REGULATION OF THE HUMAN ALPHA-2(1) PROCOLLAGEN GENE BY SEQUENCES ADJACENT TO THE CCAAT

The human, rat, mouse and chicken alpha 2(I) procollagen promoters ana lysed to date all contain an inverted CCAAT box at - 80. In this study we have examined the binding of nuclear proteins to the proximal prom oter of the human alpha 2(I) procollagen gene, where an inverted CCAAT box is flanked by a downstream GGAGG sequence and its inverted counte rpart (CCTCC) on the upstream end. Each of the GGAGG sequences is sepa rated 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 thi s DNA sequence. Methylation interference analysis and in vitro mutagen esis studies revealed that the integrity of the sequence 5'-CCTCCCATTG G-3' (the GGAGG/CCAAT-binding element or G/CBE) was important for the binding of the CCAAT-binding factor (CBF) (complex I). Competition stu dies showed that complex formation on the human G/CBE could be compete d by mouse CBE and nuclear factor-Y (NF-Y) oligonucleotides, suggestin g that mouse CBE and human G/CBE-binding proteins belong to the same f amily 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 fo rmation of a novel DNA-protein complex (complex III). The formation of this complex was not competed out by CBE or NF-Y oligonucleotides, no r was DNA-protein complex formation affected by the anti-CBF antibody. Functional analysis of G/CBE and CME elements subjected to mutagenesi s, using promoter-chloroamphenicol acetyl transferase constructs in tr ansient transfection assays, showed that both these elements were esse ntial for activity of tile human promoter. These experiments identifie d a novel regulatory element in the human alpha 2(1) procollagen gene which is not present in the rodent gene.