The cleidocranial dysplasia‐related R131G mutation in the Runt‐related transcription factor RUNX2 disrupts binding to DNA but not CBF‐β |
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Authors: | Min‐Su Han Hyo‐Jin Kim Hee‐Jun Wee Kyung‐Eun Lim Na‐Rae Park Suk‐Chul Bae Andre J. van Wijnen Janet L. Stein Jane B. Lian Gary S. Stein Je‐Yong Choi |
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Affiliation: | 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;3. Department of Biochemistry, School of Medicine, Institute for Tumor Research, Chungbuk National University, Cheongju, South Korea;4. Department of Cell Biology and Cancer Center, University of Massachusetts Medical School, Worcester, Massachusetts 01655 |
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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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Keywords: | RUNX2 RUNX2R131G core‐binding factor‐β (CBF‐β ) cleidocranial dysplasia (CCD) subcellular localization heterodimerization DNA‐binding activity transactivation |
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