Purification and functional characterization of a DNA-binding protein that interacts with a negative element in the mouse alpha 1(I) collagen promoter.
In an effort to understand the regulation of expression of the mouse type I collagen genes, we have purified to homogeneity a transcription factor, called inhibitory factor 2 (IF-2), that binds to the mouse alpha 1(I) collagen promoter upstream of the proximal CCAAT motif. IF-2 was purified to homogeneity from mouse lymphocyte nuclear extracts by ion-exchange chromatography and two different steps of DNA affinity chromatography, one using the wild-type IF-2-binding site as affinity ligand and one using a mutated IF-2-binding site that has a higher affinity for the factor. Renaturation of active DNA-binding proteins purified through several chromatography steps from sodium dodecyl sulfate-polyacrylamide gels identified two polypeptides of 120 and 100 kDa, respectively, capable of binding specifically to an IF-2-binding site. DNA transfection experiments of NIH 3T3 fibroblasts using an alpha 1(I) promoter-CAT chimeric gene in which mutations were introduced that either improved or decreased the binding affinity of IF-2 for its recognition site, strongly suggest that IF-2 acts as a transcriptional inhibitor of the mouse alpha 1(I) collagen gene. DNA-binding studies with similar concentrations of purified IF-2 and CBF (CCAAT-binding factor) indicate that CBF inhibits binding of IF-2, whereas IF-2 inhibits CBF binding more weakly.
Karsenty
G
G
Department of Molecular Genetics, University of Texas, M.D. Anderson Cancer Center, Houston 77030.
Ravazzolo
R
R
de Crombrugghe
B
B
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D002853:Chromatography, Liquid; D003094:Collagen / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D014162:Transfection
D004268:DNA-Binding Proteins; D014157:Transcription Factors; D003094:Collagen; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase