The cleidocranial dysplasia-related R131G mutation in the Runt-related transcription factor RUNX2 disrupts binding to DNA but not CBF-β

Authors

  • Min-Su Han,

    1. Department of Biochemistry and Cell Biology, School of Medicine, WCU Program, Cell and Matrix Research Institute, Kyungpook National University, Daegu 700-422, South Korea
    2. Skeletal Diseases Genome Research Center, Kyungpook National University, Daegu 700-422, South Korea
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  • Hyo-Jin Kim,

    1. Department of Biochemistry and Cell Biology, School of Medicine, WCU Program, Cell and Matrix Research Institute, Kyungpook National University, Daegu 700-422, South Korea
    2. Skeletal Diseases Genome Research Center, Kyungpook National University, Daegu 700-422, South Korea
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  • Hee-Jun Wee,

    1. Department of Biochemistry, School of Medicine, Institute for Tumor Research, Chungbuk National University, Cheongju, South Korea
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  • Kyung-Eun Lim,

    1. Department of Biochemistry and Cell Biology, School of Medicine, WCU Program, Cell and Matrix Research Institute, Kyungpook National University, Daegu 700-422, South Korea
    2. Skeletal Diseases Genome Research Center, Kyungpook National University, Daegu 700-422, South Korea
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  • Na-Rae Park,

    1. Department of Biochemistry and Cell Biology, School of Medicine, WCU Program, Cell and Matrix Research Institute, Kyungpook National University, Daegu 700-422, South Korea
    2. Skeletal Diseases Genome Research Center, Kyungpook National University, Daegu 700-422, South Korea
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  • Suk-Chul Bae,

    1. Department of Biochemistry, School of Medicine, Institute for Tumor Research, Chungbuk National University, Cheongju, South Korea
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  • Andre J. van Wijnen,

    1. Department of Cell Biology and Cancer Center, University of Massachusetts Medical School, Worcester, Massachusetts 01655
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  • Janet L. Stein,

    1. Department of Cell Biology and Cancer Center, University of Massachusetts Medical School, Worcester, Massachusetts 01655
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  • Jane B. Lian,

    1. Department of Cell Biology and Cancer Center, University of Massachusetts Medical School, Worcester, Massachusetts 01655
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  • Gary S. Stein,

    1. Department of Cell Biology and Cancer Center, University of Massachusetts Medical School, Worcester, Massachusetts 01655
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  • Je-Yong Choi

    Corresponding author
    1. Department of Biochemistry and Cell Biology, School of Medicine, WCU Program, Cell and Matrix Research Institute, Kyungpook National University, Daegu 700-422, South Korea
    2. Skeletal Diseases Genome Research Center, Kyungpook National University, Daegu 700-422, South Korea
    • Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Daegu 700-422, South Korea.
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  • Min-Su Han and Hyo-Jin Kim contributed equally to this work.

Abstract

Cleidocranial dysplasia (CCD) is caused by haploinsufficiency in RUNX2 function. We have previously identified a series of RUNX2 mutations in Korean CCD patients, including a novel R131G missense mutation in the Runt-homology domain. Here, we examine the functional consequences of the RUNX2R131G mutation, which could potentially affect DNA binding, nuclear localization signal, and/or heterodimerization with core-binding factor-β (CBF-β). Immunofluorescence microscopy and western blot analysis with subcellular fractions show that RUNX2R131G is localized in the nucleus. Immunoprecipitation analysis reveals that heterodimerization with CBF-β is retained. However, precipitation assays with biotinylated oligonucleotides and reporter gene assays with RUNX2 responsive promoters together reveal that DNA-binding activity and consequently the transactivation of potential of RUNX2R131G is abrogated. We conclude that loss of DNA binding, but not nuclear localization or CBF-β heterodimerization, causes RUNX2 haploinsufficiency in patients with the RUNX2R131G mutation. Retention of specific functions including nuclear localization and binding to CBF-β of the RUNX2R131G mutation may render the mutant protein an effective competitor that interferes with wild-type function. J. Cell. Biochem. 110: 97–103, 2010. © 2010 Wiley-Liss, Inc.

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