Regulation of osteocalcin gene expression by a novel Ku antigen transcription factor complex.
We previously described an osteocalcin (OC) fibroblast growth factor (FGF) response element (FRE) DNA binding activity as a target of Msx2 transcriptional regulation. We now identify Ku70, Ku80, and Tbdn100, a variant of Tubedown-1, as constituents of the purified OCFRE-binding complex. Northern and Western blot analyses demonstrate expression of Ku and Tbdn100 in MC3T3E1 osteoblasts. FGF2 treatment regulates Ku, but not Tbdn100, protein accumulation. Gel supershift studies confirm sequence-specific DNA binding of Ku in the OCFRE complex; chromatin immunoprecipitation assays confirm association of Ku and Tbdn100 with the endogenous OC promoter. In the promoter region -154 to -113, the OCFRE is juxtaposed to OSE2, an osteoblast-specific element that binds Runx2 (Osf2, Cbfa1). Expression of the Ku.Tbdn100 complex up-regulates both the basal and Runx2-dependent transcription driven by this 42-bp OC promoter element, reconstituted in CV-1 cells. Synergistic transactivation occurs in the presence of activated FGF receptor 2 signaling. Msx2 suppresses Ku- and Runx2-dependent transcription; suppression is dependent upon the Msx2 homeodomain NH(2)-terminal arm and extension. Pull-down assays confirm physical interactions between Ku and these co-regulatory transcription factors, consistent with the functional interactions identified. Finally, cultured Ku70 -/- calvarial cells exhibit a profound, selective deficiency in OC expression as compared with wild-type calvarial cells, confirming the biochemical data showing a role for Ku in OC transcription. In toto, these data indicate that a novel Ku antigen complex assembles on the OC promoter, functioning in concert with Msx2 and Runx2 to regulate OC gene expression.
Willis
David M
DM
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Loewy
Arleen P
AP
Charlton-Kachigian
Nichole
N
Shao
Jian-Su
JS
Ornitz
David M
DM
Towler
Dwight A
DA
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D034961:Antigens, Nuclear; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D004265:DNA Helicases; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005786:Gene Expression Regulation; D006801:Humans; D000072200:Ku Autoantigen; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010006:Osteoblasts / Q000378:metabolism*; D015675:Osteocalcin / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism*
D034961:Antigens, Nuclear; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors; D016222:Fibroblast Growth Factor 2; D015675:Osteocalcin; D005576:Colforsin; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen