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Cbfβ interacts with Runx2 and has a critical role in bone development

Nature Genetics volume 32, pages 639–644 (2002)Cite this article

Abstract

Runx2 (runt-related transcription factor 2, also known as Cbfa1, Osf2 and AML3) is essential for bone development in mice, and mutations in RUNX2 are found in 65–80% of individuals with cleidocranial dysplasia1,2. Although all Runx family members can interact with Cbfβ (core-binding factor b, encoded by Cbfb), a role for Cbfβ in bone development has not been demonstrated owing to lethality in Cbfb−/− mouse embryos at 12.5 days post coitum (d.p.c.) from hemorrhages and lack of definitive hematopoiesis3,4. Using a 'knock-in' strategy, we generated mouse embryonic stem (ES) cells that express Cbfb fused in-frame to a cDNA encoding green fluorescent protein (GFP)5. Cbfb+/GFP mice had normal life spans and appeared normal, but CbfbGFP/GFP pups died within the first day after birth. The CbfbGFP/GFP mice exhibited a delay in endochondral and intramembranous ossification as well as in chondrocyte differentiation, similar to but less severe than delays observed in Runx2−/− mice6,7. We demonstrate that Cbfβ is expressed in developing bone and forms a functional interaction with Runx2, and that CbfbGFP is a hypomorphic allele. The fusion allele maintains sufficient function in hematopoietic cells to bypass the early embryonic lethality, and identifies a new role for Cbfb in bone development. Our findings raise the possibility that mutations in CBFB may be responsible for some cases of cleidocranial dysplasia that are not linked to mutations in RUNX2.

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Figure 1: Skeletal phenotype in 18.5-d.p.c. CbfbGFP/GFP embryos.
Figure 2: Delayed ossification and decreased chondrocyte hypertrophy in the basisphenoid bone of CbfbGFP/GFP embryos.
Figure 3: Delayed chondrocyte maturation and osteoblast differentiation in the long bones of CbfbGFP/GFP embryos.
Figure 4: Expression of Cbfb in perichondrium, chondrocytes, periosteum, bone collar and primary spongiosa.
Figure 5: Physical and functional interaction of Cbfβ and Runx2.
Figure 6: Difference in expression of Cbfβ and Cbfβ–GFP in 18.5-d.p.c. calvaria and fetal liver.

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References

  1. Mundlos, S. Cleidocranial dysplasia: clinical and molecular genetics. J. Med. Genet. 36, 177–182 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Zhou, G. et al. CBFA1 mutation analysis and functional correlation with phenotypic variability in cleidocranial dysplasia. Hum. Mol. Genet. 8, 2311–2316 (1999).

    Article  CAS  Google Scholar 

  3. Wang, Q. et al. The CBFβ subunit is essential for CBFα2 (AML1) function in vivo. Cell 87, 697–708 (1996).

    Article  CAS  Google Scholar 

  4. Sasaki, K. et al. Absence of fetal liver hematopoiesis in mice deficient in transcriptional coactivator core-binding factor β. Proc. Natl Acad. Sci. USA 93, 12359–12363 (1996).

    Article  CAS  Google Scholar 

  5. Kundu, M. et al. Role of Cbfb in hematopoiesis and perturbations resulting from expression of the leukemogenic fusion gene Cbfb–MYH11. Blood 100, 2449–2456 (2002).

    Article  CAS  Google Scholar 

  6. Komori, T. et al. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89, 755–764 (1997).

    Article  CAS  Google Scholar 

  7. Otto, F. et al. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 89, 765–771 (1997).

    Article  CAS  Google Scholar 

  8. Kagoshima, H., Akamatsu, Y., Ito, Y. & Shigesada, K. Functional dissection of the α and β subunits of transcription factor PEBP2 and the redox susceptibility of its DNA binding activity. J. Biol. Chem. 271, 33074–33082 (1996).

    Article  CAS  Google Scholar 

  9. Miller, J.D., Stacy, T., Liu, P.P. & Speck, N.A. Core-binding factor β (CBFβ), but not CBFβ-smooth muscle myosin heavy chain, rescues definitive hematopoiesis in CBFβ-deficient embryonic stem cells. Blood 97, 2248–2256 (2001).

    Article  CAS  Google Scholar 

  10. Kim, I.S., Otto, F., Zabel, B. & Mundlos, S. Regulation of chondrocyte differentiation by Cbfa1. Mech. Dev. 80, 159–170 (1999).

    Article  CAS  Google Scholar 

  11. Zhang, Y.W. et al. A RUNX2/PEBP2α A/CBFA1 mutation displaying impaired transactivation and Smad interaction in cleidocranial dysplasia. Proc. Natl Acad. Sci. USA 97, 10549–10554 (2000).

    Article  CAS  Google Scholar 

  12. Harada, H. et al. Cbfa1 isoforms exert functional differences in osteoblast differentiation. J. Biol. Chem. 274, 6972–6978 (1999).

    Article  CAS  Google Scholar 

  13. Otto, F., Kanegane, H. & Mundlos, S. Mutations in the RUNX2 gene in patients with cleidocranial dysplasia. Hum. Mutat. 19, 209–216 (2002).

    Article  CAS  Google Scholar 

  14. Tahirov, T.H. et al. Structural analyses of DNA recognition by the AML1/Runx-1 Runt domain and its allosteric control by CBFβ. Cell 104, 755–767 (2001).

    Article  CAS  Google Scholar 

  15. Warren, A.J., Bravo, J., Williams, R.L. & Rabbitts, T.H. Structural basis for the heterodimeric interaction between the acute leukaemia-associated transcription factors AML1 and CBFβ. EMBO J. 19, 3004–3015 (2000).

    Article  CAS  Google Scholar 

  16. Thirunavukkarasu, K., Mahajan, M., McLarren, K.W., Stifani, S. & Karsenty, G. Two domains unique to osteoblast-specific transcription factor Osf2/Cbfa1 contribute to its transactivation function and its inability to heterodimerize with Cbfβ. Mol. Cell. Biol. 18, 4197–4208 (1998).

    Article  CAS  Google Scholar 

  17. Prince, M. et al. Expression and regulation of Runx2/Cbfa1 and osteoblast phenotypic markers during the growth and differentiation of human osteoblasts. J. Cell. Biochem. 80, 424–440 (2001).

    Article  CAS  Google Scholar 

  18. Javed, A. et al. Runt homology domain transcription factors (Runx, Cbfa, and AML) mediate repression of the bone sialoprotein promoter: evidence for promoter context-dependent activity of Cbfa proteins. Mol. Cell. Biol. 21, 2891–2905 (2001).

    Article  CAS  Google Scholar 

  19. Hanai, J. et al. Interaction and functional cooperation of PEBP2/CBF with Smads. Synergistic induction of the immunoglobulin germline Calpha promoter. J. Biol. Chem. 274, 31577–31582 (1999).

    Article  CAS  Google Scholar 

  20. Shirakabe, K., Terasawa, K., Miyama, K., Shibuya, H. & Nishida, E. Regulation of the activity of the transcription factor Runx2 by two homeobox proteins, Msx2 and Dlx5. Genes Cells 6, 851–856 (2001).

    Article  CAS  Google Scholar 

  21. Javed, A. et al. Groucho/TLE/R-esp proteins associate with the nuclear matrix and repress RUNX (CBFα/AML/PEBP2α) dependent activation of tissue-specific gene transcription. J. Cell. Sci. 113, 2221–2231 (2000).

    CAS  PubMed  Google Scholar 

  22. D'Alonzo, R.C., Selvamurugan, N., Karsenty, G. & Partridge, N.C. Physical interaction of the activator protein-1 factors c-Fos and c-Jun with Cbfa1 for collagenase-3 promoter activation. J. Biol. Chem. 277, 816–822 (2002).

    Article  CAS  Google Scholar 

  23. Gutierrez, S. et al. CCAAT/enhancer-binding proteins (C/EBP) β and Δ activate osteocalcin gene transcription and synergize with Runx2 at the C/EBP element to regulate bone-specific expression. J. Biol. Chem. 277, 1316–1323 (2002).

    Article  CAS  Google Scholar 

  24. Speck, N.A. & Gilliland, D.G. Core-binding factors in haematopoiesis and leukaemia. Nature Rev. Cancer 2, 502–513 (2002).

    Article  CAS  Google Scholar 

  25. Yoshida, T. et al. Functional analysis of RUNX2 mutations in Japanese patients with cleidocranial dysplasia demonstrates novel genotype–phenotype correlations. Am. J. Hum. Genet. 71, 724–738 (2002).

    Article  Google Scholar 

  26. Miller, J. et al. The core-binding factor β subunit is required for bone formation and hematopoietic maturation. Nature Genet. 32, 645–649 (2002).

    Article  CAS  Google Scholar 

  27. Yoshida, C.A. et al. Core-binding factor β interacts with Runx2 and is required for skeletal development. Nature Genet. 32, 633–638 (2002).

    Article  CAS  Google Scholar 

  28. St-Jacques, B., Hammerschmidt, M. & McMahon, A.P. Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev. 13, 2072–2086 (1999).

    Article  CAS  Google Scholar 

  29. Ducy, P., Zhang, R., Geoffroy, V., Ridall, A.L. & Karsenty, G. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 89, 747–754 (1997).

    Article  CAS  Google Scholar 

  30. Meyers, S., Lenny, N., Sun, W. & Hiebert, S.W. AML-2 is a potential target for transcriptional regulation by the t(8;21) and t(12;21) fusion proteins in acute leukemia. Oncogene 13, 303–312 (1996).

    CAS  PubMed  Google Scholar 

  31. Adya, N., Stacy, T., Speck, N.A. & Liu, P.P. The leukemic protein core-binding factor β (CBFβ)-smooth-muscle myosin heavy chain sequesters CBFα2 into cytoskeletal filaments and aggregates. Mol. Cell. Biol. 18, 7432–7443 (1998).

    Article  CAS  Google Scholar 

  32. Banerjee, C. et al. Differential regulation of the two principal Runx2/Cbfa1 N-terminal isoforms in response to bone morphogenetic protein-2 during development of the osteoblast phenotype. Endocrinology 142, 4026–4039 (2001).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank G. Karsenty for the osteocalcin and p6XOSE2 reporter constructs and for the Runx2 expression plasmid, S.C. Chandrasekharappa for the polyclonal antibody against MEN1, N. Speck for the polyclonal antibody against Runx1 and D. Leja for help in formatting figures. M.K. is a Damon Runyon Cancer Research Foundation Fellow. G.S.S. and J.B.L. are supported by grants from the US National Institutes of Health.

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Authors and Affiliations

  1. Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, 49 Convent Drive, Building 49, Room 3A26, Bethesda, 20892, Maryland, USA

    Mondira Kundu, Jae-Pil Jeon & P. Paul Liu

  2. Department of Cell Biology, University of Massachusetts, Worcester, Massachusetts, USA

    Amjad Javed, Jane B. Lian & Gary S. Stein

  3. Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, Maryland, USA

    Alan Horner, Lillian Shum & Glen H. Nuckolls

  4. Veterinary Resources Program, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, USA

    Michael Eckhaus

  5. Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, USA

    Maximilian Muenke

  6. Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, USA

    Yingzi Yang

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Correspondence to P. Paul Liu.

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Kundu, M., Javed, A., Jeon, JP. et al. Cbfβ interacts with Runx2 and has a critical role in bone development. Nat Genet 32, 639–644 (2002). https://doi.org/10.1038/ng1050

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