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. 2015 Feb 12;106(3):227–236. doi: 10.1111/cas.12593

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© 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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PU.1-dependent upregulation of macrophage colony-stimulating factor receptor (CSF-1R) by mixed lineage leukemia (MLL) and MLL fusions. (a) Interaction of MLL with PU.1. 293T cells were co-transfected with MLL-HA and the indicated FLAG-tagged transcription factors, including FLAG-PU.1. Anti-FLAG antibody immunoprecipitates (IP:FLAG) or cell lysates (Input) were subjected to immunoblotting with anti-HA, anti-MLL-N, or anti-FLAG antibodies. (b) Interaction between MLL-AF10 and PU.1. 293T cells were co-transfected with MLL-AF10 and FLAG-tagged WT PU.1 or PU.1/FR232A. Anti-FLAG antibody immunoprecipitates (IP:FLAG) or cell lysates (Input) were subjected to immunoblotting with anti-MLL-N or anti-PU.1 antibodies. (c) Effects of MLL, and MLL fusions on PU.1-mediated Csf1r promoter-driven transcription. SaOS2 cells were co-transfected with the Csf1r–luciferase construct and the indicated effectors. Luciferase activity was analyzed 24 h after transfection. Error bars represent SD (n = 3). (d) PU.1 binding site-dependence of MLL enhancement of Csf1r promoter-driven transcription. SaOS2 cells were transfected with the WT Csf1r–luciferase construct or its mutant lacking the PU.1-binding site, together with the indicated effectors. (e) ChIP of MLL-AF10 and PU.1. Bone marrow (BM) cells from acute myeloid leukemia mice (AML) induced by Flag-MLL-AF10, were subjected to ChIP analysis using anti-Flag (MLL-AF10), anti-PU.1, and anti-MOZ antibodies. Semiquantitative real-time PCR was carried out on the co-precipitated DNAs.