title: Regulation of the human alpha 2(1) procollagen gene by sequences adjacent to the CCAAT box.
abstract: 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.
authors.affiliation: Department of Medical Biochemistry, University of Cape Town Medical School, South Africa.
authors.forename: M
authors.initials: M
authors.lastname: Collins
authors.forename: V D
authors.initials: VD
authors.lastname: Leaner
authors.forename: M
authors.initials: M
authors.lastname: Madikizela
authors.forename: M I
authors.initials: MI
authors.lastname: Parker
mesh_terms: D002461:Cell Line, Transformed; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics; D005347:Fibroblasts; D006801:Humans; D008168:Lung; D016297:Mutagenesis, Site-Directed; D011347:Procollagen / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid
chemical_list: D004268:DNA-Binding Proteins; D011347:Procollagen