Skeletal muscle cell proliferation and differentiation are tightly regulated. Epigenetic regulation is a major component of the regulatory mechanism governing these processes. Histone modification is part of the epigenetic code used for transcriptional regulation of chromatin through the establishment of an active or repressive state for genes involved in myogenesis in a temporal manner. Here, we uncovered the function of SET domain containing 2 (Setd2), an essential histone 3 lysine 36 trimethyltransferase, in regulating the proliferation and differentiation of myoblasts. Setd2 was silenced in the skeletal muscle myoblast cell line, C2C12, using the CRISPR/CAS9 system. The mutant cells exhibited defect in myotube formation. The myotube formation marker, myosin heavy chain (MHC), was downregulated earlier in Setd2 silenced cells compared to wild-type myoblasts during differentiation. The deficiency in Setd2 also resulted in repression of Myogenin (MyoG) expression, a key myogenic regulator during differentiation. In addition to the myoblast differentiation defect, decreased proliferation rate with significantly reduced levels of histone 3 phosphorylation, indicative of cell proliferation defect, were observed in the Setd2 silenced cells; suggesting an impaired proliferation phenotype. Furthermore, compromised Gl/S- and G2/M-phase transition and decreased expression levels of major regulators of cell cycle Gl/S checkpoints, cyclin D1, CDK4, CDK6, and cyclin E2 were detected in Setd2 silenced cells. Consistent with the cell cycle arrested phenotype, cyclin-dependent kinase inhibitor p21 was upregulated in Setd2 silenced cells. Together, this study demonstrates an essential role of Setd2 in myoblast proliferation and differentiation, and uncovers Setd2-mediated molecular mechanism through regulating MyoG and p21. (C) 2017 Elsevier B.V. All rights reserved.