A COMPOSITE INTRAGENIC SILENCER DOMAIN EXHIBITS NEGATIVE AND POSITIVETRANSCRIPTIONAL CONTROL OF THE BONE-SPECIFIC OSTEOCALCIN GENE - PROMOTER AND CELL-TYPE REQUIREMENTS

The osteocalcin (OC) silencer is a unique example of exonic sequences contributing to negative transcriptional control of mammalian gene exp ression. In this paper we demonstrate, using a reporter transfection a ssay, that multiple elements reside within the OC +24/+151 domain. Thi rty-fold repression is mediated by the +49/+104 fragment, experimental ly relocated 3' of the poly(A) signal. Deletion of either the +49/+54 protein-coding sequence or the +98/+104 intronic part of this fragment results in loss of repression activity, suggesting a bipartite organi zation of the +49/+104 silencer. Of particular interest, we have mappe d an antisilencer activity to the ACCCTCTCT motif (+40/+48), found in silencers associated with several other genes. Extension of the +49/+1 04 silencer to include the +24/+48 and/or the +105/+151 sequences resu lts in increased silencer activity up to 170-fold, suggesting the pres ence of additional silencer elements within these sequences. The activ ity of the silencer contained within the +24/+151 OC sequence is direc ted to the basal promoter and is not dependent on 5' distal enhancer e lements, including those that mediate responsiveness of OC transcripti on to vitamin D. The OC silencer represses the heterologous thymidine kinase promoter and is operative in osseous (normal diploid osteoblast s, ROS 17/2.8 osteosarcoma) as well as HeLa cells. Our results, which suggest the presence of at least five regulatory elements downstream o f the OC transcription start site, indicate the complexity of sequence s that mediate repression of OC promoter activity.