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J Biol Chem, Vol. 275, Issue 18, 13907-13917, May 5, 2000

Identification of a Homeodomain Binding Element in the Bone Sialoprotein Gene Promoter That Is Required for Its Osteoblast-selective Expression*

M. Douglas BensonDagger , Jeffrey L. Bargeon§, Guozhi Xiao§, Peedikayil E. Thomas§, Ahn Kim§, Yingqi Cui§, and Renny T. FranceschiDagger §

From the Dagger  Department of Biological Chemistry, School of Medicine and the § Department of Periodontics, Prevention, and Geriatrics, School of Dentistry, University of Michigan, Ann Arbor, Michigan 48109-1078

Bone sialoprotein is a 70-kDa extracellular matrix component that is intimately associated with biomineralization, yet the cis-acting elements of the Bsp gene that restrict its expression to mineralizing cells remain uncharacterized. To identify such elements, we analyzed a 2472-base pair fragment of the murine promoter that directs osteoblast-selective expression of a luciferase reporter gene and found that the region between -338 and -178 relative to the transcriptional start is crucial for its osteoblast-selective activity. We identified an element within this region that binds a protein complex in the nuclear extracts of osteoblastic cells and is required for its transcriptional activity. Introduction of a mutation that disrupts a homeodomain binding site within this sequence eliminates both its in vitro binding and nearly all of the osteoblastic-selective activity of the 2472-base pair promoter. We further found that the Dlx5 homeoprotein, which is able to regulate the osteoblast-specific osteocalcin promoter, can bind this element and stimulate its enhancer activity when overexpressed in COS7 cells. These data represent the first description of an osteoblast-specific element within the bone sialoprotein promoter and demonstrate its regulation by a member of a family of factors known to be involved in skeletogenesis.

* This work was supported by National Institutes of Health Grants DE12211 and DE 11723, General Clinical Research Center Grant M01-RR00042, and Michigan Multipurpose Arthritis Center Grant AR 20557.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Periodontics, Prevention, and Geriatrics, School of Dentistry, University of Michigan, 1011 N. University Ave., Ann Arbor, MI 48109-1078. Tel.: 734-763-7381; Fax: 734-763-5503; E-mail: rennyf@umich.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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