**Debora Libetti, Andrea Bernardini, Sarah Sertic, Graziella Messina, Diletta Dolfini and Roberto Mantovani \***

Dipartimento di Bioscienze, Università degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy; debora.libetti@unimi.it (D.L.); andrea.bernardini@unimi.it (A.B.); sarah.sertic@unimi.it (S.S.); graziella.messina@unimi.it (G.M.); diletta.dolfini@unimi.it (D.D.)

**\*** Correspondence: mantor@unimi.it

Received: 14 February 2020; Accepted: 21 March 2020; Published: 24 March 2020

**Abstract:** 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.

**Keywords:** splicing isoforms; CRISPR-Cas9; exon deletion; NF-Y; muscle differentiation; C2C12 cells

### **1. Introduction**

Cell specification and differentiation during development of multicellular organisms is a complex set of events resulting in the formation of organs, whose physiology is maintained by a balance of cell proliferation and differentiation. A paradigmatic example of these phenomena is formation of skeletal muscle. In the case of mammals—mouse in particular—the process begins at early developmental stages, proceeding through embryonic, fetal and adult stages [1,2]. Sequence-specific transcription factors—TFs—play a central role in specifying the identities of myoblasts, their migration to different body locations, organization and the capacity to self-renew and differentiate into myotubes. These properties are key to guarantee maintenance and functionality of the different muscles throughout the lifespan of the organism, including repair after injury in adult life. A set of four key TFs —MyoD, Myf5, Myogenin, MRF4, termed myogenic regulatory factors (MRFs)—have been identified and thoroughly studied by genetic and biochemical means for their capacity to specify myoblasts identity [3,4]. During development, PAX3/7 are located upstream of MRFs [5]; downstream are many TFs, such as the MADS box MEF2A/C/D [6,7], the bHLH ID1/3 [8–10] and SNAI1 [11], the HOX SIX1/4/5 [12–15], STAT3 [16], NFIX [17,18] and the ZNF KLF2/4/5 [19,20]. Unlike MRFs, most of these TFs are not expressed predominantly in muscle cells and are equally important for development and differentiation of other tissues and organs [21–25].

NF-Y is an evolutionarily conserved heterotrimer formed by the sequence-specific NF-YA and the Histone Fold Domain—HFD—NF-YB/NF-YC [26]. The sequence recognized by NF-Y is the CCAAT box, which plays an important role in the activation of 25%–30% of mammalian genes. NF-Y has been classified as "pioneer" TF, in mammals and plants [27–31]. NF-YA is the regulatory subunit; it is alternatively spliced, generating two major isoforms "short" (NF-YAs) and "long" (NF-YAl), differing in 28 amino acids coded by exon 3 [32]. This stretch is located at the N-terminus of the protein, in the Gln-rich transactivation domain (TAD). NF-YAs and NF-YAl have identical subunits-interactions and DNA-binding properties *in vitro*; ChIP-seq from cells harboring predominantly either one of the two isoforms showed recovery of peaks enriched in CCAAT. The isoforms are expressed at various levels in different tissues and cell lines [32,33]. Importantly, no cell line has been so far described lacking NF-YA—nor the HFDs—and NF-YA inactivation was reported to be fatal to cells [28,34]. NF-YAl is the predominant isoform in muscle C2C12 cells: it is abundant in proliferating cells, but it drops to low levels following terminal differentiation to myotubes, unlike the HFD partners [35–37]. Highly reduced NF-YA protein was found in myotubes of adult mice [38]. This suggested that genes up-regulated in the terminal phases of muscle differentiation are either CCAAT-less or not NF-Y-dependent, whereas the trimer activates cell-cycle and growth-promoting genes required during the proliferative state. However, overexpression of NF-YAl led to improved differentiation of C2C12 [39], suggesting that NF-YAl does take part in the differentiation process.

For decades, C2C12 myoblast cells have represented an informative tool to identify genes involved in muscle differentiation [40]. Ablation of the whole NF-YA gene is early embryonic lethal [41], and KO in stable cell lines could not be generated so far. We investigated the role of NF-YAl by genetically ablating exon 3, leading to the production of an intact NF-YAs. We successfully generated homozygous C2C12 lines expressing only NF-YAs and went on to study differentiation properties.

#### **2. Materials and Methods**

#### *2.1. Cell Culture and Proliferation Assay*

Mouse myoblast cells (C2C12, ATCC) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS, Gibco-Thermo Fisher Scientific), 4 mM l-Glutamine, 100 units/mL penicillin and 100 μg/mL streptomycin (GM, growth medium), in a humidified 5% CO2 atmosphere at 37 ◦C. C2C12 cells differentiation was induced plating cells in DMEM with 2% horse serum (Gibco-Thermo Fisher Scientific), 4 mM l-Glutamine, 100 units/mL penicillin and 100 μg/mL streptomycin (DM, differentiation medium). Proliferation assay was performed by plating 1.5 <sup>×</sup> 105 cells into a 12-well plate and counting every 24 h for 3 days, using the Trypan Blue dye exclusion test. All data were gathered from at least three independent biological replicates. Multiple comparisons were performed using the One-way ANOVA test.
