Loss of NRMT1 allows expression of multiple differentiation pathways and alters transcription of secreted proteins in C2C12 myoblasts

Abstract

Muscle stem cells (satellite cells) retain their identity and function through expression of the paired homeobox transcription factor PAX7. PAX7 is able to both stimulate satellite cell proliferation and activate target genes involved in establishing myogenic identity, including myogenic factor 5 (MYF5) and the other myogenic regulatory factors (MRFs). Upregulation of the MRFs promotes commitment to the muscle lineage by initiating withdrawal from the cell cycle, upregulating expression of muscle-specific transcripts, and directing myoblast fusion. We have previously shown that knockout of the N-terminal methyltransferase NRMT1 in C2C12 mouse myoblasts results in significantly decreased Pax7 expression, an inability of the cells to differentiate into myotubes, and abnormal upregulation of osteogenic markers. Here, we use RNA-sequencing to more comprehensively determine how loss of NRMT1 affects the transcriptional profile of proliferating and differentiating C2C12 myoblasts. We see that upon inducing differentiation, NRMT1 knockout cells can downregulate cell cycle, DNA replication, and histone gene expression. Though they also have significantly downregulated Pax7 and Myf5 expression, other muscle-specific transcripts are significantly increased over wild type, indicating the muscle transcriptional program is not completely inhibited. However, signaling pathways involved in the differentiation of other types of mesenchymal and hematopoietic lineages are also increased with NRMT1 loss and expression of chemotactic genes is downregulated. Together, these data indicate that NRMT1 knockout cells can upregulate genes needed for cell cycle withdrawal and muscle specification but fail to suppress markers of other lineages and activate normal chemotactic signaling, which may lead to the observed differentiation phenotypes.