SRA

The maintenance of cell lineage and cell fate are essential for the function of adult stem cells. Despite this, the epigenetic mechanisms that regulate muscle stem cell (MuSC) identity are not well understood. In this study, we performed Cut&Tag of the activating histone marks H3K4me3 and H3K27ac on freshly isolated quiescent MuSCs and found that a large number of genes without transcription still maintained the permissive mark H3K4me3 but lacked the marker of active enhancers, H3K27ac. These genes included those that are essential for non-myogenic lineage determination. We found that many of the genes that were not transcribed but enriched for H3K4me3, were also enriched for the RE-1 binding motif, the motif recognized by the repressive transcription factor REST. Using a genetic mouse model where REST is conditionally knocked out of MuSCs, we further investigated the role of REST in the maintenance of quiescent MuSC cell identity. Investigation of the transcriptome and chromatin accessibility of WT and REST deficient MuSCs determined that the loss of REST results in the gain of expression of key genes of several non-myogenic tissues, particularly neuronal genes. Additionally, the loss of REST led to the dramatic reduction of the MuSC pool and the induction of muscle atrophy. The unstable cell identity caused by the genetic deletion of REST results in the MuSCs undergoing apoptosis and is the main driver of the observed loss of the MuSC pool. Together, the data presented in this study establishes a novel function of the transcription factor REST, where it safeguards the identity and myogenic lineage of MuSCs, through the repression of alternative lineages. Overall design: 1000 Muscle stem cells (MuSCs) (ITGA7+/VCAM1+/CD11b-/CD45-/CD31-/Ly6a-) were isolated by Fluorescence Activated Cell Sorting (FACS) from 3 male WT and 3 male REST-cKO mice (4-6 weeks old) and sorted directly into SMART lysis bufer for RNA-Seq of quiescent MuSCs and the remaining isolated cells were cultured for 7 days. After 1 week in culture, myoblasts were counted and 1000 myoblasts were obtained for RNA-Seq of myoblasts. RNA-Seq libraries were created using SMART-Seq to make cDNA (TAKARA,cat # 634456), followed by Nextera XT (Illumina, #cat FC-131-1024) for incorporation of Illumina sequencing adaptors. Single myofibers were isolated from 3 WT and 3 REST-cKO mice (4-6 weeks old, male) and lysed in SMART lysis buffer. Single myofiber RNA-Seq was performed as previously described (Blackburn, D.M. et al. Journal of Biological Chemistry, 2019).