Mechanisms of Notch-mediated inhibition of skeletal myogenesis .

摘要

The Notch pathway is an evolutionarily conserved signaling cascade that regulates many cell fate decisions. Recent work has revealed that Notch plays critical roles in the control of skeletal muscle development and regeneration. In the embryo, Notch maintains a pool of myogenic progenitor cells and prevents their premature differentiation. In the adult, after muscle injury, Notch signaling is essential for the initial expansion of muscle stem cells, or satellite cells.;While it has been known for over a decade that Notch activity represses myogenic differentiation, the molecular mechanisms by which this inhibition occurs are poorly defined. In this thesis, I sought to identify the key transcriptional effectors of Notch in muscle and explore how these proteins repress the myogenic program. Using the mouse myoblast cell line C2C12, I identified 82 transcripts upregulated after six hours of ligand-mediated Notch stimulation. When constitutively expressed in myoblasts, several of these genes (Nrarp, HeyL, Trib2) had no apparent impact on differentiation, while at least two of them, the canonical effector Hey1 and the novel Notch-responsive gene MyoR, were capable of recapitulating the pathway's inhibitory effects. Interestingly, siRNA knockdown of Hey1 or MyoR, or the two factors in combination, failed to rescue the differentiation of myoblasts exposed to Notch ligands. These results support a model in which Notch acts through multiple, potentially redundant pathways to repress myogenesis.;In subsequent work, I focused on the mechanistic question of how the Notch effector Hey1 interferes with myogenic transcription. My functional and biochemical data revealed that Hey1 does not target the inherent transcriptional activity of the skeletal muscle master regulator MyoD. I found that Hey1 repressed only a subset of MyoD target genes, and consistently, did not disrupt dimerization of MyoD with its obligate binding partner E47. My results indicated that Hey1 is recruited to the promoter regions of Myogenin and Mef2C, two genes whose induction is critical for differentiation. Expression of Hey1 in C2C12 myoblasts correlated with reduced recruitment of MyoD to these promoters, arguing that Hey1 inhibits myogenesis by associating with and repressing expression of key myogenic targets.