The transcriptional activity of the CCAAT-binding factor CBF is mediated by two distinct activation domains, one in the CBF-B subunit and the other in the CBF-C subunit

CBF-A, CBF-B, and CBF-C together form the heterotrimeric mammalian CCAAT- binding factor, CBF, which binds to DNA to form a CBF-DNA complex. Here we examined the transcription activation function of CBF in an in vitro reconstituted system using the three purified recombinant CBF subunits expressed in Escherichia coli. Two of the subunits, CBF-A and CBF-C, were coexpressed and purified as a CBF-A/CBF-C complex. Addition of the three wild-type recombinant CBF subunits to EL4 cell nuclear extracts depleted of CBF stimulated transcription 5-20-fold from proα2(1) collagen promoters and 10-fold from the Rous sarcoma virus long terminal repeat. Two CBF deletion mutants, one containing full-length CBF-A and CBF-C and a CBF-B lacking the NH₂-terminal residues 1-224, and the other containing full-length CBF-A and CBF-B and a CBF-C lacking the COOH-terminal residues 114-309, also stimulated transcription from these promoters, but the level of activation was reduced to half that obtained with the full-length CBF subunits. In contrast, a CBF deletion mutant protein containing full-length CBF-A and deleted forms of both CBF-B and CBF-C showed very little transcription activation from these promoters. Hence, this study demonstrates that the heterotrimeric CBF protein consists of two transcription activation domains, one present in CBF-B and the other in CBF-C, and that the two domains act additively in the in vitro assay. The activation domains of both CBF-B and CBF-C, which are rich in glutamine and hydrophobic residues, showed amino acid sequence similarities with each other and with the glutamine-rich activation domain of transcription factor Sp1.