Rat osteoblast and osteosarcoma nuclear matrix proteins bind with sequence specificity to the rat type I collagen promoter.

Abstract

The nuclear matrix mediates the 3-dimensional organization of DNA and supports DNA replication and its transcription. We hypothesize that the osteoblast nuclear matrix contributes to the transcriptional control of type I collagen (COL1A1) expression. Cis-regulatory elements of the rat COL1A1 promoter that control osteoblast expression in vivo are between -2.3 and -1.67 kilobase pairs (kb) but lie within -3.5 and -2.3 kb in cultured bone cells. This may result from differences in cell architecture between osteoblasts in tissue and those in vitro. Our aim was to identify osteoblast nuclear matrix proteins (NMPs) that associated with sequence-specificity to the COL1A1 promoter. We used osteoblasts from the rat metaphyseal femur and the rat osteosarcoma cells, ROS 17/2.8. Nuclear matrix and soluble nuclear proteins were obtained as separate subfractions. Gel mobility shift analysis, using fragments of the COL1A1 promoter, was used to identify DNA-binding proteins in the nuclear subfractions. A NMP-DNA interaction, NMP3, was observed between -2149 and -2106 nucleotide in both osteoblasts and osteosarcoma cells. NMP4 was detected between -3518 to -3406 nucleotide. Therefore, osteoblast NMPs recognize sequences in regulatory regions of the COL1A1 promoter and may link cell structure and the transcriptional regulation of this protein.