Dlx5, a member of the Dlx family of homeodomain proteins, plays a critical role in bone development and fracture healing. To understand the molecular mechanism underlying the transcriptional regulation by Dlx5, we performed yeast two-hybrid screening and isolated a novel protein, Dlxin-1, that binds Dlx5 and regulates its transcriptional function. Dlxin-1 cDNA encodes a 775-amino acid protein that has a partial homology with necdin at the C terminus and 25 repeats of hexapeptides (WQXPXX) in the middle region. Dlxin-1 mRNA is expressed in various adult tissues, but not the spleen, and also in osteoblastic and chondrogenic cell lines. During embryogenesis, a strong signal for Dlxin-1 mRNA was found in cell layers surrounding cartilaginous elements in bone rudiment during digit formation. Dlxin-1 binds not only Dlx5 but also Dlx7 and Msx2 and forms homomultimers in vivo. Transfection and reporter gene assays indicate that Dlxin-1 activates the transcriptional function of Dlx5. Therefore, Dlxin-1 may act as a regulator of the function of Dlx family members in bone formation.The Dlx gene family, which comprises at least six mammalian homologues of the Drosophila homeodomain protein Distal-less, is expressed predominantly in forebrain, limbs, and branchial arches during fetal development (1, 2). Among this family, Dlx5 is expressed in most developing skeletal elements and is induced during the process of bone fracture healing (2-6), suggesting that it may play a crucial role in bone development and formation. Msx2 is expressed in skeletal elements and many other organs (7-9). The expression of Dlx5 mRNA, as well as Msx2, is induced by bone morphogenetic protein (5, 7, 10). It has been suggested that Dlx5 is a transcriptional activator and regulates osteoblastic functions positively (4,5,11,12). In contrast, Ryoo et al. (13) have demonstrated, using a reporter gene assay, that Dlx5 is a potential negative regulator of the expression of the rat osteocalcin gene, which is specifically expressed in bone. Recently, Acampora et al. (14) and Depew et al. (15) have reported the phenotype of mice homozygous for targeted deletion of the dlx5 gene, in which the expression of the osteocalcin gene in osteoblasts was markedly increased, suggesting that Dlx5 represses the expression of the osteocalcin gene in vivo (14).To understand the molecular mechanism(s) underlying the transcriptional regulation involving Dlx5, we attempted to identify a molecule that binds Dlx5 and regulates its function. Here we report the isolation and characterization of Dlxin-1, a novel protein that binds Dlx5 and regulates its transcriptional function. Dlxin-1 is a unique member of the necdin/melanomaassociated antigen (MAGE) 1 family (16 -18).
MATERIALS AND METHODS
Cells-KUSA/A1(19), a mouse osteoblastic cell line, and HT1080, a human fibrosarcoma cell line, were kindly provided by Drs. A. Umezawa (Keio University, Japan) and A. Fukamizu (University of Tsukuba, Japan), respectively. A yeast strain, Y153, was a gift from Dr.
S. Kato (University of Tok...