Characterizing the Mechanisms Regulating Enhancer Commissioning by MyoD in Muscle Cells

Abstract

Myogenesis refers to the cellular process required for muscle formation and repair of damaged muscle tissue. During this process, the activation of muscle gene expression is tightly regulated to ensure efficient transitions between cell fates. This activation is promoted by large groups of neighbouring transcriptional enhancer regions known as super-enhancers. Enhancer regions are opened and commissioned by master transcription factors such as MyoD. While MyoD is crucial for muscle development and differentiation due to its role in initiating muscle-specific gene expression, the mechanisms used to regulate MyoD function at different stages of myogenesis remain poorly understood. To better understand how MyoD activity is regulated in muscle cells, we first utilize proximity labelling coupled with mass spectrometry to identify potential coactivators of MyoD. Our results reveal a link between MyoD and the Mediator complex, a key component of enhancer-promoter looping. Next, using phenotypic assays and transcriptomics, we demonstrate that knocking down Mediator function severely impedes muscle differentiation by down-regulating essential muscle genes. Establishing that MyoD associates with Mediator at enhancers to facilitate the expression of muscle functional genes during myogenesis. Additionally, we address the importance of modulating the amount of MyoD available in the cell to control myogenesis. Using a CRISPR-modified mouse model where the canonical degradation pathway of MyoD is suppressed, our analysis revealed that prolonged MyoD stability delays terminal differentiation, and results in poor regeneration efficiency. Thus, muscle cells must have the ability to turnover MyoD proteins for efficient transitioning between the proliferative and differentiating states. Taken together, our results show that MyoD functions with the coactivator Mediator to activate gene expression, and that MyoD degradation modulates transcriptional output in muscle cells. These findings advance our understanding of the role of MyoD in myogenesis, paving the way for novel therapeutic approaches in muscle diseases.