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The reduced and altered activities of PAX5 are linked to the protein–protein interaction motif (coiled-coil domain) of the PAX5–PML fusion protein in t(9;15)-associated acute lymphocytic leukemia

Oncogene volume 30, pages 967–977 (2011)Cite this article

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Abstract

The paired box domain of PAX5 was reported to fuse with the sequence of promyelocytic leukemia (PML) to produce PAX5–PML chimeric protein in two patients with B-cell acute lymphoblastic leukemia. In the present studies, we found, by gel shift assays, that PAX5–PML bound to a panel of PAX5-consensus sequence acts as a homodimer with reduction of its DNA-binding affinities in comparison with wild-type PAX5. In transient transfection assays using 293T and HeLa cells, and retrovirus transduction of murine hematopoietic stem/progenitor cells together with quantitative real-time polymerase chain reaction analysis, PAX5–PML inhibited wild-type PAX5 target gene transcriptional activity. Studies comparing PAX5–PML with PAX5–PML(ΔCC) demonstrated that the coiled-coil (CC) protein interaction domain located within the PML moiety was required for PAX5–PML homodimer complex formation and partial transcriptional repression of genes controlled by PAX5. Fluorescent microscopic examination of transiently expressed YFP-tagged proteins in HeLa and 293T cells demonstrated that YFP–PAX5–PML and YFP–PAX5–PML(ΔCC) exhibited a diffuse granular pattern within the nucleus, similar to PAX5 but not PML. By fluorescent recovery after photobleach (FRAP), we have shown that PAX5–PML fusion protein has reduced intranuclear mobility compared with wild-type PAX5. Furthermore, the dimerization domain (CC) of PML was responsible for the reduced intranuclear mobility of PAX5–PML. These results indicate that the CC domain of PAX5–PML is important for each of the known activities of PAX5–PML fusion proteins.

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Acknowledgements

We thank Dr M Busslinger (Research Institute of Molecular Pathology, Vienna, Austria) for the CD19-luciferase and PAX5 expression vectors, Dr AS Belmont (University of Illinois Champagne-Urbana) for the A03_1 cell line, and Dr CWE So (King's College London, London, UK) for critical reading of the manuscript. This work was supported in part, by funds from the Albert and Margaret alkek Foundation and Leukemia Research Foundation (LRF).

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  1. H Chu & X Jiang

    Present address: 2Current address: The Central Hospital of Wuhan, Wuhan Second Hospital, Wuhan, China,

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  1. Department of Medicine and Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX, USA

    J J Qiu, H Chu, X Lu, X Jiang & S Dong

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Qiu, J., Chu, H., Lu, X. et al. The reduced and altered activities of PAX5 are linked to the protein–protein interaction motif (coiled-coil domain) of the PAX5–PML fusion protein in t(9;15)-associated acute lymphocytic leukemia. Oncogene 30, 967–977 (2011). https://doi.org/10.1038/onc.2010.473

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