS) and alveolar (A-RMS). E-RMS has its highest incidence in infants and young children whereas A-RMS is more frequent in older children and adolescents. A-RMS is distinguished from E-RMS by one of two recurrent chromosome translocations: t(2;13) or t(1;13), which result in fusion of PAX3 or PAX7 to FOXO1, respectively [4]
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