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

ROLE OF NF-YA ISOFORMS IN MOUSE EMBRYONIC STEM CELLS AND MYOBLASTS DIFFERENTIATION

The Nuclear Factor Y (NF-Y) is a Transcription Factor (TF) composed by three different subunits, NF-YA, NF-YB and NF-YC, all necessary to recognize and bind the CCAAT box, a DNA- regulatory region highly enriched in active enhancers in human and mouse Embryonic Stem cells (ESCs). NF-YB and NF-YC subunits have a histone-like domain (related to core histones H2B and H2A respectively) that associate forming a dimer required to interact with the NF-YA subunit. In particular, NF-YA has an evolutionary conserved domain in the C-terminal part responsible for NF-YB/NF-YC interaction and DNA binding, while the N-terminal domain is characterized by a glutamine rich (Q- rich) trans-activation domain. NF-YA comes in two isoforms, the long (NF- YAl) and the short (NF-YAs), differing for 28 amino acids coded by exon 3. Mouse ESCs preferentially express the short isoform, but after differentiation by Embryoid bodies (EBs) formation, a complete switch from the short to the long isoform is observed. Moreover, the progenitor muscle C2C12 cells express the long isoform, but after complete differentiation into muscle tissue, NF-YA protein is not detected. The first project of my PhD thesis aims to shed light on the function of NF-YAs and NF-YAl in controlling cell differentiation by generating mES and C2C12 cell lines with a genomic deletion of exon 3 through the CRISPR/Cas9 Nickase system. Two isolated homozygous clones for each cell line were obtained and they expressed only the NF-YA short isoform (NF-YAl-KO), as expected. Both cell lines, after exon 3 deletion, maintained the same morphology of wild type cells, but after differentiation stimuli, NF-YAl-KO clones and wild type cells showed different responses. In mESCs, NF-YAl-KO clones maintained the typical stem cell morphology with high levels of stemness genes and low levels of differentiation markers, compared to the wild type cells. In C2C12, after differentiation induction, wild type cells originated the classical myotubes, while the NF-YAl-KO clones maintained the myoblast identity. These results were supported by a low expression levels of muscle-associated genes in NF-YAl-KO clones, compared to wild type cells. These data confirm a different role of the two NF-YA isoforms in stemness maintenance and in particular suggest that the long one has a pivotal role during the differentiation process. The second project has been inspired by the study of NF-YA protein 3D structure that highlighted the presence of two features typical of CPPs in the evolutionary conserveddomain. Previous studies have shown that the GST-TAT-NF-YA short fusion protein stimulated Hematopoietic Stem cells (HSCs) growth enhancing cell proliferation. Moreover, mESCs transfected with the GST-TAT-NF-YA short fusion protein maintained their pluripotency identity even after Leukemia Inhibitory Factor (LIF) withdrawal, which is necessary for mESCs to maintain stemness identity in culture media. The work presented in this thesis demonstrated the NF-YA capability to enter cells and to translocate into nuclei in a TAT- independent manner. Moreover, the differentiation induction of myoblast C2C12 cells after NF-YA short recombinant protein transfection inhibited myotubes formation demonstrating the functionality of the transduced recombinant protein