Analysis of the Runx2 promoter in osseous and non-osseous cells and identification of HIF2A as a potent transcription activator.
Little is known about the upstream regulator of Runx2, a master regulator of osteoblast differentiation in bone tissues. To elucidate the molecular mechanism of Runx2 gene expression, we analyzed Runx2 promoter activity in osseous (MC3T3-E1, KS483, Kusa) and non-osseous (NIH3T3, C3H10T1/2, mouse embryonic fibroblasts) cells and also identified Runx2 upstream regulator using a Runx2 promoter-derived luciferase reporter system. After cloning 15 serial deletion constructs from -6832 bp/+390 bp to -37 bp/+390 bp of the Runx2-P1 promoter, we performed a transient transfection assay in osseous and non-osseous cells. A reduction in Runx2 promoter activity was observed in two regions; one was between -3 kb and -1 kb, and the other was between -155 bp and -75 bp. The step-down pattern in promoter activity between -3 kb and -1 kb was observed only in osseous cells. Interestingly, the step-down pattern between -155 bp and -75 bp was revealed in both cell types. Consistently, beta-galactosidase staining in axial skeleton of -3 kb-Runx2-P1-LacZ transgenic mice was positive, but that of all skeletal tissues of -1 kb-Runx2-P1-LacZ transgenic mice was negative. To identify upstream regulators of the Runx2-P1 promoter, we screened 100 transcription factors using Runx2-P1-luciferase reporter constructs in NIH3T3 fibroblasts and HeLa cells. Among them, HIF2A was identified as the strongest activator of Runx2-P1 promoter activity. A HIF2A-responsive site on the Runx2 promoter was identified between -106 bp and -104 bp by mutation analysis. An electrophoretic mobility shift assay and chromatin immunoprecipitation assay confirmed the binding of HIF2A to the Runx2-P1 promoter in vitro and in vivo, respectively. Knock-down using siRNA against HIF2A confirmed that HIF2A is an important regulator of Runx2 gene expression. Collectively, these results suggest that the region between -3 kb and -1 kb is required for the minimal skeletal tissue-specific expression of Runx2, and that the region between -155 bp and -75 bp is important for its basal transcription, which may be in part mediated by HIF2A in bone tissues.
Tamiya
Hiroyuki
H
Center for Disease Biology and Integrative Medicine, Faculty of Medicine, the University of Tokyo, Tokyo, 113-8655, Japan.
Ikeda
Toshiyuki
T
Jeong
Jae-Hwan
JH
Saito
Taku
T
Yano
Fumiko
F
Jung
Youn-Kwan
YK
Ohba
Shinsuke
S
Kawaguchi
Hiroshi
H
Chung
Ung-Il
UI
Choi
Je-Yong
JY
D000818:Animals; D001483:Base Sequence; D001842:Bone and Bones / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics*; D004252:DNA Mutational Analysis; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000502:physiology*; D051379:Mice; D008822:Mice, Transgenic; D011401:Promoter Regions, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D050659:Core Binding Factor Alpha 1 Subunit; D051795:Hypoxia-Inducible Factor 1, alpha Subunit