NF-YA enters cells through cell penetrating peptides.

Cell Penetrating Peptides -CPPs- are short aminoacidic stretches present in proteins that have the ability to translocate the plasma membrane and facilitate delivery of various molecules. They are usually rich in basic residues, and organized as alpha helices. NF-Y is a transcription factor heterotrimer formed by two Histone Fold Domain -HFD- subunits and the sequence-specific NF-YA. NF-YA possesses two α-helices rich in basic residues. We show that it efficiently enters cells at nanomolar concentrations in the absence of carrier peptides. Mutagenesis identified at least two separate CPPs in the A1 and A2, which overlap with previously identified nuclear localization signals (NLS). The half-life of the transduced protein is short in human cancer cells, longer in mouse C2C12 myoblasts. The internalized NF-YA is capable of trimerization with the HFD subunits and binding to the target CCAAT box. Functionality is further suggested by protein transfection in C2C12 cells, leading to inhibition of differentiation to myotubes. In conclusion, NF-YA contains CPPs, hinting at novel -and unexpected- properties of this subunit.

LTR12 promoter activation in a broad range of human tumor cells by HDAC inhibition.

A considerable proportion of the human genome consists of transposable elements, including the long terminal repeats (LTRs) of endogenous retroviruses. During evolution, such LTRs were occasionally inserted upstream of protein-coding genes, contributing to their regulation. We previously identified the LTR12 from endogenous retrovirus 9 (ERV9) as a regulator of proapoptotic genes such as TP63 or TNFRSF10B. The promoter activity of LTR12 is largely confined to the testes, silenced in testicular carcinoma, but reactivated in testicular cancer cells by broad-range histone deacetylase (HDAC) inhibitors. Here we show that inhibition of HDAC1-3 is sufficient for LTR12 activation. Importantly, HDAC inhibitors induce LTR12 activity not only in testicular cancer cells, but also in cells derived from many additional tumor species. Finally, we characterize the transcription factor NF-Y as a mediator of LTR12 promoter activity and HDAC inhibitor-induced apoptosis, in the context of widespread genomic binding of NF-Y to specific LTR12 sequences. Thus, HDAC inhibitor-driven LTR12 activation represents a generally applicable means to induce proapoptotic genes in human cancer cells.

NF-Y activates genes of metabolic pathways altered in cancer cells.

The trimeric transcription factor NF-Y binds to the CCAAT box, an element enriched in promoters of genes overexpressed in tumors. Previous studies on the NF-Y regulome identified the general term metabolism as significantly enriched. We dissect here in detail the targeting of metabolic genes by integrating analysis of NF-Y genomic binding and profilings after inactivation of NF-Y subunits in different cell types. NF-Y controls de novo biosynthetic pathways of lipids, teaming up with the master SREBPs regulators. It activates glycolytic genes, but, surprisingly, is neutral or represses mitochondrial respiratory genes. NF-Y targets the SOCG (Serine, One Carbon, Glycine) and Glutamine pathways, as well as genes involved in the biosynthesis of polyamines and purines. Specific cancer-driving nodes are generally under NF-Y control. Altogether, these data delineate a coherent strategy to promote expression of metabolic genes fuelling anaerobic energy production and other anabolic pathways commonly altered in cancer cells.