AbstractThe genome exists as an organized, three-dimensional (3D) dynamic architecture, and each cell type has a unique 3D genome organization that determines its cell identity. An unresolved question is how cell type-specific 3D genome structures are established during development. Here, we analyzed 3D genome structures in muscle cells from mice lacking the muscle lineage transcription factor (TF), MyoD, versus wild-type mice. We show that MyoD functions as a “genome organizer” that specifies 3D genome architecture unique to muscle cell development, and that H3K27ac is insufficient for the establishment of MyoD-induced chromatin loops in muscle cells. Moreover, we present evidence that other cell lineage-specific TFs might also exert functional roles in orchestrating lineage-specific 3D genome organization during development.