During skeletal development, chondrocytes go through several stages of differentiation. The last stage, chondrocyte hypertrophy, occurs in areas of endochondral ossification. Mature hypertrophic chondrocytes differ from immature chondrocytes in that they become postmitotic, increase their cellular volume up to eightfold, and synthesize a unique set of matrix molecules. One such molecule is a short collagenous protein, collagen X. Previous studies have shown that collagen X is not expressed by other cell types and that its specific expression in hypertrophic chondrocytes is controlled by transcriptional mechanisms. To define these mechanisms, plasmid constructs containing the chicken collagen X gene promoter and 5' flanking regions fused to a reporter gene (chloramphenicol acetyl transferase, CAT) were transfected into primary cultures of collagen X-expressing and nonexpressing cells. A construct containing a short (558 bp) promoter exhibited high levels of CAT activity in all cell types (fibroblasts, immature, and hypertrophic chondrocytes). Adding a 4.2-kb fragment of 5' flanking DNA to this construct resulted in a dramatic reduction of CAT activity in fibroblasts and immature chondrocytes, but had no effect in hypertrophic chondrocytes. Addition of three subfragments of the 4.2-kb fragment to the initial construct, either individually or in various combinations, showed that all subfragments reduced CAT activity somewhat in non-collagen X-expressing cells, and that their effects were additive. Unrelated DNA had no effect on CAT activity. The results suggest that multiple, diffuse upstream negative regulatory elements act in an additive manner to restrict transcription of the collagen X gene to hypertrophic chondrocytes.

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