Molecular cloning and characterization of the mouse Ecm1 gene and its 5' regulatory sequences.

The mouse Ecm1 (extracellular matrix protein 1) gene codes for an extracellular protein of 85kDa. We have determined the primary structure of this gene and analysed 1665 bases of its 5' upstream regulatory region. The gene is approximately 5kb long and contains 11 exons. The exons range in size from 45 to 375bp, whereas the intron sizes ranges from 95 to 1115bp. All splice donor/acceptor sites conform to the GT/AG rule. The 5' upstream sequences contain a TATA-box, a CCAAT-box and an inverted CCAAT-box. We have analysed the Ecm1 regulatory elements by reporter gene constructs and transient transfections in the stromal osteogenic cell line MN7. Progressive deletion of the Ecm1 promoter revealed the presence of a region with a repressive activity between -110 and -317 and showed that a 110-bp fragment, containing potential binding sites for AP1, Sp1, GATA and Ets family of transcription factors, is sufficient for CAT expression in MN7 cells. Except for the GATA binding site, these regulatory sequences are conserved in the human promoter. Point mutation analysis revealed that the AP1, Sp1 and Ets binding sites are absolutely necessary for Ecm1 expression in MN7.