Interactions between p300 and multiple NF-Y trimers govern cyclin B2 promoter function

The CCAAT box is one of the most common elements in eukaryotic promoters and is activated by NF-Y, a conserved trimeric transcription factor with histone-like subunits. Usually one CCAAT element is present in promoters at positions between -60 and -100, but an emerging class of promoters harbor multiple NF-Y sites. In the triple CCAAT-containing cyclin B2 cell-cycle promoter, all CCAAT boxes, independently from their NF-Y affinities, are important for function. We investigated the relationships between NF-Y and p300. Chromatin immunoprecipitation analysis found that NF-Y and p300 are bound to the cyclin B2 promoter in vivo and that their binding is regulated during the cell cycle, positively correlating with promoter function. Cotransfection experiments determined that the coactivator acts on all CCAAT boxes and requires a precise spacing between the three elements. We established the order of in vitro binding of the three NF-Y complexes and find decreasing affinities from the most distal Y1 to the proximal Y3 site. Binding of two or three NF-Y trimers with or without p300 is not cooperative, but association with the Y1 and Y2 sites is extremely stable. p300 favors the binding of NF-Y to the weak Y3 proximal site, provided that a correct distance between the three CCAAT is respected. Our data indicate that the precise spacing of multiple CCAAT boxes is crucial for coactivator function. Transient association to a weak site might be a point of regulation during the cell cycle and a general theme of multiple CCAAT box promoters.