Distinct rat proteins can recognize CCAAT-homologous sequences of the metallothionein promoter and trans-activate this promoter

Abstract

We have previously purified and characterized a rat liver protein C′BP-1 that is either identical or closely related to C/EBPδ (). The mouse metallothionein-I (MT-I) promoter contains two C′BP-1 binding sites, one of which includes the MRE-c′ region (−135 to −110). The C′BP-1 binding activity was detected by EMSA as a major activity for MRE-c′ in nonproliferating adult liver cells but not in rat hepatoma cells. ln this study, we purified and characterized a factor, C′BP-2, which had a dominant binding activity for MRE-c′ in Morris hepatoma 3924A, a poorly differentiated, fast-growing tissue. C′BP-2 is a 28 kDa protein which exists in solution as a monomer. As observed for C′BP-1, affinity-purified C′BP-2 stimulated transcription from the mMT-I gene promoter. DNase I footprinting revealed two C′BP-2 binding sites in the regions that overlap with the CCAAT homologies of the C′BP-1 binding sites on the mMT-I promoter. C′BP-2 made essential contacts with the CCAAT homology and in the region upstream of this sequence. Competition electrophoretic mobility shift assay and methylation interference analysis revealed that C′BP-2 is a protein closely related, but not identical, to CP2. These data suggest that C′BP-1 and C′BP-2 may play a role in hepatocyte proliferation and/or differentiation.