LOCALIZATION OF SILENCER AND ENHANCER ELEMENTS IN THE HUMAN TYPE-X COLLAGEN GENE

Collagen type X is a short, network-forming collagen expressed tempora lly and spatially tightly controlled in hypertrophic chondrocytes duri ng endochondral ossification. Studies on chicken chondrocytes indicate that the regulation of type X collagen gene expression is regulated a t the transcriptional level. In this study, we have analyzed the regul atory elements of the human type X collagen (Col10a1) by reporter gene constructs and transient transfections in chondrogenic and nonchondro genic cells. Four different promoter fragments covering up to 2,864 bp of 5'-flanking sequences, either including or lacking the first intro n, were linked to luciferase reporter gene and transfected into 3T3 fi broblasts, HT1080 fibrosarcoma cells, prehypertrophic chondrocytes fro m the resting zone, hypertrophic chondrocytes, and chondrogenic cell l ines. The results indicated the presence of three regulatory elements in the human Col10a1 gene besides the proximal promoter. First, a nega tive regulatory element located between 2.4 and 2.8 kb upstream of the transcription initiation site was active in all nonchondrogenic cells and in prehypertrophic chondrocytes. Second, a positive, but also non -tissue-specific positive regulatory element was present in the first intron. Third, a cell-type-specific enhancer element active only in hy pertrophic chondrocytes was located between -2.4 and -0.9 kb confirmin g a previous report by Thomas et al. [(1995): Gene 160:291-296]. The e nhancing effect, however, was observed only when calcium phosphate was either used for transfection or included in the culture medium after lipofection. These findings demonstrate that the rigid control of huma n Col10a1 gene expression is achieved by both positive and negative re gulatory elements in the gene and provide the basis for the identifica tion of factors binding to those elements. (C) 1997 Wiley-Liss, Inc.