Structural and Functional Analysis of MouseMsx1Gene Promoter: Sequence Conservation with HumanMSX1Promoter Points at Potential Regulatory Elements

摘要

VertebrateMsxgenes are related to one of the most divergent homeobox genes ofDrosophila, the muscle segment homeobox (msh) gene, and are expressed in a well-defined pattern at sites of tissue interactions. This pattern of expression is conserved in vertebrates as diverse as quail, zebrafish, and mouse in a range of sites including neural crest, appendages, and craniofacial structures. In the present work, we performed structural and functional analyses in order to identify potentialcis-acting elements that may be regulatingMsx1gene expression. To this 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 assays with fragments of regulatory sequences driving the expression of the bacteriallacZreporter gene showed that a region of 4 kb upstream of the transcription start site contains positive and negative elements responsible for controlling gene expression. Interestingly, a fragment of 130 bp seems to contain the minimal elements necessary for gene expression, as its removal completely abolishes gene expression in cultured cells. These results are reinforced by comparison of this region with the humanMsx1gene promoter, which shows extensive conservation, including many consensus binding sites, suggesting a regulatory role for the