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