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 promot er DNA between -1719 and -1670 base pairs is necessary for transcripti on of the rat COL1A1 gene in transgenic mouse calvariae, In this study , we further define this element to the 13-base pair region between -1 683 and -1670. This element contains a TAAT motif that binds homeodoma in-containing proteins, Site-directed mutagenesis of this element in t he context of a COL1A1-chloramphenicol acetyltransferase construct ext ending to -3518 base pairs decreased the ratio of reporter gene activi ty in calvariae to tendon from 3:1 to 1:1, suggesting a preferential e ffect on activity in calvariae, Moreover, chloramphenicol acetyltransf erase-specific immunofluorescence microscopy of transgenic calvariae s howed that the mutation preferentially reduced levels of chloramphenic ol acetyltransferase-protein in differentiated osteoblasts, Gel mobili ty shift assays demonstrate that differentiated osteoblasts contain a nuclear factor that binds to this site, This binding activity is not p resent in undifferentiated osteoblasts, Ne show that Msx2, a homeodoma in protein, binds to this motif; however, Northern blot analysis revea led that Msx2 mRNA is present in undifferentiated bone cells but not i n fully differentiated osteoblasts, In addition, cotransfection studie s in ROS 17/2.8 osteosarcoma cells using an Msx2 expression vector sho wed that Msx2 inhibits a COL1A1 promoter-chloramphenicol acetyltransfe rase construct, Our results suggest that high COL1A1 expression in bon e is mediated by a protein that is induced during osteoblast different iation. This protein may contain a homeodomain; however, it is distinc t from homeodomain proteins reported previously to be present in bone.