Identification of a TAAT-containing motif required for high level expression of the COL1A1 promoter in differentiated osteoblasts of transgenic mice.
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.
Dodig
M
M
Department of Pediatrics, University of Connecticut Health Center, Farmington, Connecticut 06030, the Department.
Kronenberg
M S
MS
Bedalov
A
A
Kream
B E
BE
Gronowicz
G
G
Clark
S H
SH
Mack
K
K
Liu
Y H
YH
Maxon
R
R
Pan
Z Z
ZZ
Upholt
W B
WB
Rowe
D W
DW
Lichtler
A C
AC
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001842:Bone and Bones / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D003094:Collagen / Q000096:biosynthesis* / Q000235:genetics*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000096:biosynthesis; D018398:Homeodomain Proteins / Q000096:biosynthesis; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D012886:Skull / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C084119:MSX2 protein; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D003094:Collagen; D015500:Chloramphenicol O-Acetyltransferase