Helix-loop-helix transcription factor Ascl4 induces myogenic program in pluripotent stem cells Yusaku Kodakaa, Shuichi Watanabe, Hikaru Ito, Mayank Verma, Tomohide Takaya, Yoko Asakura, Michael Kyba, Atsushi Asakura Stem Cell institute, bPaul & Sheila Wellstone Muscular Dystrophy Center, cDepartment of Neurology, dDepartment of Pediatrics, University of Minnesota Medical School, Minneapolis, MN The basic helix-loop-helix (bHLH) transcription factors play central roles in developmental processes including cell fate specification such as MyoD family for myogenesis and Achaete-scute complex-like 1 (Ascl1) for neurogenesis. Here, we found that all Ascl family (Ascl1-5) has the ability to induce myogenic program when overexpressed in embryonic stem cells (ESCs). Among them, we noticed that Ascl4 expression is detected in dermomyotome, a place for myogenic progenitor cells during mouse embryogenesis. In both ESCs and induced Pluripotent Stem Cells (iPSCs), overexpression of Ascl4 efficiently induces MyoD/Pax7-positive myogenic cells followed by myosin heavy chain positive terminally differentiated myocytes when Ascl4 expression was withdrawn. Integrative analysis of RNA-seq and ChIP-seq data revealed that Ascl4 is able to induce MyoD expression through the binding of novel MyoD enhancer region termed embryonic enhancer region (EER) located at 60 kb upstream of its start site. Together, these findings imply that Ascl4 may mediate activation of MyoD as a key regulator of somitic myogenesis.