SP1-BINDING ELEMENTS, WITHIN THE COMMON METAXIN-THROMBOSPONDIN-3 INTERGENIC REGION, PARTICIPATE IN THE REGULATION OF THE METAXIN GENE

Metaxin (Mtx) is an essential nuclear gene which is expressed ubiquito usly in mice and encodes a mitochondrial protein, The gene is located upstream and is transcribed divergently from the thrombospondin 3 (Thb s3) gene; 1352 nucleotides separate the putative translation start sit es, Although the Mtx and Thbs3 genes share a common intergenic region, transient transfection experiments in rat chrondro-sarcoma cells and in NIH-3T3 fibroblasts demonstrated that the elements required for exp ression of the Mtx gene are situated within a short proximal promoter and have no major effect on the transcription of Thbs3, The metaxin -3 77 bp promoter contains four clustered GC boxes between nucleotides -1 46 and -58 and an inverted GT box between nucleotides -152 and -161, b ut does not contain TATA or CCAAT boxes, Like many genes regulated by a TATA-less promoter, the transcription start site of metaxin is heter ogeneous, The major start site is only 13 bp upstream from the putativ e translation start site, Electrophoretic mobility shift, competition and supershift assays showed that the ubiquitous transcription factor, Spl, and, to a lesser extent, the Spl-related protein, Sp3, bind to f our of these Spl-binding motifs, Co-transfection of metaxin promoter-l uciferase constructs and an Spl expression vector into Schneider Droso phila cells, which do not synthesize Spl, demonstrated that the metaxi n gene is activated by Spl, Deletion of the four upstream Spl-binding elements, on the other hand, demonstrated that these motifs are superf luous in context of the larger Mtx promoter, Thus, despite the potenti al for common regulatory mechanisms, the available evidence indicates that the Mtx minimal promoter does not significantly affect Thbs3 gene expression.