The cleidocranial dysplasia-related R131G mutation in the Runt-related transcription factor RUNX2 disrupts binding to DNA but not CBF-beta.
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.
Department of Biochemistry and Cell Biology, School of Medicine, WCU Program, Cell and Matrix Research Institute, Kyungpook National University, Daegu 700-422, South Korea.
Min-Su
MS
Han
Hyo-Jin
HJ
Kim
Hee-Jun
HJ
Wee
Kyung-Eun
KE
Lim
Na-Rae
NR
Park
Suk-Chul
SC
Bae
Andre J
AJ
van Wijnen
Janet L
JL
Stein
Jane B
JB
Lian
Gary S
GS
Stein
Je-Yong
JY
Choi
D020816:Amino Acid Motifs; D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D016466:CHO Cells; D002467:Cell Nucleus / Q000378:metabolism; D002973:Cleidocranial Dysplasia / Q000235:genetics*; D050659:Core Binding Factor Alpha 1 Subunit / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D050676:Core Binding Factor Alpha 2 Subunit / Q000737:chemistry / Q000378:metabolism; D050658:Core Binding Factor beta Subunit / Q000737:chemistry / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000187:drug effects* / Q000235:genetics; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D015533:Transcriptional Activation / Q000235:genetics
C493692:CBFB protein, human; D050659:Core Binding Factor Alpha 1 Subunit; D050676:Core Binding Factor Alpha 2 Subunit; D050658:Core Binding Factor beta Subunit; D050505:Mutant Proteins; C493728:RUNX1 protein, human; C493709:RUNX2 protein, human; D004247:DNA