A remote upstream element regulates tissue-specific expression of the rat aggrecan gene

The regulation of chondrogenesis and of the genes expressed as markers of chondrocyte differentiation is poorly understood. The hyaluronan-binding proteoglycan aggrecan is an essential and specific component of cartilage, but the aggrecan proximal promoter is expressed in an unregulated fashion in vitro. DNA comprising the rat aggrecan gene (83 kb including the 30-kb first intron) was surveyed for active elements, which would impart selective expression to the aggrecan promoter in transfection assays in vitro. A 4.7-kb DNA fragment (P3) with cell-specific enhancer activity was discovered approximately 12 kb upstream of the transcription start site; this active DNA fragment is position- and orientation-independent, and strongly stimulates aggrecan promoter expression in chondrocytes, while weakly suppressing transcription in fibroblasts. Most of this activity has been localized to P3-7, a 2.3-kb internal fragment of P3. Another enhancer element (A23), which is not tissue-specific, was discovered about 70 kb downstream of the transcription start site. Several lines of transgenic mice were created using combinations of these DNA elements to drive the lacZ reporter gene. Neither a short (900 bp) nor a long (3.7 kb) promoter alone showed detectable expression in 14.5-day embryos, whereas placing the P3 tissue-specific enhancer together with P0 gave strong expression restricted to embryonic cartilage of transgenic mice. The A23 downstream enhancer in conjunction with P0 did not confer expression. This is the first report of a gene control region which confers authentic tissue-specific regulation of aggrecan in vitro or in vivo and should greatly facilitate understanding the coordinate regulation of chondrocytic genes.