*3.2. Characterization of C2-YAl-KO Cells*

The two C2-YAl-KO clones were characterized first for expression of NF-YA. We performed qRT-PCR analysis with oligos specific for the individual isoforms [46]; Figure 2A shows that the NF-YAl mRNA is absent in the C2-YAl-KO clones. Extracts were prepared and Western blots performed: as expected, the parental C2C12 cells show expression of NF-YAl (Figure 2B). Instead, the clones express uniquely the NF-YAs isoform. We exposed the blots for long times to verify that no NF-YAl is visible in the two KO clones. Note that the levels of the two isoforms in parental cells—NF-YAl—and edited clones—NF-YAs—are essentially identical, as are the levels of NF-YB and NF-YC: since there is an important level of autoregulation among NF-Y subunits [47], this result indicates that HFD subunits are available for trimer formation and DNA-binding in C2C12 and C2-YAl-KO cells. In summary, genetic ablation of exon 3 in C2C12 was effective, leading to generation of clones that express uniquely the short isoform of NF-YA at physiological levels.

**Figure 2.** C2-YAl-KO clone characterization. (**A**) Gene expression analysis of NF-YA short and long levels in ctr and C2-YAl-KO clones (#83 and #117) in growth medium (GM) condition. Error bars represent the SD of three independent experiments. P-values were calculated using the one-sample t-test. (**B**) Western blot analysis of NF-Y protein subunits (NF-YA, NF-YB, NF-YC) in ctr cells and C2-YAl-KO clones (#83 and #117) in GM condition. For NF-YA isoforms analysis, short and long exposures are shown. Vinculin was used as loading control. (**C**) Phase contrast analysis of myoblast cells (ctr and C2-YAl-KO clones) morphology in GM condition. Scale bar 200 μm. (**D**) Proliferation assay performed in GM condition counting every 24 h for 3 days using the Trypan Blue dye exclusion test. Error bars represent the SD of three independent experiments. P-values were calculated using the one-way ANOVA test. (**E**) Gene expression analysis of key cell-cycle regulators in ctr and C2-YAl-KO clones (#83 and #117) in GM condition. Error bars represent the SD of three independent experiments. P-values were calculated using the one-sample t-test.

Next, we started to analyze the phenotype of the KO clones: they are stable upon repeated cycles of freezing and thawing and their morphology looks apparently similar to the parental C2C12 cells (Figure 2C). In mouse embryonic stem cells, expression of NF-YAs is associated with growth, and NF-YAl to differentiation [43]: in theory, NF-YAs-expressing C2C12 clones could be enhanced in proliferation. Cells were compared for growth under standard conditions: Figure 2D shows that

growth curves are similar, with the two edited clones being marginally slower. In FACS analysis, we did notice some differences: a higher number of S-phase and G2/M cells in the two clones (Figure S2, 21% and 28%, with respect to 18% in C2C12). We checked the mRNA levels of PCNA, Cyclin B1/B2: a slight increase of Cyclin B1 and PCNA in the KO clones is observed (Figure 2E); although not statistically significant, this is consistent with the FACS data. The most noticeable difference, however, was the lower number of sub-G1 cells: 6%–7% in the two clones compared to 12% in the parental C2C12 cells (Figure S2): such non cycling cells are possibly undergoing cell death, suggesting that the switch to NF-YAs is not provoking negative effects on cellular vitality, and, if anything, the opposite. In summary, C2-YAl-KO clones expressing NF-YAs have an apparently normal morphology, grow well, but not faster, with the expected partitioning in cell cycle phases, bar slightly elevated G2/M and decreased sub-G1 populations.

#### *3.3. C2-YAl-KO Cells Fail to Di*ff*erentiate and Fuse into Myotubes*

The levels of NF-YAl drop following terminal differentiation of C2C12 cells and myotubes of mouse muscles show low-to-nil levels of NF-YAl [35–39]. To ascertain whether NF-YAs-expressing cells could form myotubes, we switched the parental C2C12 and the two C2-YAl-KO clones at 70%–80% confluence to a differentiation medium. Before and after 72 h, we monitored cell morphology, performed Immunofluorescence experiments and derived whole cell extracts. Figure 3A shows that parental C2C12 form well organized, multinucleated myotubes, as expected (Upper Panels). The average number of nuclei per fiber is 15, in keeping with an efficient process (Figure 3B). On the other hand, the two edited clones showed a dramatic lack of myotubes formation: cells did not fuse; they were disorganized (Figure 3A, lower panels). We reasoned that the process could be simply slower in these cells and prolonged differentiation up to 5 days: this did not lead to formation of myotubes, nor cell fusion in the C2-YAl-KO clones (not shown). Immunofluorescence and Western blot data are consistent: the MyHCs marker is clearly visible in IFs (Figure 3A, right panels) and WB (Figure 3C) in C2C12 cells after differentiation, but not in the two edited clones. Interestingly, the levels of Myogenin and MyoD were substantially lower both in growing cells and at these late stages of differentiation in C2-YAl-KO clones. As previously reported, NF-YAl, in C2C12, and NF-YAs, in the edited clones, are down-regulated after 72 h of differentiation; NF-YB remained unchanged (Figure 3C). In summary, we conclude that terminal differentiation is completely blocked in C2C12 cells expressing NF-YAs instead of NF-YAl.

**Figure 3.** C2-YAl-KO clones fail to differentiate into myotubes. (**A**) Phase contrast analysis of myoblast cells (ctr and C2-YAl-KO clones) before and after 72 h of differentiation (differentiation medium (DM) condition) and immunofluorescence analyses after 72 h of differentiation. Antibody against all sarcomeric MyHCs and DAPI were used. (**B**) Fusion index was calculated as the number of nuclei in each myotube (with three or more nuclei). (**C**) Western blot analysis of key muscle differentiation regulators (MyHCs, MyoD), NF-YA isoforms (NF-YAl, NF-YAs) and NF-YB proteins, before (GM) and after 72 h of differentiation (72 h DM). Vinculin was used as loading control. The experiment was performed three times.

#### *3.4. Expression of TFs in C2-YAl-KO*

We analyzed expression of MRFs and TFs with a proven role in differentiation, in the parental and in the C2-YAl-KO cells under growing conditions and 24 h after differentiation. Profiling experiments established this as an early time point to detect significant changes in gene expression [48]. Note that most of the TFs analyzed have CCAAT in promoters and some formally shown to be under NF-Y control. First, we verified expression levels of MRFs in parental C2C12 (Figure S3): Myogenin is robustly induced; MyoD is modestly increased; Myf5 is modestly decreased after differentiation; Mef2C, but not Mef2D, is robustly increased. These changes are in agreement with expectations [49]. At the same time, we analyzed other TFs shown to be important for muscle differentiation: Six1/4/5, Snai1, Stat3 and Klf5 are all increased upon C2C12 differentiation, Id1/3 are modestly decreased, Pax3 is unchanged (Figure S3). These results are also in agreement with published data. Having established that our differentiation program runs normally in C2C12 cells, we monitored expression of these genes in the C2-YAl-KO clones. The results are shown in Figure 4A for growing conditions and Figure 4B for differentiation. MRFs show the most conspicuous differences: Myogenin is almost undetectable in growing C2-YAl-KO clones and marginally increased upon differentiation. MyoD basal levels are normal, but induction is reduced upon differentiation, compared to parental C2C12. Myf5 expression is basally similar in the edited clones and higher after differentiation (Figure 4A,B). Mef2C levels are similar in growing conditions, but lower after differentiation: note that the levels are very low basally and differences with parental C2C12 cells are not statistically significant. Mef2D expression is identical in C2C12 and edited clones. As for the other TFs, Six1/4/5, Klf5 and Pax3 show similar expression patterns (Figure 4A,B). Minor changes are observed in growing conditions for Snai1, Stat3 and Id1 (one clone only) and for Id1 (same clone) after differentiation. Finally, Id3 shows somewhat higher levels before and after differentiation, but again, these changes are variable in the three experiments and thus not statistically significant.

**Figure 4.** MRFs are downregulated in C2-YAl-KO clones. (**A**) Gene expression analysis of key muscle differentiation regulators (left panel) and other TFs shown to be important for muscle differentiation (right panel) in GM condition. Error bars represent the SD of three independent experiments. P-values were calculated using the one-sample t-test. (**B**) Gene expression analysis of key muscle differentiation regulators (left panel) and other TFs shown to be important for muscle differentiation (right panel) 24 h after differentiation (24 h DM). Error bars represent the SD of three independent experiments. P-values were calculated using the one-sample t-test. (**C**) Western blot analysis of key muscle differentiation regulators (Myogenin, MyoD, Myf5), NF-YA isoforms (NF-YAl, NF-YAs) and NF-YB proteins and other TFs shown to be important for muscle differentiation (Pax3, Snai1), in GM and 24 h DM. Vinculin was used as loading control.

To substantiate these results, protein expression of selected TFs was monitored by Western Blot analysis. Figure 4C shows that Myogenin levels are consistent with the mRNA data, being much lower in C2-YAl-KO clones than in parental cells, both in growing cells and after 24 h of differentiation. MyoD is substantially reduced in growing and differentiating clones, compared to parental C2C12. Note that protein levels were far lower than expected based on the mRNA levels, especially under growing conditions: this calls for post-transcriptional control in edited clones. Myf5 protein is downregulated in C2C12 after differentiation, as expected; in edited clones, it shows lower levels in growing cells, but higher after induction. NF-YA and NF-YB show the expected patterns; Pax3 is very modestly increased in C2-YAl-KO clones and Snai1 is unchanged. In summary, C2-YAl-KO cells have substantial differences in MRFs levels with respect to C2C12 cells, both before and after differentiation, whereas the other TFs showed rather minor changes.

#### *3.5. Expression of Myomaker and Myomixer Is Activated by Myogenin and It Is Impaired in C2-YAl-KO*

We were intrigued by the lack of cell fusion of the C2-YAl-KO clones after induction of differentiation. Myomaker—Mymk—and Myomixer—Mymx—are genes induced transcriptionally during muscle terminal differentiation, including in the C2C12 system [50,51]. Specifically, their expression is essential for the process of myocytes fusion [52]. We checked expression by qRT-PCR in parental C2C12 and in the two edited clones 24 h after differentiation. Figure 5A shows a strong induction—20-fold—of both Myomaker and Myomixer in C2C12 cells. C2-YAl-KO have much lower levels in growing cells (Figure 5B) and even more after differentiation (Figure 5C).

The obvious hypothesis was that these genes are under direct NF-Y control. We surveyed their promoter sequences and verified that no bona fide CCAAT box is present, notably within the evolutionary conserved areas: given the specificity of NF-Y CCAAT recognition, we considered unlikely that it acts directly on their expression. Genetic experiments in zebrafish have recently shown that Myomaker and Myomixer are directly activated by Myogenin [53]. We analyzed ENCODE datasets of C2C12 cells and found that Myogenin and MyoD target both promoters. Myomixer has apparently one promoter, Myomaker has two promoters, some 4 kb distant from each other: Figure 5D shows the overlapping peaks of Myogenin and MyoD. Myogenin binds exclusively after 24 h of differentiation, in accordance with its induced expression. One MyoD peak is visible already under growing conditions on Myomaker, and two additional peaks are found at 24 h. Importantly, the regions bound by MyoD and Myogenin in these two promoters are conserved across vertebrates, as shown by PhastCons data in Figure S4A: this corroborates the functional relevance proven in zebrafish [53]. To verify whether Myogenin activates Myomaker and Myomixer, we overexpressed it in parental C2C12 and in one of the C2-YAl-KO clones (#83) and induced to differentiate: Western blot of Figure 5E shows the increased levels of Myogenin compared to cells transfected with an Empty vector control; q-RT-PCR of Figure 5F shows that Myogenin overexpression has negligible effects on expression of the endogenous Myomaker and Myomixer in parental C2C12, but it increases expression of both genes in the C2-YAl-KO cells. Finally, morphological observation of the edited cells shows —incomplete—improvement in differentiation (Figure S4B).

In essence, we find that the marginal levels of Myogenin in C2-YAl-KO cells could result in lack of induction of the Myomaker and Myomixer targeted genes, entailing lack of cell fusion in NF-YAs-expressing clones.

**Figure 5.** Myogenin directly regulates Myomaker and Myomixer expression. (**A**) Relative expression levels of Myomaker (Mymk) and Myomixer (Mymx) in C2C12 cells before and after 24 h of differentiation (24 h DM). Error bars represent the SD of three independent experiments. P-values were calculated using the one-sample t-test. (**B**,**C**) Relative expression levels of Myomaker (Mymk) and Myomixer (Mymx) in C2C12 cells before (**B**) and after 24 h of differentiation (**C**) in ctr and the two C2-YAl-KO clones. Error bars represent the SD of three independent experiments. P-values were calculated using the one-sample t-test. (**D**) ChIP-seq peaks of MyoD and Myogenin on Mymk and Mymx promoters in GM and after 24 h of differentiation (24 h DM) (UCSC-genome browser available tracks). Vertical viewing range Mymk: min 0, max 5.5. Vertical viewing range Mymx: min 0, max 8. (**E**) Western blot analysis of Myogenin protein levels in C2C12 cells transfected with a control plasmid (pEmpty) and the Myogenin-overexpressing plasmid (pMyog) 96 h after differentiation induction. Vinculin was used as loading control. (**F**) Relative expression levels of Mymk and Mymx in C2C12 Myog-overexpressing cells after 96 h of differentiation. Error bars represent the SD of three independent experiments. *p*-values were calculated using the one-sample t-test.

#### *3.6. Myogenin and MyoD Are—Indirectly—Regulated by NF-Y*

The results shown above beg the question as to whether NF-Y directly regulates MRFs. Myogenin and Myf5 promoters do not contain CCAAT boxes, MyoD does [54]. To verify the NF-Y dependence of these genes, we transitorily inactivated NF-Y activity. In our hands, NF-YA inactivations by shRNA or siRNA were rather inefficient in C2C12 cells (not shown). We thus turned to NF-YB by treating C2C12 cells with an siRNA previously shown to be active and very specific, including in profiling experiments [29]. NF-YB is a necessary component of the DNA-binding trimer: this allows us to inhibit CCAAT-binding activity, upon siRNA treatment. Most importantly, unlike NF-YA, NF-YB inactivation does not trigger apoptosis [29,34], making this a suitable choice for long differentiation processes. Figure 6 shows the results of experiment 1, Figure S5 those of experiment 2: in both, RT-qPCR (Figure 6A and Figure S5A) and Western blots (Figure 6B and Figure S5B) show far lower expression of NF-YB in C2C12 cells treated with NF-YB siRNA, with respect to the control siRNA. In mRNA analysis, Myogenin, MyoD and Mef2C, but not Myf5 nor Mef2D, are substantially downregulated upon NF-Y inactivation; Myomaker and Myomixer are also reduced. Six1/4/5 are reduced: for Six4, this in keeping with an NF-Y dependence predicted from previous data on NF-Y binding to a canonical promoter CCAAT [39]. As for Id1 and Id3, they are somewhat reduced, but the results are borderline significant: Id1 in experiment 2 and Id3 in experiment 1. We conclude that NF-Y removal entails a reduction of MRFs, which, in turn, could explain the observed drop of Myomaker and Myomixer. We also show that members of the Six family are NF-Y targets. Analysis of proteins levels in extracts of siRNA-inactivated cells by Western blots confirmed these results: the levels of NF-YB were lower (although not to the extent of the mRNA) and paralleled by somewhat lower levels of NF-YA. Myogenin is substantially decreased and MyoD is also affected, to a lesser extent (Figure 6B and Figure S5B). We conclude that NF-Y regulates the expression of MyoD and Myogenin in C2C12 cells.

The Myogenin promoter is CCAAT-less and was not bound by NF-Y in C2C12 cells [39] and, despite the presence of a canonical CCAAT, the MyoD promoter was also not bound [39]. To understand whether the positive effect of NF-Y on MyoD is direct, we checked the parental C2C12 cells for the presence of NF-Y in ChIP experiments. Three independent experiments are shown in Figure 6C and Figure S5C. The absence of enrichment of NF-Y on MyoD is indeed confirmed, whereas the Stard4 positive control promoter is clearly bound. Equally positive was the promoter of Id1, but not that of Id3. Note that there is variability in the fold-enrichments in the three experiments: as this is high (from 60 to 800-folds), we consider quantitative changes difficult to interpret, especially when compared to completely negative promoters such as MyoD and Id3. Therefore, we conclude that NF-Y does not regulate MyoD directly—and despite promoter binding—NF-Y has modest effects on Id1 transcription in C2C12 cells.

**Figure 6.** Analysis of NF-Y involvement in muscle specific genes expression. (**A**) Gene expression analysis of NF-YB and key muscle differentiation regulators in C2C12 cells 72 h after NF-YB silencing (siNF-YB) and scrambled siRNA control. Error bars represent the SD of two different RT-qPCR 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 siRNA control. Vinculin was used as loading control. (**C**) ChIP experiment performed on C2C12 ctr cells in GM condition using NF-YB and negative control (nc) antibodies. The unrelated region (ur) and Stard4 were used as negative and positive control, respectively. Results are represented as the input percentage of each sample normalized to the input percentage of the nc antibody.

#### **4. Discussion**

By genome editing, we derived clones of C2C12 cells that express NF-YAs instead of NF-YAl. We verified that NF-YAs—and companion HFDs—are expressed at comparable levels and that it decreases after differentiation. The edited C2C12 clones are stable, grow normally, yet they are completely deficient in differentiation. We report defects of basal and induced expression of Myomaker and Myomixer, early response-genes likely responsible for lack of cell fusion. Their promoters are

targeted by MyoD and Myogenin. In turn, we find low—basal and induced—levels of MyoD and Myogenin in the NF-YAs-expressing clones. Finally, expression of both MRFs are indirectly controlled by NF-Y.
