The MAGED1 promoter is activated during myogenic differentiation and contains two E-boxes required for MyoD-dependent transcription. (A) Three promoter fragments of the human MAGED1 gene were amplified using PCR and inserted upstream from the firefly Luc reporter gene into pGL3-basic. Regions of the promoter conserved in mammals are boxed. E-box consensus sequences are indicated by vertical boxes (black if conserved in mammals). Coordinates are shown with respect to the first nucleotide of the MAGED1 transcript. (B) The 3 reporter constructs (pMAGED1-1977+80-luc, pMAGED1-971+80-luc, pMAGED1-531+80-luc) were used to transfect C2C12 myoblasts. Twenty-four hours after transfection, cells were shifted to DM and cultivated for another 24 hours before luciferase activity quantification. Firefly luciferase activity was normalized by the Renilla luciferase activity specified by a Renilla luciferase expression plasmid (pRL-CMV) co-transfected with the MAGED1-luc constructs. Results are presented as relative luciferase activity with respect to the activity of the MAGED1-1977+80-luc construct at the time of serum starvation (t = 0 h). (C) Effect of mutation of the -227 and -118 E-boxes on the luciferase activity of pMAGED1-531+80-luc. Experimental procedure was the same as in B. (D) Effect of MyoD on the promoter activity of the -531+80 fragment with intact or mutated -227 and -118 E-boxes. 3T3 fibroblasts were transfected with the MAGED1-luc constructs with or without the MyoD expression plasmid pEMSV-MyoD. Results are presented as relative luciferase activities with respect to the activity of the promoter fragments in absence of MyoD.