The Switch from NF-YAl to NF-YAs Isoform Impairs Myotubes Formation.

NF-YA, the regulatory subunit of the trimeric transcription factor (TF) NF-Y, is regulated by alternative splicing (AS) generating two major isoforms, "long" (NF-YAl) and "short" (NF-YAs). Muscle cells express NF-YAl. We ablated exon 3 in mouse C2C12 cells by a four-guide CRISPR/Cas9n strategy, obtaining clones expressing exclusively NF-YAs (C2-YAl-KO). C2-YAl-KO cells grow normally, but are unable to differentiate. Myogenin and-to a lesser extent, MyoD- levels are substantially lower in C2-YAl-KO, before and after differentiation. Expression of the fusogenic Myomaker and Myomixer genes, crucial for the early phases of the process, is not induced. Myomaker and Myomixer promoters are bound by MyoD and Myogenin, and Myogenin overexpression induces their expression in C2-YAl-KO. NF-Y inactivation reduces MyoD and Myogenin, but not directly: the Myogenin promoter is CCAAT-less, and the canonical CCAAT of the MyoD promoter is not bound by NF-Y in vivo. We propose that NF-YAl, but not NF-YAs, maintains muscle commitment by indirectly regulating Myogenin and MyoD expression in C2C12 cells. These experiments are the first genetic evidence that the two NF-YA isoforms have functionally distinct roles.

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.