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 extracell ular protein of 85 kDa. We have determined the primary structure of this ge ne and analysed 1665 bases of its 5' upstream regulatory region. The gene i s approximately 5 kb long and contains 11 exons. The exons range in size fr om 45 to 375 bp, whereas the intron sizes ranges from 95 to 1115 bp. All sp lice donor/acceptor sites conform to the GT/AG rule. The 5' upstream sequen ces contain a TATA-box, a CCAAT-box and an inverted CCAAT-box. We have anal ysed the Ecm1 regulatory elements by reporter gene constructs and transient transfections in the stromal osteogenic cell line MN7. Progressive deletio n 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, containin g potential binding sites for AP1, Sp1, GATA and Ets family of transcriptio n factors, is sufficient for CAT expression in MN7 cells. Except for the GA TA binding site, these regulatory sequences are conserved in the human prom oter. Point mutation analysis revealed that the AP1, Sp1 and Ets binding si tes are absolutely necessary for Ecm1 expression in MN7. (C) 1999 Elsevier Science B.V. All rights reserved.