3 CLASSES OF MUTATIONS IN THE A-SUBUNIT OF THE CCAAT-BINDING FACTOR CBF DELINEATE FUNCTIONAL DOMAINS INVOLVED IN THE 3-STEP ASSEMBLY OF THECBF-DNA COMPLEX

The mammalian CCAAT-binding factor CBF (also called NF-Y or CP1) consi sts of three subunits, CBF-A, CBF-B, and CBF-C, all of which are requi red for DNA binding and present in the CBF-DNA complex, In this study we first established the stoichiometries of the CBF subunits, both in the CBF molecule and in the CBF-DNA complex, and showed that one molec ule of each subunit is present in the complex. To begin to understand the interactions between the CBF subunits and DNA, we performed a muta tional analysis of the CBF-h subunit, This analysis identified three c lasses of mutations in the segment of CBF-A that is conserved in Sacch aromyces cerevisiae and mammals. Analysis of the first class of mutant s revealed that a major part of the conserved segment was essential fo r interactions with CBF-C to form a heterodimeric CBF-A/CBF-C complex, The second class of mutants identified a segment of CBF-A that is nec essary for interactions between the CBF-A/CBF-C heterodimer and CBF-B to form a CBF heterotrimer. The third class defined a domain of CBF-A involved in binding the CBF heterotrimer to DNA. The second and third classes of mutants acted as dominant negative mutants inhibiting the f ormation of a complex between the wild-type CBF subunits and DNA. The segment of CBF-A necessary for DNA binding shelved sequence homology t o a segment of CBF-C, Interestingly, these sequences in CBF-A and CBF- C were also homologous to the sequences in the histone-fold motifs of histones H2B and H2A, respectively, and to the archaebacterial histone -like protein HMf-2. We discuss the functional domains of CBF-A and th e properties of CBF in light of these sequence homologies and propose that an ancient histone-like motif in two CBF subunits controls the fo rmation of a heterodimer between these subunits and the assembly of a sequence-specific DNA-protein complex.