Pax7 is essential for the function of muscle satellite cells. It was previously determined that Pax7 methylation is essential for its transcriptional activity and function in satellite cells. We investigated whether Pax7 displays other post-translational modifications playing a role in its function. By mass spectrometry using immunoprecipitated FLAG-Pax7, we identified two lysine residues (K105 and K193) within the Pax7 protein that are acetylated. Pax7 transcriptional activity was monitored using a luciferase reporter under the control of Myf5, a Pax7 target gene. Treatment with Trichostatin A, a histone deacetylase inhibitor, increased significantly luciferase activity, but this activity was progressively loss when the created Pax7 mutants (K105R, K193R) were introduced. This suggests that acetylation plays a role in Pax7 transcriptional activity. To identify the acetyltransferase modulating Pax7 activity, we used a candidate approach. Myst1 is expressed in muscle satellite cells. Myst1 is known for its interaction with Wdr5 and MLL1/2, a known Pax7 partner. We detected an interaction between Pax7 and Myst1 by co-immunoprecipitation in fibroblasts and in primary myoblasts. Myst1 knockdown decreases Pax7 acetylation status suggesting Myst1 as Pax7’s acetylase. Myst1 siRNA knockdown negatively impacts many Pax7’s target genes as well as primary myoblast proliferation. Moreover, primary myoblasts treated with siMyst1 express higher levels of MyoD. In satellite cells Myst1 reduction through siRNA knockdown significantly reduces satellite cells progenitor expansion as well as increases MyoD expression. In all, Myst1 modulating Pax7 activity through acetylation represents a novel mechanism in muscle stem cell biology.

Rhabdomyosarcoma (RMS) is a highly malignant form of pediatric cancer that originates from skeletal muscle cells. While the normal skeletal muscle cells are generated via a highly regulated process called myogenesis that depends on myogenic regulatory factors (MRFs), the RMS cells fail to differentiate as a result of impaired myogenesis due to abnormal MRF activity. We found that TCEA3 regulates myogenin (an essential MRF member) activity at the gene expression level. Our work showed that depletion of TCEA3 in normal myoblast cells results in an inhibition of differentiation and downregulation of MRFs. TCEA3 confers myogenic activity and results in differential recruitment of RNAPII to target promoters. While its role in ovarian cancer is well understood, its role in RMS is yet unclear. Thus, our work focused on investigating the role of TCEA3 in both the RMS subtypes, ARMS and ERMS. The results revealed that TCEA3 is downregulated in both the RMS subtypes, and further, its overexpression resulted in a decrease in migration and inhibition of anchorage-independent growth. It also is shown to induce apoptosis through intrinsic and extrinsic pathways. Additionally, we found that over expressing TCEA3 in RMS cells could sensitize these cells to chemotherapeutic drugs. Similar to TCEA3, TCEAL7 is downregulated in various types of cancers, including over 90% of ovarian epithelial cancers, and has shown tumor suppressing properties in ovarian cancers. Our work on RMS showed that TCEAL7 is highly deregulated in both of the immortal cell lines representing ARMS and ERMS, and highly expressed in the normal myoblast cells. Further, deletion of Tceal7 inhibits the proliferation of myoblasts suggesting that TCEAL7 is involved in promoting cell cycle progression in myoblast cells. Deletion of TCEAL7 also revealed a loss of MyHC expression upon differentiation and overexpression of TCEAL7 in RMS promotes differentiation in RMS cells. Additionally, the overexpression of TCEAL7 has led to a decrease in expression of the oncogene, Cyclin D1. Overall, the overexpression of TCEAL7 in RMS cell induces apoptosis and sensitizes cells to TRAIL induced cell apoptosis.