Expression screening of factors binding to the osteocalcin bone-specific promoter element OC Box I: Isolation of a novel osteoblast differentiation-specific factor

Abstract

Contributing to bone-specific expression of the osteocalcin gene is the promoter element OC Box I (-99 to -76), which binds both Hox proteins and another nonhomeodomain factor (designated OCBP for osteocalcin-box binding protein) (Hoffmann et al. [1996] J Cell Biochem 61:310-324). OCBP correlates with increased promoter activity and may, therefore, be important to development or maintenance of the osteoblast phenotype. To identify OCBP candidates, we used a multimerized OC Box I sequence to screen a gammagt11 cDNA expression library, constructed from the rat osteosarcoma osteoblastic ROS 17/2.8 cell line, for cDNA clones encoding factors that recognize this element. Mutant OC Box I sequences that do not bind OCBP and/or homeodomain proteins were used to counterscreen the cDNA isolates. Clones showing binding specificity were sequenced and further characterized for patterns of expression in different tissues and cell lines. Among the characterized nonhomeodomain-related isolates, we identified a nucleolin, a clone encoding rCAP2 that is related to myogenic differentiation and more importantly, a cDNA clone containing a previously uncharacterized gene that has been designated as a cell differentiation-related factor (DRF). DRF mRNA is highly expressed in ROS 17/2.8 cells and in a developmentally regulated pattern during osteoblast differentiation, being upregulated at the postproliferative maturation transition and coinciding with the induction of osteocalcin gene expression. The 7.7-kb transcript encoded by clone 44 is abundantly expressed in osteoblasts, but the mRNA was not present at detectable levels in bone and soft tissues by Northern blot analysis. However, related expressed sequence tags were recently reported in cDNA libraries of rat lung and mouse sympathetic ganglion. The identification of DRF represents a novel osteoblast differentiation-specific marker related to osteocalcin expression. The identification of DRF may further facilitate definition of gene regulatory mechanisms mediating the final stages of bone cell differentiation

CCAAT/Enhancer-binding Proteins (C/EBP) β and δ Activate Osteocalcin Gene Transcription and Synergize with Runx2 at the C/EBP Element to Regulate Bone-specific Expression

“…Previous studies using promoter deletion constructs of the rat OC gene have shown that the initial 200 bp of the promoter can confer tissue-specific expression (38). This region contains a Runx-responsive motif and a homeodomain box that also binds an osteoblast-specific complex (54). Sequence analysis of this A, total RNA was isolated from various skeletal and soft rat tissues as described under "Materials and Methods."…”

Functional Implication of Nucleolin in the Mouse First Molar Development

“…However, our present study showed that the reduction of MK mRNA level because of the suppression of nucleolin transcription by the application of nucleolin antisense probe was about 50%. This phenomenon seems to be caused by following several reasons as follows: 1) transcriptional reduction of nucleolin was not complete even by the antisense probe application, probably because the nucleolin was a ubiquitously expressed gene in cells; 2) it is unknown whether the transcriptional regulation of MK by nucleolin is direct or indirect, and there may be another transcriptional pathway for the MK expression besides nucleolin (39,40); 3) translational disturbance of nucleolin induced by adding antisense probe may influence the stabilization of MK mRNA as shown in previous reports (41,42). It therefore seems important to disclose the molecular interaction between nucleolin and MK in the cellular biology of tooth germ development.…”

Dlx3 Transcriptional Regulation of Osteoblast Differentiation: Temporal Recruitment of Msx2, Dlx3, and Dlx5 Homeodomain Proteins to Chromatin of the Osteocalcin Gene

“…The OC box binding protein (30,31), a previously characterized non-HD protein complex in ROS 17/2.8 cells, was not observed in nuclear extracts from the normal ROB cells at any stage (Fig. 4A).…”

“…EMSA reaction mixtures were prepared using 10 fmol of radiolabeled probe and 2.5 to 5 g of nuclear extract according to the procedure developed for optimal HD protein binding (30). Briefly, the DNA-protein binding reactions were carried out at room temperature for 10 min.…”