CONSTANS Imparts DNA Sequence Specificity to the Histone Fold NF-YB/NF-YC Dimer

NF-Y is a heterotrimeric transcription factor that binds CCAAT elements. The NF-Y trimer is composed of a Histone Fold Domain (HFD) dimer (NF-YB/NF-YC) and NF-YA, which confers DNA sequence-specificity. NF-YA shares a conserved domain with the CCT (CONSTANS, CONSTANS-LIKE, TOC1) proteins. We show that CONSTANS (CO/BBX1), a master flowering regulator, forms a trimer with Arabidopsis NF-YB2/NF-YC3 to efficiently bind the CORE element of the FT promoter. We term this complex NF-CO. Using saturation mutagenesis EMSAs and RNA-Seq profiling of co, nf-yb, and nf-yc mutants, we identify CCACA elements as the core NF-CO binding site. CO physically interacts with the same HFD surface required for NF-YA association, as determined by mutations in NF-YB2 and NF-YC9, and tested in vitro and in vivo. The co-7 mutation in the CCT domain, corresponding to an NF-YA arginine directly involved in CCAAT recognition, abolishes NF-CO binding to DNA. In summary, a unifying molecular mechanism of CO function relates it to the NF-YA paradigm, as part of a trimeric complex imparting sequence-specificity to HFD/DNA interactions. It is likely that members of the large CCT family participate in similar complexes with At-NF-YB and At-NF-YC, broadening HFD combinatorial possibilities in terms of trimerization, DNA-binding specificities and transcriptional regulation.