Two different negative and one positive regulatory factors interact with a short promoter segment of the alpha 1 (I) collagen gene.
Type I collagen, a heterotrimeric protein composed of two alpha 1 chains and one alpha 2 chain, is a major specialized biosynthetic product of fibroblastic cells. We performed a functional dissection of a mouse alpha 1 (I) collagen promoter segment (between -222 and -80) that displays strong activity in vitro and in DNA transfection experiments. Four binding sites for factors present in nuclear extracts of NIH-3T3 fibroblasts were identified in this promoter segment. One factor, which has the same binding characteristics as CBF, a heterodimeric CCAAT binding factor that also binds and activates the coordinately expressed alpha 2(I) collagen promoter, interacts with the proximal of two CCAAT motifs. A second factor, designated IF1, binds to two more upstream, adjacent sites (-190 to -170 and -160 to -130). A third factor, designated IF2, makes contact with several G residues in the distal unit of a 12-base pair exact G-rich repeat that brackets the proximal CCAAT motif. Binding studies with mutant oligonucleotides and experiments with purified CBF indicate that the binding of IF2 is inhibited by CBF. IF2 is a metalloprotein that requires zinc cations for efficient binding to its recognition site. DNA transfection experiments using point mutations or small substitution mutations that abolish binding of the transacting factors to their cognate elements were performed. A mutation in the proximal IF1 binding site increases promoter activity 4-fold, a mutation in the IF2 binding site increases this activity 10-fold and a mutation in the CBF binding site decreases this activity 4-5-fold. This suggests that IF1 and IF2 act as transcriptional inhibitors whereas CBF acts as an activator of the alpha 1(I) collagen promoter. We propose that mutually competitive binding of IF2 and CBF could play a role in the control of the alpha 1(I) collagen promoter.
Karsenty
G
G
Department of Molecular Genetics, University of Texas M.D. Anderson Cancer Center, Houston 77030.
de Crombrugghe
B
B
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D003094:Collagen / Q000235:genetics*; D004247:DNA / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I; D005796:Genes; D007700:Kinetics; D046911:Macromolecular Substances; D008745:Methylation; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D009154:Mutation; D009710:Nucleotide Mapping; D015345:Oligonucleotide Probes; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D003094:Collagen; D004247:DNA; D003850:Deoxyribonuclease I