Cellular transformation of NIH3T3 fibroblasts by CIZ/NMP4 fusions

Molecular cloning of the translocations t(12;22)(p13;q12) and t(12;17)(p13;q11) in acute leukaemia showed that either EWSR1 or its homologue TAF15 are fused to the transcription factor CIZ. EWSR1 and TAF15 belong to the TET family (TLS/FUS, EWSR1 and TAF15) of proteins. TET fusions have been identified in both solid tumours and acute myeloid leukaemia. The novel 12p translocations directly implicated TET fusions in acute lymphoblastic leukaemia as well, and demonstrated the involvement of CIZ in haematopoietic malignancies. In addition, a new fusion E2A-CIZ was recently cloned as a result of a t(12;19)(p13;p13) in a patient with acute lymphoblastic leukaemia. NIH3T3 cells stably expressing TET-CIZ fusions display a transformed phenotype in a focus formation assay. We show here that E2A-CIZ also transforms 3T3 fibroblasts, suggesting that the addition of a transactivation domain to the CIZ protein is involved in this phenotype. An artificial VP16-CIZ construct reveals similar transforming properties, supporting this. We have then analysed the domains within TAF15-CIZ that are necessary for 3T3 fibroblast transformation. Deletion of the zinc fingers of CIZ resulted in loss of both DNA-binding and transforming properties of TAF15-CIZ, whereas deletion of the other functional domains of CIZ had no effect. Fusion of a transactivation domain to CIZ is suggestive for a transactivating function in transformation. Luciferase experiments indeed showed that E2A-CIZ as well as VP16-CIZ transactivates the MMP7 promoter. Taken together, our results reported here suggest that transformation of 3T3 fibroblasts by CIZ fusions is dependent on DNA-binding and might involve transactivation of CIZ target genes.