Project Summary Muscle stem cells, also known as satellite cells (SC), are a progenitor population required to replenish muscle damaged by exercise or wasting caused by disease or aging. The ability to generate muscle progenitors from embryonic stem cells (ESC) would represent a major advance in regenerative medicine. However, despite recent progress, it remains very challenging to differentiate mouse and human ESC into muscle progenitors, and SC represent a very small population within muscle tissue. These limitations have significantly hampered progress toward understanding muscle stem cells, and the critical regulatory processes that govern SC identity. The transcription factor, Pax7, plays a critical role in establishing SC identity, but given the limitations associated with obtaining SC, our understanding of its molecular function remains incomplete. We have taken advantage of a system in which Pax7 expression in ESC generates myogenic precursors able to repopulate the SC niche, and we have begun investigating the chromatin landscape and epigenetic features of this population to understand its unique properties. With this system, we have shown that Pax7 can modulate enhancer function and genome topology. Our goal is to understand how a specific chromatin environment specifies satellite cell identity using state-of-the-art genomic, biochemical, and proteomic approaches. In two Aims, we will: (I) investigate how Pax7 re-models the genome, packaging chromatin for long-range interactions; (II) explore how Pax7 collaborates with other factors to establish gene expression and genome re-wiring in myogenic precursors in vitro and in vivo. We will inactivate a subset of Pax7-associated factors, enhancers, and identified regulatory elements and examine their functional impact on genome architecture, gene expression, cell identity, and differentiation. It is known that satellite cells are less efficiently renewed during aging, and given that muscle tumors (rhabdomyosarcomas) aberrantly express Pax7 fusion proteins to maintain muscle in a pre-differentiated state, our proposal will shed important light on pathological states resulting from aberrant Pax7 activity and stem cell function.