Meox Homeodomain Proteins Are Required for Bapx1 Expression in the Sclerotome and Activate Its Transcription by Direct Binding to Its Promoter

Abstract

The axial skeleton of vertebrates derives from the sclerotomal compartment of the somites. Genetic analysis has demonstrated that the transcription factors Pax1, Pax9, Meox1, Meox2, and Bapx1 are all required for sclerotomal differentiation. Their hierarchical relationship is, however, poorly understood. Because Bapx1 expression in the somites starts slightly later than that of the Meox genes, we asked whether Bapx1 is one of their downstream targets. Our analysis of Meox1; Meox2 mutant mice supports this hypothesis, as Bapx1 expression in the sclerotome is lost in the absence of both Meox proteins. Using transient-transfection assays, we show that Meox1 activates the Bapx1 promoter in a dose-dependent manner and that this activity is enhanced in the presence of Pax1 and/or Pax9. Furthermore, by electrophoretic mobility shift and chromatin immunoprecipitation experiments, we demonstrate that Meox1 can bind the Bapx1 promoter. The palindromic sequence TAATTA, present in the Bapx1 promoter, binds the Meox1 protein in vitro and is necessary for Meox1-induced transactivation of the Bapx1 promoter. Our data demonstrate that the Meox genes are required for Bapx1 expression in the sclerotome and suggest that the mechanism by which the Meox proteins exert this function is through direct activation of the Bapx1 gene.

We thank R. E. Hill for the Bapx1 probe and J. Gerber for providing the Pax9 expression plasmid. We are grateful to E. Ballestar for helpful technical advice on the ChIP technique.

I.R. held a Marie Curie fellowship from the European Community, program Human Potential, under contract number HPCF-CT-1999-00112. This study was supported by grants from the Deutsche Forschungsgemeinschaft and the GSF-National Research Center for Environment and Health to K.I., from the Spanish MCyT (BMC-2001-0818) to P.B., and from the Medical Research Council to B.S.M.