Purification and characterization of a rat liver protein that recognizes CCAAT-homologous sequences of the metallothionein promoter and trans-activates this promoter.
C'BP-1, a protein that binds to the MRE-c' region (-135 to -110) of the mouse metallothionein-I (MT-I) gene in metal-independent manner, was purified from rat liver nuclear extract by ion exchange and affinity chromatography. Analysis by SDS-PAGE, UV cross-linking, and glycerol gradient sedimentation, taken together, showed that C'BP-1 is a dimer of the 34-kDa polypeptides. Affinity-purified C'BP-1 could significantly stimulate transcription from mouse MT-I gene promoter. DNase I footprinting with the purified protein identified two binding sites for C'BP-1 located at positions -135 to -100 and -210 to -175 with respect to the start site of MT-I gene transcription. Both C'BP-1 binding sequences were found to contain imperfect dyad of the CCAAT homology. C'BP-1 was shown to make critical contacts with the CCAAT homology by methylation interference analysis and competition electrophoretic mobility shift assay with mutants harboring alterations in the CCAAT homology. An antibody that specifically recognizes C/EBP delta partially supershifted C'BP-1/MRE-c' complex, suggesting that C'BP-1 is identical to C/EBP delta or is closely related to C/EBP delta.
Aniskovitch
L P
LP
Department of Pharmacology and Molecular Biology, Chicago Medical School, Illinois 60064, USA.
Jacob
S T
ST
D000818:Animals; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D016384:Consensus Sequence; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000276:immunology; D008099:Liver / Q000737:chemistry*; D008114:Liver Neoplasms, Experimental / Q000737:chemistry; D008668:Metallothionein / Q000235:genetics*; D051379:Mice; D009363:Neoplasm Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D009687:Nuclear Proteins / Q000276:immunology; D011401:Promoter Regions, Genetic; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D015534:Trans-Activators / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D015533:Transcriptional Activation
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D015534:Trans-Activators; D008668:Metallothionein