DNA hypermethylation preceded by H3K27 trimethylation is linked to downregulation of gene expression in disuse muscle atrophy in male mice

Disuse muscle atrophy can result in downregulated gene expression vital to muscle integrity, yet the mechanisms driving this downregulation remain unclear. Epigenetic alterations regulate transcriptional potential, with repressive changes suppressing gene expression. This study explored epigenetic mechanisms of gene downregulation during disuse muscle atrophy. Male C57BL/6J mice underwent hindlimb suspension for 3 or 7 days. The vastus intermedius (VI) muscle was analyzed, showing unchanged mass on day 3, but on day 7, decreased mass and reduced fiber size were assessed via immunohistochemistry. Corresponding to this atrophy timing, qPCR analysis revealed nine downregulated genes on day 7, which were selected for epigenetic analysis; collectively, they showed no downregulation on day 3. Among the nine genes, methylated DNA immunoprecipitation revealed significantly elevated DNA methylation (hypermethylation) in the upstream regions of transcription start sites (TSS) on day 7, which overall negatively correlated with gene expression. Histone marks (H3K27me3, H3K4me3, H3.3, and total H3) were also assessed using chromatin immunoprecipitation, revealing that the repressive histone mark H3K27me3 increased in the regions on day 3 but decreased on day 7. These findings suggest that DNA hypermethylation in the upstream regions preceded by H3K27me3 enrichment contributes to the downregulation of gene expression during disuse muscle atrophy.