Differentially expressed genes in myotubes derived from MBNL knockouts hiPSCs generated by CRISPR/Cas9.

Alternative splicing has emerged as a fundamental mechanism not only for the diversification of protein isoforms but also for the spatiotemporal control of development. Therefore, a better understanding of how this mechanism is regulated has the potential not only to elucidate fundamental biological principles, but also to decipher pathological mechanisms implicated in diseases where normal splicing networks are misregulated. Here, we took advantage of human pluripotent stem cells to decipher during human myogenesis the role of MBNL proteins, a family of tissue-specific splicing regulators whose loss of function is associated with Myotonic Dystrophy type 1, an inherited neuromuscular disease. Thanks to the CRISPR/Cas9 technology, we generated human-induced pluripotent stem cells (hiPSCs) depleted in MBNL proteins and evaluated the molecular and functional consequences of this loss on the generation of skeletal muscle cells. Our results indicated that MBNL proteins are specifically required for the late myogenic maturation but not for early myogenic commitment. By a transcriptomic analysis, we further demonstrated that MBNL proteins are not only important for the regulation of alternative splicing but also for the regulation of gene expression. Together, our study reveals the temporal requirement of MBNL proteins in human myogenesis and should facilitate the identification of new therapeutic strategies capable to cope the loss of function of these MBNL proteins. Overall design: 12 samples were analyzed with 3 replicates per condition: 3 WT hiPSC-derived myotubes (control), 3 DM1 hiPSC-derived myotubes, 3 MBNL1(-/-) hiPSC-derived myotubes and 3 MBNL1(-/-); MBNL2(-/-) DKO hiPSC-derived myotubes.