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

doi:10.1074/jbc.271.27.16422

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

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

^j To whom correspondence should be addressed:
Dept. of Pediatrics, MC1515 University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030.
Tel.: 203-679-2461; Fax: 203-679-1047; E-mail: lichtler{at}panda.uchc.edu

↵^b Permanent address: University of Zagreb, School of Medicine, Salata 3b, 41000 Zagreb, Croatia.

Abstract

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.

Footnotes

↵^f Supported in part by the Medical Research Service, Department of Veterans Affairs.
↵* This work was supported in part by National Institutes of Health Grants AR29983 (to A. C. L.), AR38933 (to B. E. K., D. W. R., S. H. C., and A. C. L.), and AR29850 (to B. E. K.), by a grant from NASA, and by American Heart Association Grant AHA92015860 (to D. W. R.). 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.
↵¹ The abbreviations used are:

bp

base pair(s)

kb

kilobases

CAT

chloramphenicol acetyltransferase

PBS

phosphate-buffered saline

FCS

fetal calf serum

CMV

cytomegalovirus.
↵² M. Dodig and A. Lichtler, unpublished observations.
- Received November 30, 1995.
- Revision received March 11, 1996.