grantor:
University of TorontoIn response to osteo-inductive signals generated by the bone morphogenetic proteins (BMPs), undifferentiated mesenchymal stem cells can differentiate into osteoblasts. However, the molecular mechanisms that coordinate and direct osteogenesis are largely unknown. Differential display was utilized to identify a gene, provisionally named 'AJ18', whose expression is responsive to BMP-7 and up-regulated during osteoblastic differentiation in fetal rat calvarial cells (FRCCs). 'AJ18' encodes a novel protein comprising a Kr没ppel-associated box (KRAB) domain, which is a well-characterized transcription repressor motif, and 11 successive C2H2 zinc finger motifs. Using a target detection assay, a consensus DNA-binding site with the sequence 5'-CCACA-3' was revealed. This sequence is present in the enhancer for 'Runx2' (OSE2, osteoblast-specific element 2; 5'-ACCACA-3 '), a master gene for osteogenesis. By using a transient transfection assay AJ18 expression was found to abrogate the transactivation activity of Runx2 in a dose-dependent manner. To study the regulation of 'AJ18' gene expression, a rat genomic library was screened to obtain the 5'-flanking region of 'AJ18'. Mapping of the transcription start site showed that the 'AJ18' gene contains an unusually long 2.3 kb 5 '-untranslated region (5'-UTR) with potential for strong secondary structure. Chimeric constructs encompassing the immediate promoter region (77 to +177) revealed strong transcriptional activity when ligated to a luciferase reporter gene in transient transfection assays. Addition of ~2 kb of upstream sequence did not increase this activity. Comparison of mouse and rat 'AJ18' promoters however revealed high sequence identity that included several responsive elements for proteins such as Runx2, NF[kappa]B, Smads, Sp1, and Ets1. The expression of AJ18 mRNA and protein at various stages of mouse development revealed high levels in brain, kidney, and mineralized tissues during embryonic development. In developing endochondral bone, AJ18 staining was strong in proliferating and pre-hypertrophic chondrocytes, and osteoblasts, with low or no staining in hypertrophic chondrocytes. Nuclear staining was also observed in differentiating cells that form the mineralized tissues of teeth. Notably, the expression of AJ18 was similar to the expression of BMP-7 consistent with its perceived role as a transcriptional factor that regulates developmental processes downstream of BMP-7. These studies, therefore, have identified the first KRAB/C2H 2 zinc finger gene to be implicated in skeletal development, and have characterized the first promoter sequence for a gene belonging to the large KRAB/C2H2 zinc finger family.Ph.D
