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

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 RUNX2(R131G) mutation, which could potentially affect DNA binding, nuclear localization signal, and/or heterodimerization with core-binding factor-beta (CBF-beta). Immunofluorescence microscopy and western blot analysis with subcellular fractions show that RUNX2(R131G) is localized in the nucleus. Immunoprecipitation analysis reveals that heterodimerization with CBF-beta 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 RUNX2(R131G) is abrogated. We conclude that loss of DNA binding, but not nuclear localization or CBF-beta heterodimerization, causes RUNX2 haploinsufficiency in patients with the RUNX2(R131G) mutation. Retention of specific functions including nuclear localization and binding to CBF-beta of the RUNX2(R131G) mutation may render the mutant protein an effective competitor that interferes with wild-type function.

• 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
• Journal: Journal of cellular biochemistry (J Cell Biochem) Vol. 110 Issue 1 Pg. 97-103 (May 2010) ISSN: 1097-4644 [Electronic] United States
• PMID: 20225274 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
• Copyright: (c) 2010 Wiley-Liss, Inc.

Chemical References

• CBFB protein, human
• Core Binding Factor Alpha 1 Subunit
• Core Binding Factor Alpha 2 Subunit
• Core Binding Factor beta Subunit
• Mutant Proteins
• RUNX1 protein, human
• RUNX2 protein, human
• DNA

Topics

• Amino Acid Motifs
• Amino Acid Substitution (genetics)
• Animals
• CHO Cells
• Cell Nucleus (metabolism)
• Cleidocranial Dysplasia (genetics)
• Core Binding Factor Alpha 1 Subunit (chemistry, genetics, metabolism)
• Core Binding Factor Alpha 2 Subunit (chemistry, metabolism)
• Core Binding Factor beta Subunit (chemistry, metabolism)
• Cricetinae
• Cricetulus
• DNA (metabolism)
• HeLa Cells
• Humans
• Mutant Proteins (chemistry, metabolism)
• Mutation (drug effects, genetics)
• Protein Binding
• Protein Multimerization
• Protein Structure, Tertiary
• Protein Transport
• Transcriptional Activation (genetics)