Multiple proteins are involved in the protein-DNA complex in the proximal promoter of the human α1(III) collagen gene (COL3A1)

We have characterized the proximal promoter of the human α1(III) collagen gene (COL3A1). Transient transfection assays using a series of chimeric constructs linked to the luciferase gene indicated that the segment from −96 to −34 is necessary to activate transcription. Electrophoretic mobility shift assays (EMSAs) showed that the multiple proteins form the DNA-protein complex in different combinations depending on the cell types. A competition assay using mutant oligonucleotides showed that the sequence 5′-GCTCTCATATTTCAGAA-3′ (−79 to −63 bp) is critical for DNA-protein complex formation. This sequence is contained in the B element of mouse α1(III) collagen gene (Col3a1) reported by Ruteshouse and de Crombrugghe (J. Biol. Chem., 1993). In the rhabdomyosarcoma cell line, A204, at least two proteins of 92-118 kDa and 40-52 kDa are involved in the DNA-protein complex bound to this motif.