MODULATION OF DNA-BINDING IN TRANSCRIPTION FACTORS: THE CASE OF NF-Y AND USF1

Transcription is ultimately driven by DNA-binding events involving regulatory proteins known as transcription factors (TFs). NF-Y is a ubiquitous eukaryotic TF composed by three different subunits, all necessary to specifically recognize and bind CCAAT-box DNA. NF-Y plays a central role in shaping the architecture of promoters and distal regulatory regions, both by influencing local chromatin environment and working as a pioneering binding-platform for several TFs in different genomic contexts. Still, the way NF-Y activity itself is modulated, and the molecular determinants at the basis of NF-Y function as transcriptional organizer pose many open questions. The aims of my doctoral thesis were, in general, to provide new structure-function correlations of NF-Y activity, and in specific to (I) uncover the molecular role of two NF-YA subunit phosphorylation sites and (II) dissect the molecular interplay between NF-Y and the E- box TF USF1 on different DNA configurations. NF-YA was shown to be a cell-cycle modulated phospho-protein, with two C-terminal residues (Ser320 and Ser326), located close to the conserved DNA-binding domain of the subunit, targeted as phosphorylation sites by CDK2. Still, their molecular function remained elusive. We designed a panel of NF-YA Ser320 and Ser326 phospho-ablative (Ser to Ala) and phospho- mimicking (Ser to Glu) mutants, in order to assess the phosphorylation state of the protein in vivo, and to characterize their behaviour in DNA-binding and transcriptional assays. First, we detected Ser320-P as the major NF-YA phospho-isotype in asynchronous HeLa cells, strongly increasing upon nocodazole-induced mitotic arrest. Second, we found that while none of the phospho-mutant recombinant proteins affected the overall affinity of the NF-Y heterotrimer for DNA, Ser320 (but not Ser326) mutations kinetically destabilized the DNA-bound complex, greatly increasing its off- rate. Third, Ser320 phospho-mutants impaired NF-Y dependent transcriptional activation of the RHOB and MDR1 target promoters in reporter assays. Taken together, our results point at Ser320- phosphorylation as a major regulatory switch for NF-Y activity, directly affecting its residence time on DNA. In the second project we aimed to dissect the USF1 protein regions which mediate DNA- binding cooperativity with NF-Y, in order to uncover the molecular bases of one of the genome- wide NF-Y-directed regulatory modules. We detected transcriptional synergy in reporter assays using four distinct target promoters characterized by the recurrent 10-12 bp spacing between E- box and CCAAT-box elements. Moreover, we identified the USR, an intrinsically disordered domain of USF1, as the region responsible for cooperative interaction with NF-Y on DNA. Notably, depending on the underlying DNA configuration of the binding sites, distinct portions of this context-specific transactivation domain mediate the protein-protein interactions.