CHARACTERIZATION OF A GENOMIC CLONE COVERING THE STRUCTURAL MOUSE MYOD1 GENE AND ITS PROMOTER REGION

We have isolated the mouse MyoD1 gene flanked by its promoter region by screening a genomic library with synthetic oligonucleotides. The structural gene is interrupted by two G + C rich introns. Transfection of the cloned gene inserted into an expression vector converts fibroblasts to myoblasts. Sequence analysis of about 650 bp of the 5' upstream region revealed the presence of several potential regulatory elements such as a TATA-box, an AP2-box, two SP1-boxes and a CAAT-box. In addition, there are three half palindromic estrogen response elements, a potential cAMP response element and various muscle specific elements such as a muscle-specific CAAT-box (MCAT) and four potential binding sites for MyoD1. Using S1 protection analysis the major start site of transcription in muscle and myoblast cells was mapped 3 bp upstream of the published cDNA 5' end. Promoter activity of the 650 bp upstream fragment was tested by in vitro transcription and by transfection analysis of myoblasts and fibroblasts. In all promoter test systems used, MyoD1 promoter activity was detected in myoblasts as well as in fibroblasts. Furthermore, DNA methylation was found to turn off MyoD1 promoter activity both in myoblasts and in fibroblasts.