Cooperative Interactions between RUNX2 and Homeodomain Protein-binding Sites Are Critical for the Osteoblast-specific Expression of the Bone Sialoprotein Gene

Hernan Roca,Renny T. Franceschi,Mattabhorn Phimphilai,Guozhi Xiao,Rajaram Gopalakrishnan

doi:10.1074/jbc.m503942200

Hernan Roca, Renny T. Franceschi + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m503942200

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Abstract

The bone sialoprotein (Bsp) gene provides an excellent model for studying mechanisms controlling osteoblast-specific gene expression. Although the RUNX2 transcription factor directly regulates many osteoblast-related genes, its function in Bsp expression remains uncertain. By using chromatin immunoprecipitation (ChIP) analysis in MC3T3-E1 (clone MC-4) preosteoblast cells, RUNX2 was shown to bind a chromatin fragment containing the proximal Bsp promoter. Two putative RUNX2-binding sites (R1 and R2) were identified within this region of the mouse, rat, and human genes and were shown to bind RUNX2 in vitro and in vivo (by ChIP assay). Site-specific mutagenesis established that both sites act as osteoblast-specific transcriptional enhancers and together account for nearly two-thirds of the total promoter activity. In addition, functional cooperativity was observed between the R2 site and an adjacent homeodomain protein-binding site previously characterized by this laboratory (the C site). All three sites (R1, R2, and C) are necessary for maximal promoter activity in osteoblasts. DLX5 in MC-4 cell nuclear extracts binds to the C site in vitro. Furthermore, ChIP assays revealed that DLX5 is selectively associated with chromatin in the vicinity of the C site only when Bsp is transcriptionally active. Finally, co-immunoprecipitation assays detected a physical complex containing DLX5 and RUNX2. Taken together, our data show that RUNX2 is a direct regulator of Bsp in osteoblasts and that it functions in cooperation with DLX5 or a related factor to activate osteoblast-specific gene expression.

Highlights

The bone sialoprotein (Bsp) gene provides an excellent model for studying mechanisms controlling osteoblast-specific gene expression
To begin exploring the mechanism used by RUNX2 to regulate Bsp, the activity of this gene was examined in the murine C3H10T1/2 mesenchymal cell line
This study explores functional interrelationships between a previously described homeodomain protein-binding site in the murine Bsp promoter and two newly identified cryptic RUNX2binding sites

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—The isolation of preosteoblastic clone 4 cells (MC-4) from the parent MC3T3-E1 line was described previously [5, 25]. In the case of C3H10T1/2 or COS7 cells, they were transfected using 400 ng of Bsp-luciferase reporter construct and 400 ng of RUNX2 expression (pCMV5-Runx2) or control (pCMV5-LacZ) vector plus 40 ng of Renilla luciferase vector. For the co-immunoprecipitation studies, COS7 cells were plated in 100-mm dishes and transfected with 2.5 ␮g of DLX5 (pDlx5) and RUNX2 (pCMV5-Runx2) expression plasmids and were harvested after 48 h for the preparation of nuclear extracts. One primer (PBL1) was selected from the last 19 bp of Bsp promoter fragment (Ϫ2472 to ϩ41) cloned in pGL3Basic, and the second primer (PBL2) includes 21 bp of the specific plasmid sequence located just 8 bp before the start of luciferase coding region (see Fig. 5A and Table I) In this case the PCR proceeded for 23 cycles of amplification. Statistical significance was assessed using a Tukey-Kramer multiple comparisons test with an Instat 4.0 software package

RESULTS

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