Runx proteins are tissue-specific transcriptional scaffolds that organize and assemble regulatory complexes at strategic sites of target gene promoters and at intranuclear foci to govern activation or repression. During interphase, fidelity of intranuclear targeting supports the biological activity of Runx1 and Runx2 proteins. Both factors regulate genes involved in cell cycle control and cell growth (e.g., rRNA genes), as well as lineage commitment. Here, we have examined the subcellular regulatory properties of the third Runx member, the tumor suppressor protein Runx3, during interphase and mitosis. Using in situ cellular and biochemical approaches we delineated a subnuclear targeting signal that directs Runx3 to discrete transcriptional foci that are nuclear matrix associated. Chromatin immunoprecipitation results show that Runx3 occupies rRNA promoters during interphase. We also find that Runx3 remains associated with chromosomes during mitosis and localizes with nucleolar organizing regions (NORs), reflecting an interaction with epigenetic potential. Taken together, our study establishes that common mechanisms control the subnuclear distribution and activities of Runx1, Runx2, and Runx3 proteins to support RNA polymerase I and II mediated gene expression during interphase and mitosis. J. Cell. Physiol. (c) 2008 Wiley-Liss, Inc.

The coordinated activity of Runx2 and BMP/TGFbeta-activated Smads is critical for formation of the skeleton, but the precise structural basis for the Runx2/Smad interaction has not been resolved. By deletion mutagenesis, we have defined the Runx2 motif required for physical and functional interaction with either BMP or TGFbeta responsive Smads. Smad responsive transcriptional activity was retained upon deletion of the C-terminus to amino acid (aa) 432 but lost with deletion to aa 391. Thus the Smad interacting domain (SMID) of Runx2 (432-391) is embedded in the well-defined nuclear matrix targeting signal (NMTS) that mediates intranuclear trafficking. The SMID suffices as an interacting module when fused to the heterologous Gal-4 protein. Formation of the Runx2 and Smad complex is dependent on Runx2 phosphorylation through the MAPK signaling pathway, as determined by co-immunoprecipitation studies. We established that all SMID/NMTS deficient Runx2 mutants do not show in situ association with Smad in the nucleus nor do they support BMP2-mediated osteogenic induction of the mesenchymal C2C12 cell line. Thus, we provide direct evidence that the SMID/NMTS domain (391-432) of Runx2 is essential for BMP2-mediated osteoblast differentiation. Our findings suggest that TGFbeta/ BMP2 signaling, MAPK dependent phosphorylation, and Runx2 subnuclear targeting converge to induce the osteogenic phenotype.