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Volume 271, Number 27, Issue of July 5, 1996 pp. 16422-16429
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Identification of a TAAT-containing Motif Required for High Level Expression of the COL1A1 Promoter in Differentiated Osteoblasts of Transgenic Mice

(Received for publication, November 30, 1995, and in revised form, March 11, 1996)

From the Departments of ^a Pediatrics, ^c Medicine, ^d Orthopaedic Surgery, ⁱ BioStructure and Function, and the ^e Division of Rheumatic Diseases, Department of Medicine, University of Connecticut Health Center, Farmington, Connecticut 06030, the ^g Department of Veterans Affairs Medical Center, Newington, Connecticut 06111, and the ^h Department of Biochemistry and Molecular Biology, Kenneth R. Norris Hospital and Institute, University of Southern California School of Medicine, Los Angeles, California 90033

Our previous studies have shown that the 49-base pair region of promoter DNA between 1719 and 1670 base pairs is necessary for transcription of the rat COL1A1 gene in transgenic mouse calvariae. In this study, we further define this element to the 13-base pair region between 1683 and 1670. This element contains a TAAT motif that binds homeodomain-containing proteins. Site-directed mutagenesis of this element in the context of a COL1A1-chloramphenicol acetyltransferase construct extending to 3518 base pairs decreased the ratio of reporter gene activity in calvariae to tendon from 3:1 to 1:1, suggesting a preferential effect on activity in calvariae. Moreover, chloramphenicol acetyltransferase-specific immunofluorescence microscopy of transgenic calvariae showed that the mutation preferentially reduced levels of chloramphenicol acetyltransferase protein in differentiated osteoblasts. Gel mobility shift assays demonstrate that differentiated osteoblasts contain a nuclear factor that binds to this site. This binding activity is not present in undifferentiated osteoblasts. We show that Msx2, a homeodomain protein, binds to this motif; however, Northern blot analysis revealed that Msx2 mRNA is present in undifferentiated bone cells but not in fully differentiated osteoblasts. In addition, cotransfection studies in ROS 17/2.8 osteosarcoma cells using an Msx2 expression vector showed that Msx2 inhibits a COL1A1 promoter-chloramphenicol acetyltransferase construct. Our results suggest that high COL1A1 expression in bone is mediated by a protein that is induced during osteoblast differentiation. This protein may contain a homeodomain; however, it is distinct from homeodomain proteins reported previously to be present in bone.

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