A FAR UPSTREAM, CELL-TYPE-SPECIFIC ENHANCER OF THE MOUSE THROMBOSPONDIN-3 GENE IS LOCATED WITHIN INTRON-6 OF THE ADJACENT METAXIN GENE

Thrombospondin 3 (TSP3) is a secreted, pentameric glycoprotein whose r egulation of expression and function are not well understood. Mouse Th bs3 is located just downstream from the divergently transcribed metaxi n gene (Mtx), which encodes an outer mitochondrial membrane import. pr otein. Although Thbs3 and Mtx share a common promoter region, previous studies showed that Mtx is regulated by proximal elements that had Li ttle effect on Thbs3 expression. In this study, transient transfection . of rat chondrosarcoma cells and NIH-3T3 fibroblasts demonstrated tha t Thbs3 is regulated in a cell type-specific manner by a position- and orientation-independent far upstream enhancer located within intron 6 of Mtx. Despite its greater proximity to the transcription start site of Mtx, the Thbs3 enhancer did not have a significant effect on Mtx e xpression. Two DNA-protein complexes, which were both required for act ivity, were identified when nuclear extracts were assayed with a probe containing the enhancer sequence. The protein ire one of these comple xes was identified as Spl, while the other DNA-protein complex remains uncharacterized. A B-kilobase pair promoter containing the enhancer w as able to direct specific expression of the E. coli lacZ gene in tran sgenic mice, whereas a 2-kilobase pair promoter that lacked the enhanc er was inactive. Thus, despite their close proximity, the genes of the Mtx/Thbs3 gene cluster are regulated independently.