Recombinant rat CBF-C, the third subunit of CBF/NFY, allows formation of a protein-DNA complex with CBF-A and CBF-B and with yeast HAP2 and HAP3.
The CCAAT binding factor CBF is a heteromeric transcription factor, which binds to functional CCAAT motifs in many eukaryotic promoters. cDNAs for the A and B subunits of CBF (CBF-A and CBF-B) and for their yeast homologues HAP3 and HAP2 have been previously isolated, but the purified recombinant CBF-A and CBF-B together are unable to bind to CCAAT motifs in DNA. Here we report the isolation of a cDNA coding for rat CBF-C, demonstrate that recombinant CBF-C is required together with CBF-A and CBF-B to form a CBF-DNA complex, and show that CBF-C is present in this protein-DNA complex together with the other two subunits. We further show that CBF-C allows formation of a complex between the purified recombinant yeast HAP2 and HAP3 polypeptides and a CCAAT-containing DNA and is present in this complex, implying the existence of a CBF-C homologue in yeast. We show that CBF-A and CBF-C interact with each other to form a CBF-A-CBF-C complex and that CBF-B does not interact with CBF-A or CBF-C individually but that it associates with the CBF-A-CBF-C complex. Our results indicate that CBF is a unique evolutionarily conserved DNA binding protein.
Sinha
S
S
Department of Molecular Genetics, University of Texas, M.D. Anderson Cancer Center, Houston 77030.
Maity
S N
SN
Lu
J
J
de Crombrugghe
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D023081:CCAAT-Binding Factor; D003001:Cloning, Molecular; D050660:Core Binding Factors; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins; D012045:Regulatory Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
D023081:CCAAT-Binding Factor; D050660:Core Binding Factors; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C490431:HAP3 protein, S cerevisiae; D046911:Macromolecular Substances; D009363:Neoplasm Proteins; D009838:Oligodeoxyribonucleotides; D010455:Peptides; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors