Dlx5 specifically regulates Runx2 type II expression by binding to homeodomain-response elements in the Runx2 distal promoter.
Two major isoforms of the Runx2 gene are expressed by alternative promoter usage: Runx2 type I (Runx2-I) is derived from the proximal promoter (P2), and Runx2 type II (Runx2-II) is produced by the distal promoter (P1). Our previous results indicate that Dlx5 mediates BMP-2-induced Runx2 expression and osteoblast differentiation (Lee, M.-H., Kim, Y-J., Kim, H-J., Park, H-D., Kang, A-R., Kyung, H.-M., Sung, J-H., Wozney, J. M., Kim, H-J., and Ryoo, H-M. (2003) J. Biol. Chem. 278, 34387-34394). However, little is known of the molecular mechanisms by which Dlx5 up-regulates Runx2 expression in BMP-2 signaling. Here, Runx2-II expression was found to be specifically stimulated by BMP-2 treatment or by Dlx5 overexpression. In addition, BMP-2, Dlx5, and Runx2-II were found to be expressed in osteogenic fronts and parietal bones of the developing cranial vault and Runx2-I and Msx2 in the sutural mesenchyme. Furthermore, Runx2 P1 promoter activity was strongly stimulated by Dlx5 overexpression, whereas Runx2 P2 promoter activity was not. Runx2 P1 promoter deletion analysis indicated that the Dlx5-specific response is due to sequences between -756 and -342 bp of the P1 promoter, where three Dlx5-response elements are located. Dlx5 responsiveness to these elements was confirmed by gel mobility shift assay and site-directed mutagenesis. Moreover, Msx2 specifically suppressed the Runx2 P1 promoter, and the responsible region overlaps with that recognized by Dlx5. In summary, Dlx5 specifically transactivates the Runx2 P1 promoter, and its action on the P1 promoter is antagonized by Msx2.
Lee
Mi-Hye
MH
Department of Biochemistry, School of Dentistry and Skeletal Diseases Genome Research Center, School of Medicine, Kyungpook National University, Daegu, Korea.
Kim
Youn-Jeong
YJ
Yoon
Won-Joon
WJ
Kim
Jee-In
JI
Kim
Byung-Gyu
BG
Hwang
Yoo-Seok
YS
Wozney
John M
JM
Chi
Xin-Zi
XZ
Bae
Suk-Chul
SC
Choi
Kang-Young
KY
Cho
Je-Yoel
JY
Choi
Je-Yong
JY
Ryoo
Hyun-Mo
HM
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D050659:Core Binding Factor Alpha 1 Subunit / Q000096:biosynthesis*; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism; D018398:Homeodomain Proteins / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D017403:In Situ Hybridization; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000737:chemistry; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D020218:Response Elements; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism / Q000502:physiology*; D015533:Transcriptional Activation; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism; D015854:Up-Regulation
C525718:BMP2 protein, human; C525719:Bmp2 protein, mouse; C525720:Bmp2 protein, rat; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D050659:Core Binding Factor Alpha 1 Subunit; C100596:DLX5 protein, human; D017931:DNA Primers; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C084119:MSX2 protein; D009841:Oligonucleotides; D020033:Protein Isoforms; D011994:Recombinant Proteins; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D004247:DNA; D008156:Luciferases