DETERMINATION OF FUNCTIONAL DOMAINS IN THE C-SUBUNIT OF THE CCAAT-BINDING FACTOR (CBF) NECESSARY FOR FORMATION OF A CBF-DNA COMPLEX - CBF-BINTERACTS SIMULTANEOUSLY WITH BOTH THE CBF-A AND CBF-C SUBUNITS TO FORM A HETEROTRIMERIC CBF MOLECULE

The mammalian CCAAT-binding factor (CBF; also called NF-Y and CP1) is a heterotrimeric protein consisting of three subunits, CBF-A, CBF-B, a nd CBF-C, all of which are required for DNA binding and all of which a re present in the CBF-DNA complex. In this study using cross-linking a nd immunoprecipitation methods, we first established that CBF-B intera cts simultaneously with both subunits of the CBF-A-CBFG heterodimer to form a heterotrimeric CBF molecule. We then performed a mutational an alysis of CBF-C to define functional interactions with the other two C BF subunits and with DNA using several in vitro assays and an in vivo yeast two-hybrid system. Our experiments established that the evolutio narily conserved segment of CBF-C, which shows similarities with the h istone-fold motif of histone H2A, was necessary for formation of the C BF-DNA complex. The domain of CBF-C which interacts with CBF-A include d a large portion of this segment, one that corresponds to the segment of the histone-fold moth in H2A used for interaction with H2B. Two cl asses of interactions involved in formation of the CBF-A-CBF-C heterod imer were detected; one class, provided by residues in the middle of t he interaction domain, was needed for formation of the CBF-A-CBF-C het erodimer. The other, provided by sequences flanking those of the first class was needed for stabilization of the heterodimer. Two separate d omains were identified in the conserved segment of CBF-C for interacti on with CBF-B; these were located on each side of the CBF-A interactio n domain. Since our previous experiments identified a single CBF-B int eraction domain in the histone-fold motif of CBF-A, we propose that a tridentate interaction domain in the CBF-A-CBF-C heterodimer interacts with the 21-amino-acid-long subunit interaction domain of CBF-B. Toge ther with our previous mutational analysis of CBF-A (S. Sinha, L-S. Ri m, K.-Y. Sohn, B. de Crombrugghe, and S. N. Maity, Mol. Cell. Biol. 16 :328-337, 1996), this study demonstrates that the histone fold-motifs of CBF-A and CBF-C interact with each other to form the CBF-A-CBF-C he terodimer and generate a hybrid surface which then interacts with CBF- B to form the heterotrimeric CBF molecule.