Runx (Cbfa͞AML) transcription factors are critical for tissue-specific gene expression. A unique targeting signal in the C terminus directs Runx factors to discrete foci within the nucleus. Using Runx2͞CBFA1͞AML3 and its essential role in osteogenesis as a model, we investigated the fundamental importance of fidelity of subnuclear localization for tissue differentiating activity by deleting the intranuclear targeting signal via homologous recombination. Mice homozygous for the deletion (Runx2⌬C) do not form bone due to maturational arrest of osteoblasts. Heterozygotes do not develop clavicles, but are otherwise normal. These phenotypes are indistinguishable from those of the homozygous and heterozygous null mutants, indicating that the intranuclear targeting signal is a critical determinant for function. The expressed truncated Runx2⌬C protein enters the nucleus and retains normal DNA binding activity, but shows complete loss of intranuclear targeting. These results demonstrate that the multifunctional N-terminal region of the Runx2 protein is not sufficient for biological activity. We conclude that subnuclear localization of Runx factors in specific foci together with associated regulatory functions is essential for control of Runx-dependent genes involved in tissue differentiation during embryonic development.F actors that mediate transcription, processing of gene transcripts, DNA replication, and DNA repair are organized as discrete domains within the nucleus (1-13). The osteogenic and hematopoietic Runx transcription factors contain a unique and conserved amino acid motif in the C terminus designated the nuclear matrix targeting signal that directs Runx proteins to subnuclear foci where gene regulatory complexes are assembled in situ (14)(15)(16)(17)(18)(19)(20). Thus the Runx transcription factors provide a paradigm for pursuing mechanisms that coordinate the compartmentalization of regulatory proteins in intranuclear sites. In this study we directly addressed the fundamental question of whether subnuclear targeting and the associated regulatory activities of Runx factors are obligatory for in vivo function.The Runx (CBFA͞AML͞PEBP2␣) ʈ family of transcription factors are critical for cellular differentiation and organ development (21-23). Key studies have established that Runx2͞ CBFA1͞AML3 is required for osteoblast differentiation and in vivo bone formation (24-28). Ablation of the Runx2 gene in mice results in a complete absence of intramembranous and endochondral bone that is attributed to the maturational arrest of hypertrophic chondrocytes and osteoblasts (26,29,30). Haploinsufficiency of this gene results in the human syndrome cleidocranial dysplasia, a dominantly inherited developmental disorder of bone (24,25). A basis is thereby provided for examining the biological consequences resulting from absence of the Cterminal targeting function.Runx factors share multiple functional domains. The Nterminal runt homology DNA binding domain (RHD), which interacts with many coregulatory factors and chromatin m...

The Runx2 (CBFA1/AML3/PEBP2␣A) transcription factor promotes skeletal cell differentiation, but it also has a novel cell growth regulatory activity in osteoblasts. We addressed here whether Runx2 activity is functionally linked to cell cycle-related mechanisms that control normal osteoblast proliferation and differentiation. We found that the levels of

␤ig-h3 is a TGF-␤-induced matrix protein known to mediate the adhesion of several cell types. In this study, we found that all four of the fas-1 domains in ␤ig-h3 mediate MRC-5 fibroblast adhesion and that this was specifically inhibited by a function-blocking monoclonal antibody specific for the ␣v␤5 integrin. Using deletion mutants of the fourth fas-1 domain revealed the MRC-5 cell adhesion motif (denoted the YH motif) is located in amino acids 548 -614. Experiments with substitution mutants showed that tyrosine 571, histidine 572, and their flanking leucine and isoleucine amino acids, which are all highly conserved in many fas-1 domains, are essential for mediating MRC-5 cell adhesion. A synthetic 18-amino acid peptide encompassing these conserved amino acids could effectively block MRC-5 cell adhesion to ␤ig-h3. Using HEK293 cells stably transfected with the ␤5 integrin cDNA, we confirmed that the ␣v␤5 integrin is a functional receptor for the YH motif. In conclusion, we have identified a new ␣v␤5 integrininteracting motif that is highly conserved in the fas-1 domains of many proteins. This suggests that fas-1 domain-containing proteins may perform their biological functions by interacting with integrins. ␤ig-h3 is an extracellular matrix protein whose expression in several cell types, including fibroblasts, is strongly induced by TGF-␤.1 The gene encoding ␤ig-h3 was first identified by Skonier et al. (1), who isolated it by screening a cDNA library made from a human lung adenocarcinoma cell line (A549) that had been treated with TGF-␤. The ␤ig-h3 protein comprises 683 amino acids containing four homologous internal repeat domains. These domains are homologous to similar motifs in the Drosophila protein fasciclin-I and thus are denoted fas-1 domains. The fas-1 domain has highly conserved sequences found in secretory and membrane proteins of several species, including mammals, insects, sea urchins, plants, yeast, and bacteria (2).Mutations in ␤ig-h3 have been shown to be responsible for 5q31-linked human autosomal dominant corneal dystrophies. It has a fibrillar structure and interacts with several extracellular matrix proteins such as fibronectin and collagen (3). In addition, ␤ig-h3 has been reported to be involved in cell growth and differentiation, wound healing, and cell adhesion (4 -9). ␤ig-h3 mediates the adhesion of many different cell types, including corneal epithelial cells, chondrocytes, and fibroblasts (8 -10). We reported previously that ␤ig-h3 mediates corneal epithelial cell adhesion by binding to ␣3␤1 integrin. Two motifs interacting with the ␣3␤1 integrin were located within the second and the fourth fas-1 domains of ␤ig-h3. Interestingly, however, we found that these two motifs are not involved in ␤ig-h3-mediated fibroblastic cell adhesion. Furthermore, all four fas-1 domains of ␤ig-h3 mediate fibroblastic cell adhesion, whereas corneal epithelial cell adhesion is supported by just the second and the fourth fas-1 domains. This suggests that ␤ig-h3 has additional motifs that can mediate the ...

The bone morphogenetic protein (BMP)-2 is a potent osteoinductive signal, inducing bone formation in vivo and osteoblast differentiation from non-osseous cells in vitro. The runt domain-related protein Cbfa1/PEBP2alphaA/AML-3 is a critical component of bone formation in vivo and transcriptional regulator of osteoblast differentiation. To investigate the relationship between the extracellular BMP-2 signal, Cbfa1, and osteogenesis, we examined expression of Cbfa1 and osteoblastic genes during the BMP-2 induced osteogenic transdifferentiation of the myoblastic cell line C2C12. BMP-2 treatment completely blocked myotube formation and transiently induced expression of Cbfa1 and the bone-related homeodomain protein Msx-2 concomitant with loss of the myoblast phenotype. While induction of collagen type I and alkaline phosphatase (AP) expression coincided with Cbfa1 expression, Cbfa1 mRNA was strikingly downregulated at the onset of expression of osteopontin (OPN) and osteocalcin (OCN) genes, reflecting the mature osteoblast phenotype. TGF-beta1 treatment effectively suppressed myogenesis and induced Cbfa1 expression but was insufficient to support osteoblast differentiation reflected by the absence of ALP, OPN, and OCN. We addressed whether induction of Cbfa1 in response to BMP-2 results in the transcriptional activation of the OC promoter which contains three enhancer Cbfa1 elements. Transfection studies show BMP-2 suppresses OC promoter activity in C2C12, but not in osteoblastic ROS 17/2.8 cells. Maximal suppression of OC promoter activity in response to BMP-2 requires sequences in the proximal promoter (up to nt -365) and may occur independent of the three Cbfa sites. Taken together, our results demonstrate a dissociation of Cbfa1 expression from development of the osteoblast phenotype. Our findings suggest that Cbfal may function transiently to divert a committed myoblast to a potentially osteogenic cell. However, other factors induced by BMP-2 appear to be necessary for complete expression of the osteoblast phenotype.