Regulation of the human alpha 2(1) procollagen gene by sequences adjacent to the CCAAT box

The human, rat, mouse and chicken alpha 2(1) procollagen promoters analysed to date all contain an inverted CCAAT box at -80. In this study we have examined the binding of nuclear proteins to the proximal promotor of the human alpha 2(1) procollagen gene, where an inverted CCAAT box is flanked by a downstream GGAGG sequence and its inverted counterpart (CCTCC) on the upstream end. Each of the GGAGG sequences is separated from the inverted CCAAT box by a single pyrimidine nucleotide (5'-CCTCCCATTGGTGGAGGCCCTTTT-3'). Electrophoretic mobility-shift assays (EMSAs) revealed that two distinct DNA-protein complexes formed on this DNA sequence. Methylation interference analysis and in vitro mutagenesis studies revealed that the integrity of the sequence 5'-CCTCCCATTGG-3' (the GGAGG/CCAAT-binding element or G/CBE) was important for the binding of the CCAAT-binding factor (CBF) (complex I). Competition studies showed that complex formation on the human G/CBE could be competed by mouse CBE and nuclear factor-Y (NF-Y) oligonucleotides, suggesting that mouse CBE and human G/CBE-binding proteins belong to the same family of CCAAT box binding proteins. Furthermore, antibodies to mouse CBF specifically supershifted the G/CBE complex (complex I) in EMSAs. The downstream GGAGG and 3'-flanking sequences (5'-GGAGGCCCTTTT-3') or collagen modulating element (CME), however, were important for the formation of a novel DNA protein complex (complex III). The formation of this complex was not competed out by CBE or NF-Y oligonucleotides, nor was DNA-protein complex formation affected by the anti-CBF antibody. Functional analysis of G/CBE and CME elements subjected to mutagenesis, using promoter-chloroamphenicol acetyl transferase constructs in transient transfection assays, showed that both these elements were essential for activity of the human promoter. These experiments identified a novel regulatory element in the human alpha 2(1) procollagen gene which is not present in the rodent gene.