Purification and functional characterization of a DNA-binding protein that interacts with a negative element in the mouse α1(I) collagen promoter

Gerard Karsenty, Roberto Ravazzolo, Benoit De Crombrugghe

Research output: Contribution to journalArticlepeer-review

Abstract

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 α1(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 α1(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 α1(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.

Original languageEnglish
Pages (from-to)24842-24848
Number of pages7
JournalJournal of Biological Chemistry
Volume266
Issue number36
Publication statusPublished - Dec 25 1991

ASJC Scopus subject areas

  • Biochemistry

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