Hemizygous mutation of <i>Jmjd3</i> in muscle stem cells increases H3K27 methylation on <i>Pax7</i> leading to impaired myogenesis

Development of myogenic cells, called satellite cells, is determined by transcription factors that regulate their quiescence (e.g., Pax7), activation (e.g., MyoD), and terminal differentiation (e.g., myogenin). Demethylation of lysine 3 on histone 27 (H3K27) activates expression of <i>Myod</i> and <i>Myog.</i> In this investigation, we investigated the effects of a satellite cell-targeted, hemizygous mutation of the H3K27 demethylase <i>Jmjd3</i> in healthy muscle. Using sequencing of chromatin fragments precipitated from <i>Jmjd3</i> mutant and control satellite cells using anti-H3K27me3, we found that the <i>Pax7</i> promoter was the only chromatin that experienced significantly increased H3K27 methylation in mutant cells. However, RNA sequencing showed that 143 genes were downregulated in mutant cells, including <i>Myod</i>, a direct target of <i>Pax7</i>, and at least 72 other genes that contained E-boxes targeted by <i>MyoD</i>. Gene ontology analysis showed enrichment of genes involved in myogenesis in the downregulated genes. Reduced expression of <i>Pax7</i>, <i>Myod</i>, and <i>Myog</i> was confirmed by quantitative PCR (qPCR), Western blots, and immunohistochemistry. Mutant muscles also had smaller diameter fibers, fewer myonuclei, and diminished myogenic cell fusion, indicating impaired growth and differentiation. These findings show that Jmjd3 affects demethylation of H3K27 at the <i>Pax7</i> promoter, and increased H3K27 methylation reduces expression of <i>Pax7</i> and its target genes, disrupting muscle growth.<b>NEW & NOTEWORTHY</b> This investigation reveals a new mechanism that regulates the development of muscle. Mutating <i>Jmjd3</i> produced epigenetic modifications to the transcription factor <i>Pax7</i>, reducing its expression and impairing muscle growth.