Two silencers regulate the tissue-specific expression of the collagen II gene

Collagen II, the major component of cartilage, is synthesized primarily by chondrocytes and by certain cells in the eye. Previously, we have studied the regulatory regions of the collagen II gene by DNA transfection assays (Horton, W., Miyashita, T., Kohno, K., and Yamada, Y. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8864-8868). These studies show that both the promoter and an enhancer sequence in the first intron are required for high transcriptional activity in chondrocytes. These elements do not show significant activity in cells which do not synthesize collagen II, such as in muscle cells and fibroblasts. In this report, we have constructed plasmids containing various deletions of the promoter of the collagen II gene, fused to a reporter gene for chloramphenicol acetyltransferase (CAT) and transfected them into both chick embryonic fibroblasts and HeLa cells. We have found that silencer elements in the collagen II promoter region reduce CAT activity 11-fold in fibroblasts, while not affecting the enhancer-mediated transcription in chondrocytes. Deletions in the promoter showed that most of the silencing activity was localized in two sites, between -360 and -460 base pairs and between -620 and -700 base pairs. Furthermore, a fragment containing these two sequences in a thymidine kinase promoter CAT construct reduced the activity of the promoter in an orientation independent fashion. Sequence analysis revealed that the two silencer regions are homologous and contain consensus motifs for silencer elements found in other genes. Gel retardation experiments showed that nuclear factors from HeLa cells bind specifically to a DNA fragment containing the silencer, whereas chondrocyte nuclear extracts did not show any activity. Thus, our study indicates that the expression of the collagen II gene is controlled by both negative and positive elements to ensure that the gene is only expressed in suitable cells.