Skeletal muscle (SkM) is one of the most plastic tissues in the human body with an amazing ability to adapt to imposed demands and regenerate after injuries. The regenerative capacity of muscle is largely driven by the muscle stem cell, more commonly referred to as satellite cells (SCs) 1, 2. SCs account for ∼1–4% of all the nuclei in SkM tissue and reside mainly in a quiescent state in homeostatic muscle, mainly identified by the SC-specific transcription factor (TF) Paired Box 7 (PAX7). After an injury, however, SCs become activated, downregulate PAX7, reenter the cell cycle, and ultimately differentiate to aid in SkM regeneration [3]. The potential of SCs to regenerate damaged muscle tissues has led multiple labs to develop in vitro strategies to generate SCs in a dish 4, 5, 6. These strategies often begin with pluripotent stem cells (PSCs) or induced PSCs and introduce small molecules and/or growth factors that mimic in vivo signaling cascades found in human/mouse muscle development. Currently, many of these protocols produce PAX7+ skeletal muscle progenitor cells (SMPCs), which can proliferate and differentiate to form myotubes, but are distinct from SCs 4, 5, 6. SMPCs represent a more fetal-like progenitor and do not regenerate injured SkM to the same degree as SCs 7, 8. Many lineages derived from PSCs are plagued with similar quandaries, including cardiomyocytes, neurons, and hematopoietic stem cells 9, 10, 11, 12, 13, 14. These common observations have uncovered interesting questions regarding the factors that control human development, cell state and identity dynamics, as well as the roadblocks that may be at play in restricting the desired cell states. One underexplored avenue is the role that genome folding plays in development, stem cell regulation, or maturation. Indeed, recent studies in hematopoietic stem cells have highlighted the importance of the three-dimensional genome (3D) in transition from quiescence to differentiation [15].

The 3D genome is arranged in hierarchical levels [16]. At the chromosomal level, each chromosome occupies unique territories and interacts with the nuclear envelope to establish these territories [17]. On the scale of hundreds to tens of kilobases (KBs), large collections of chromatin called topologically associating domains (TADs) are observed [18]. TADs are 3D structures in which DNA that is within a TAD interacts more frequently than between TADs 18, 19. At finer resolutions of hundreds of base pairs to tens of KBs, DNA loops are formed, largely by specific proteins that include CCCTC-Binding Factor (CTCF) and Cohesin [19]. DNA loops are responsible for bringing gene enhancers closer to their target genes as well as restricting spurious enhancer–promoter (E–P) contacts allowing regulatory control of gene expression 20, 21, 22, 23, 24, 25, 26, 27. At the finest resolution, DNA is wrapped around histones, which can restrict access to regulatory sequences (i.e. gene promoters, enhancers). Various proteins and protein complexes can modify, exchange, and/or evict histones to provide accessibility to these regulatory sequences (Figure 1). Access to these sequences, namely enhancers, influences cell fate and state via the binding of lineage-specific TFs resulting in the increase of gene expression of the target gene [28].

Recent work in neural crest cells, adipocytes, and sarcomas has highlighted how mutations to either enhancers or histone modification complexes that regulate access to enhancers can cause gene misexpression resulting in developmental disorders and/or disease 29, 30, 31•, 32. Given the importance of enhancers in regulating cell and tissue development, understanding how enhancers regulate their target genes could allow us to modulate gene expression in a cell-specific way and thus improve disease treatment as well as cellular reprograming 33, 34, 35. Below, we review recent studies that describe how enhancers are regulated in developing SkM and SCs with a particular focus on muscle-specific TFs and their ability to establish and activate enhancers as well as their ability to modify the 3D genome to upregulate muscle-specific genes. We then discuss how these findings could impact SkM in vitro differentiation strategies for gene and stem cell-based therapies. Given the difficulties in profiling enhancers in human SCs, many of the studies below are focused on mouse SC investigations. While innovative and extremely insightful, not all enhancers are conserved between mouse and human [36]. New technologies utilizing low cell numbers, such as Cleavage Under Targets & Tagmentation and low-input High throughput chromosome conformation capture, will allow for identification of human-specific enhancers from hard-to-procure samples and provide insight into controlling cell state in vitro [37].