*4.1. Role of NF-YA Alternative Splicing in Muscle Cells*

Specific isoforms of TFs have long been known to impact heavily on transcriptional regulation. Paradigmatic examples are the members of the p53/p63/p73 families, whose isoforms, produced by multiple promoters and alternative splicing, have different targets and often opposing transcriptional effects [55]. The muscle system is no exception [56,57]. Mef2C and Mef2D undergo alternative splicing during muscle differentiation [57,58]: a muscle-specific isoform of Mef2D contains exon α2 rather than α1, both expressed in muscle cells. Growing and early differentiating cells harbors MEF2Dα1; the switch to MEF2Dα2 occurs in terminal stages of C2C12 differentiation, leading to activation of late genes. MEF2Dα1 is phosphorylated at two serines by PKA [59], which mediate association with HDACs, resulting in repression. MEF2Dα2 lacks these residues, functioning as a transcriptional activator. Parallel molecular mechanisms appear to be operating for the related MEF2Cα1/α2 alternative splicing isoforms [58]. The key issue in Mef2 splicing regulation is involvement in late stages of differentiation. Alternative splicing was reported for the master TFs of muscle commitment PAX3 and PAX7, but the functional roles of the single isoforms are less well characterized [60–64].

We show here that a switch from NF-YAl to NF-YAs causes a major difference in the differentiation properties of C2C12 cells. The major NF-YA isoforms, originally reported decades ago [32], are only recently attracting the attention they deserve. In part, this was due to the elusive logic of their expression patterns: in some systems, cells have NF-YAs before—and NF-YAl after—differentiation; in others, such as in muscle cells, NF-YAl is mostly found. In part, it was because of the rather unimpressive nature of the exon 3 amino acids incorporated into NF-YAl: a short stretch rich in glutamines and hydrophobic residues amid the larger transactivation domain. Overexpression experiments suggested differences in gene activation [39,65], but these experiments are to be taken with a grain of salt, because of the large amount of proteins produced, targeting the large number of potential NF-Y sites in the genome. NF-YA AS is likely more complex than what is shown here. First, NF-YAx is another alternatively spliced isoform, recently reported in glioblastomas, devoid of exons 3 and 5: this greatly reduces the activation domain, with important functional consequences [66]. Expression of NF-YAx will have to be monitored in normal cells, to verify whether it is specific for glioblastomas. Second, there are micro differences—6 amino acids—produced in many cell types within the acceptor site of exon 5. Third, some cells show the inclusion of an additional Gln residue at the acceptor splicing site of exon 3, producing a 29 amino acids insertion [32]. Note that a similar situation was reported for PAX3, in which an extra Gln causes differences in DNA-binding affinity [59]. Precise editing techniques, as we have started to use here in C2C12 cells, could sort out the functionality of the various isoforms.

#### *4.2. NF-Y Does Not Target Directly Genes Involved in C2C12 Di*ff*erentiation*

Sequence-specific TFs target specific genomic sites, driven by the discriminatory power of their DNA-binding Domains. However, they are also known to be binding indirectly, being tethered by other TFs or complexes: analysis of genomic locations by ENCODE has shown that this latter mechanism is far from marginal [67]. In addition to ENCODE, several independent ChIP-seq of TFs—and cofactors—identified binding to CCAAT locations [68]. One such example regards the orphan receptor Rev-Erb, important for muscle regeneration, targeting NF-Y sites in C2C12 cells [69]. The reverse, namely NF-Y being tethered to CCAAT-less locations by other TFs, has yet to be described. The issue could theoretically be relevant, since the genes down-regulated after NF-Y removal, or by switching from NF-YAl to NF-YAs, have generally no CCCAT in promoters. The effects appear to be largely indirect, but we do not favor the promoter tethering hypothesis. Rather, we report binding of Myogenin and MyoD to the promoters of Myomaker and Myomixer and show that Myogenin overexpression leads to recovery of their expression in C2-YAl-KO cells. This extends to mouse cells

genetic experiments made in zebrafish [53]. It also indicates that NF-Y does not regulate other TFs essential for expression of these two genes. In summary, NF-Y/CCAAT interactions in promoters, which are structurally identical for NF-YAl and NF-YAs, are likely not crucial for genes induced during myotubes formation: rather, the focus is shifted to the control of MRFs, or other TFs.

We have analyzed expression of TFs involved in myoblast/C2C12 differentiation. The majority are not dramatically altered in edited clones. Mef2C induction is impaired, but previous studies indicated that NF-Y is bound to the Mef2D, not to Mef2C promoter [39]. We find that Mef2C, not Mef2D, is regulated by NF-YB RNAi interference. Note that these TFs are also targeted by MyoD and Myogenin, as they play a role in the final stages of differentiation [7,59]. This suggests indirect regulation by NF-Y via MRFs. Id1/Id3 do have bona fide functional CCAAT in promoters [70], bound in cancer cells as per ENCODE data (M. Ronzio, A.B., D.D., R.M., in preparation) and in NTera2 cells [71]: Id1, but not Id3, is bound in vivo by NF-Y in C2C12, parental cells and edited clones. The levels are decreased in C2-YAl-KO upon differentiation, but NF-Y-inactivation brings very marginal decrease in Id1 expression. PAX3, which acts upstream of MyoD, shows variable, somewhat increased mRNA levels in the edited clones, but this is not supported by analysis of protein levels. In summary, there is no clear CCAAT-driven TF that could explain the phenotype: instead, we propose that the decrease of Myogenin and MyoD expression entails a cascade of transcriptional events leading to failure of differentiation (Figure 7).

**Figure 7.** NF-YA isoforms involvement in regulation of expression of muscle genes. Model for NF-YA isoforms mediated regulation of expression of muscle genes in growth condition (left panel) and differentiation condition (right panel).

#### *4.3. NF-Y Regulates MRFs Expression Indirectly*

Switching from NF-YAl to NF-YAs—and NF-YB inactivation—negatively affects MRFs expression. Myf5 is moderately down in growing cells, remaining somewhat higher after differentiation. NF-Y-inactivation leads to a severe drop in Myogenin expression and a decrease of MyoD, which indicates an impact of NF-Y on their expression. The regulation appears to be transcriptional for Myogenin, not for MyoD, whose mRNA levels are variable, but overall similar. The Myogenin promoter is CCAAT-less and an indirect effect of NF-Y must be invoked. As for MyoD, the promoter harbors a high affinity NF-Y site, extremely conserved in evolution [54] and at the expected position (at -70 from TSS). Yet, NF-Y is not bound in vivo (Figure 6C). This is the only such example in nearly 200 promoters for which genetic analysis was reported [72]. The combination of an evolutionarily conserved, canonical CCAAT in a standard promoter position might function through NF-Y somewhen during the physiological activation of MyoD in development, while it has become expendable in the C2C12 system. Thus, down-regulation of MyoD in NF-YAs-expressing cells is also an indirect effect. It was proposed that MyoD serves as "pioneer" TF predisposing chromatin configurations for Myogenin to act as powerful activator of terminal differentiation genes and repressor of cell-cycle genes [73]. The latter function might be robustly counteracted by NF-YAs, but we have no evidence of that (Figure 2). It is now clear that the focus is set on transcriptional regulation of the MyoD and Myogenin units and on which activator TF(s)—or cofactor(s)—is under NF-YAl—but not NF-YAs—direct control. For the time being, the "candidate" TFs approach used here failed to offer a plausible explanation on how NF-YAl regulates MRFs expression, thereby muscle differentiation. We must resolve to more systematic analysis, such as RNA-seq, to identify potential NF-Y-mediated regulators in C2C12. In light of the low intrinsic levels of muscle-commitment by MRFs in C2-YAl-KO clones, such analysis could also shed light on the actual identity of these cells.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4409/9/3/789/s1, Figure S1: CRISPR/Cas9n system and ablation of NF-YA exon 3 in C2C12 cells. (A) Schematic representation of plasmids construction, following the Multiplex CRISPR/Cas9n Assembly System Kit protocol (Yamamoto lab) [40]. (B) Sequencing of the two C2-YAl-KO clones (#83, #117) compared to the control (ctr). Deleted sequence, targeted sequence and exon 3 sequence are highlighted. Figure S2: Cell-cycle analysis of C2-YAl-KO clones. Flow cytometry analysis of ctr C2C12 cells and the two C2-YAl-KO clones in GM condition. The analysis of three independent experiments and the average of percentage of cells in each cell-cycle phase are shown. Figure S3: Gene expression analysis of TFs in growing and differentiated C2C12 cells. Gene expression analysis by RT-qPCR of key muscle differentiation regulators (left panel) and other TFs shown to be important for muscle differentiation (right panel) in GM condition and 24 h after differentiation in C2C12 ctr cells. Error bars represent the SD of three different experiments. P-values were calculated using the one-sample t-test. Figure S4: Myomaker and Myomixer expression are regulated by MyoD and Myogenin. (A) UCSC view of Mymk and Mymk *loci* showing alignment of ChIP-Seq data and DNA regulatory motifs conserved across Vertebrates by PhastCons. (B) Phase-contrast analysis of C2C12 cells (ctr and #83) morphology transfected with pEmpty or pMyog, 96 h after differentiation. Figure S5: Analysis of NF-Y involvement in muscle specific genes expression. (A) Gene expression analysis by RT-qPCR of NF-YB and key muscle differentiation regulators in C2C12 cells 72 h after NF-YB silencing (siNF-YB) and the scrambled siRNA control (II◦ experiment). Error bars represent the SD of two different q-PCR replicates. *p*-values were calculated using the one-sample t-test. (B) Western blot analysis of NF-YB, NF-YA and key muscle differentiation regulators (Myogenin, MyoD) protein levels 72 h after NF-YB silencing (siNF-YB) and the scrambled control. Vinculin was used as loading control. (C) Analysis of II◦ and III◦ ChIP experiments performed on C2C12 ctr cells in GM condition using NF-YB antibody and the negative control (nc). The unrelated region (ur) and Stard4 were used as negative and positive control, respectively. Results are represented as the input percentage of sample normalized to the nc. Table S1: Off-targets analysis. Analysis of possible off-target sites of each gRNA using the online tool https://crispr.cos.uni-heidelberg.de. For each gRNA the off-target gene name, gene id, position (intronic, intergenic, exonic), mismatches (MM) and the PAM sequence are reported. Table S2: Primers used. The specific sequence of each primer (forward and reverse) used for RT-qPCR and ChIP analysis are reported.

**Author Contributions:** D.L.: investigation, formal analysis and visualization; A.B.: data curation and methodology; S.S.: investigation; G.M.: resources, writing (review and editing); D.D.: methodology, data curation, writing (review and editing); R.M.: supervision, funding acquisition, writing (Original draft and editing). All authors have read and agreed to the published version of the manuscript.

**Funding:** The work was supported by AFM Téléthon Trampoline Grant (n.16408), AIRC Grant (n.19050) and PRIN 2015 to R.M. and AFM Téléthon Research Grant (n. 18364) to G.M.

**Acknowledgments:** The authors would like to thank prof. Carol Imbriano (Università degli Studi di Modena e Reggio Emilia) for providing antibodies against Myod and Myf5.

**Conflicts of Interest:** The authors declare no conflicts of interest.
