Transcriptional regulation is achieved by the finely orchestrated control of Transcription Factor (TFs) interactions and their recognition of target sequences on gene promoters and regulatory regions. NF-Y is a sequence specific TF composed of a Histone Fold Domain (HFD) subunits dimer, NF-YB/NF-YC, and a third regulatory subunit, NF-YA, which provides sequence specificity to the trimer for the recognition of CCAAT elements. In plants, the large expansion of NF-Y subunit genes have provided the means of functional diversification, although the molecular bases of their vast combinatorial potential in trimeric complexes, and their functional relevance, are yet to be understood. NF-Y genes are implicated in numerous processes in Arabidopsis and relevant crop species, including embryo development, responses to biotic and environmental signals, and regulation of flowering time. Several reports have also provided genetic and biochemical evidences of direct interactions of NF-Y subunits with CONSTANS, CO-Like, TOC1 (CCT) domain family of transcriptional regulators. Such interaction is based on the conserved CCT domain, which shares similarity with the NF-YA subunit homology domain.
We could recently show that the NF-Y HFD subunits can engage in a novel trimeric complex, termed NF-CO, by recruiting the master regulator of flowering CONSTANS (CO) to specifically recognise the CO-responsive (CORE) DNA element of the FT florigen gene promoter. Importantly, mutant alleles in the CCT domain which determine flowering time defects in Arabidopsis and rice, are functionally impaired in DNA-binding complex formation. These data represent the keystone of a unifying model whereby the NF-YB/NF-YC dimeric subunits can serve as a platform for multiple transcriptional regulators to recognise their relative cognate DNA element as trimeric units. Our main goal is to provide a detailed understanding of the shared features and specificities of the proposed NF-Y-based trimeric complexes which include CCT proteins impacting on flowering time, such as the circadian clock and vernalization. This project specific aims are primarily focused on the biochemical and molecular characterization of the trimerization properties of selected members of the CO-like and TOC1 subfamilies of CCT proteins, which have been shown to interact with NF-YB/NF-YC partners. Target DNA element(s) will be determined for each trimer with molecular genetics approaches by saturation mutagenesis of the CORE element bound by NF-CO. Structural determination of the DNA-bound NF-Y complex composed of canonical or divergent subunits, will be instrumental for understanding intrinsic properties of the plant TF, and the potential bases for functional diversification of the trimers. Crystallization of NF-CO/CORE complex(es) will be an added advantage for the establisment of the DNA binding strategies and trimerization properties of the TF, that we expect to be akin to the NF-Y trimer, although with substantial distinctions. The gained informations on bound elements, critical residues in DNA recognition and combinatorial specificities will be functionally validated in cell-based transcriptional assays by transient expression in mesophyll protoplasts. Promoter elements analysis and mutagenesis will also provide substantial evidences on the relative contribution of the distinct TFs on transcriptional regulation of relevant genes.
An additional aim pertaining the multimeric assembly of TF complexes is to gain insights into evolutionary conserved binary interactions of NF-Ys with a distinct familiy of bZIP TFs which synergise in regulation of stress response genes, associating on spatially arranged promoter elements. Detailed knowledge on the human partnership with the ER stress TF ATF6 allows us to extend our analyses on the plant bZIP28 homolog which parallels the functional cooperativity and direct interactions. For this objective, we will conduct molecular analyses on DNA-binding properties of T