STRUCTURAL AND FUNCTIONAL-ANALYSIS OF MOUSE MSX1 GENE PROMOTER - SEQUENCE CONSERVATION WITH HUMAN MSX1 PROMOTER POINTS AT POTENTIAL REGULATORY ELEMENTS

Vertebrate Msx genes are related to one of the most divergent homeobox genes of Drosophila, the muscle segment homeobox (msh) gene, and are expressed in a well-defined pattern at sites of tissue interactions, T his pattern of expression is conserved in vertebrates as diverse as qu ail, zebrafish, and mouse in a range of sites including neural crest, appendages, and craniofacial structures. In the present work, we perfo rmed structural and functional analyses in order to identify potential cis-acting elements that may be regulating Msx1 gene expression. To t his end, a 4.9-kb segment of the 5'-flanking region was sequenced and analyzed for transcription-factor binding sites. Four regions showing a high concentration of these sites were identified, Transfection assa ys with fragments of regulatory sequences driving the expression of th e bacterial lacZ reporter gene showed that a region of 4 kb upstream o f the transcription start site contains positive and negative elements responsible for controlling gene expression, Interestingly, a fragmen t of 130 bp seems to contain the minimal elements necessary for gene e xpression, as its removal completely abolishes gene expression in cult ured cells. These results are reinforced by comparison of this region with the human Msx1 gene promoter, which shows extensive conservation, including many consensus binding sites, suggesting a regulatory role for them.