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

Dipartimento di Bioscienze, Università degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy
Author to whom correspondence should be addressed.
Cells 2020, 9(3), 789;
Received: 14 February 2020 / Revised: 19 March 2020 / Accepted: 21 March 2020 / Published: 24 March 2020
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. View Full-Text
Keywords: splicing isoforms; CRISPR-Cas9; exon deletion; NF-Y; muscle differentiation; C2C12 cells splicing isoforms; CRISPR-Cas9; exon deletion; NF-Y; muscle differentiation; C2C12 cells
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Libetti, D.; Bernardini, A.; Sertic, S.; Messina, G.; Dolfini, D.; Mantovani, R. The Switch from NF-YAl to NF-YAs Isoform Impairs Myotubes Formation. Cells 2020, 9, 789.

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