Nuclear factor Y (NF-Y) is a transcription factor trimer binding to the functionally important CCAAT box, present in promoters of growth-promoting and cell cycle-regulated genes. The regulatory nuclear factor YA (NF-YA) subunit confers sequence-specificity to the histone-like nuclear factor YB/YC dimer. NF-YA harbors 2 serines-Ser320 and Ser326-shown to be phosphorylated by cyclin-dependent kinase 2. High-throughput proteomics data indicate that they are phosphorylated in vivo. Specifically, Ser320 makes structural contacts with the DNA phosphate backbone; Ser320-P is the major NF-YA phosphorylation isoform following overexpression in HeLa cells, increasing upon mitotic arrest. EMSA with recombinant Ala and Glu mutants confirm a role of Ser320, but not Ser326, in stabilization of DNA binding. Transactivation assays of the CCAAT-dependent MDR1 and RHOB promoters show loss in transcription function for Ser320Glu and Ser320Ala NF-YA mutants. Phylogenetic analysis of NF-YA proteins indicates that Ser320 is indeed evolutionarily conserved. We conclude that phosphorylation of this residue belongs to the core mechanisms of DNA-binding control, possibly driven by the necessity to unfasten binding of or to evict NF-Y from CCAAT sites under specific conditions of growth regulation.-Bernardini, A., Lorenzo, M., Nardini, M., Mantovani, R., Gnesutta, N. The phosphorylatable Ser320 of NF-YA is involved in DNA binding of the NF-Y trimer.