Regulation of Gax Homeobox Gene Transcription by a Combination of Positive Factors Including Myocyte-Specific Enhancer Factor 2

Homeobox-containing genes play an essential role in basic processes during embryogenesis and development, but little is known about the regulation of their expression. To elucidate regulatory networks that govern homeobox gene expression, we defined the core promoter of the mouse Gax homeobox gene and characterized its interactions with cellular proteins. Transient transfection experiments revealed Gax promoter activity in several cell types. Deletion analysis defined a 138-bp minimal promoter fragment between positions -125 and +13 relative to the transcription initiation site. Mutagenesis and protein-DNA binding assays suggested that at least three positive factors interact with this fragment and are required for transcriptional activity. One of these factors, HRF-1, recognizes a cis element consisting of an inverted palindromic motif. A second factor is Sp1, that binds to a G/C-rich element. The third is the MADS box factor referred to as MEF2 or RSRF. Mutations in the MEF2/RSRF site had the greatest effect on transcription in cell types that expressed the highest levels of endogenous MEF2 activity. Conversely, overexpression of MEF2A transactivated the Gax promoter more efficiently in cells lacking endogenous MEF2. These data provide evidence for a direct transcriptional link between members of the MADS and homeobox families of transcription factors.