BIOCHEMICAL AND FUNCTIONAL ANALYSIS OF NF-YA CELL PENETRATING PEPTIDE PROPERTIES IN DIFFERENT CELL SYSTEMS

1. Abstract The transcription factor NF-Y is a trimeric complex composed by three subunits NF-YA, NF-YB and NF-YC, which specifically recognizes and binds the CCAAT box sequence, present in almost 30% of eukaryotic promoters. NF-YB and NF-YC contain a histone fold domain, structurally related to H2A and H2B and they dimerize head to tail; NF-YA exists in two different isoforms and it is thought to be the regulatory subunit of the trimer. NF-Y is a key regulator of cell cycle progression and it regulates different metabolic pathways in proliferating cells. In the last years, it also emerged as important component of stemeness circuitry. The projects performed during my PhD were focused on the analysis and characterization of different biological and functional properties of NF-Y. My main project has been inspired by the inspection of NF-YA 3D structure that suggested us the presence, in the evolutionary conserved domain of the protein, of Cell Penetrating Peptide (CPP) features: the presence of two alpha helices and the abundance of arginines residues. Previous works used a functional recombinant GST-TAT-NFYA fusion protein (TAT is a well-known CPP), to transfer NF-YA in hematopoietic stem cells (HSCs), demonstrating its ability to repopulate the bone marrow of immunocompromized animals and, in mESCs, demonstrating its cruciality to maintain cell pluripotency. In this study, we demonstrated NF-YA capability to enter cells and translocate into the nuclei in a TAT-independent manner. Mutational analyses, in the evolutionary conserved domain of the protein, led to the identification of basic aminoacids required for NF-YA cellular uptake. Thanks to the stability of recombinant NF-YA, after transduction, in NIH3T3 and mESCs, we provided evidences about the functionality of the protein in vivo. In NIH3T3, NF-YA affects the expression of CCAAT cell cycle progression genes causing cell growth delay and induces the activation of the apoptotic pathway mediated by E2F1; mESCs, instead, after recombinant NF-YA transduction, maintain their pluripotency properties when grown without leukemia inhibitory factor (LIF). In the two last decades, the use of CPPs has become an important tool for therapeutic applications. In this context, the discovered NF-YA properties, to overpass cell membranes, could be exploited to make recombinant NF-YA a new reagent to study its biological effects in different cell contexts, avoiding limitations of canonical methods of gene delivery, and laying the groundwork for its potential application in clinical trials. The cruciality of NF-Y in mESCs pluripotency maintenance, and the possibility to use recombinant NF-YA in stem cell systems, pushed us to investigate NF-Y also in human stem cells. We reprogrammed adult human fibroblasts to induced pluripotent stem cells (iPSCs). Preliminary data suggest that, similarly to mESCs, NF-Y is involved in stemness circuitry regulation of human stem cells. Indeed, we observed a down modulation of master stemness genes after NF-Y subunits silencing in human iPSCs. The second project was focused on a detailed analysis on the regulation of NF-Y metabolic target genes, altered in cancer cells. By integrating different studies on NF-Y genomic binding with data of gene expression profiling, coming from different cell types, after NF-Y subunits inactivation, we demonstrated that NF-Y plays an important role in the regulation of genes involved in lipid pathways interacting with master lipogenic TFs, SREBPs. Moreover, we found that NF-Y directly and positively regulates genes involved in glycolysis, and in the biosynthesis of amino acids (serine, glycine and glutamine), purines and polyamines. Differently from its activity in yeast metabolic pathways, in mammalian cells NF-Y enhances energy production through anaerobic pathways; the analysis performed in this study, indeed, highlighted that NF-Y negatively regulates genes involved in TCA cycle. Most of metabolic genes regulated by NF-Y, identified in this study, are altered in different tumors. This work contributes to unravel the regulatory circuitry of cancer cells and to better understand how the alteration of metabolic pathways affects the progression and growth of cancer cells.