Cut&Run of freshly isolated quiescent muscle stem cells (MuSCs) for H3K27me3 and Cut&Run of myoblasts for REST/NRSF

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. For Cut&Run of H3K27me3 on MuSCS, 45000 MuSCs (ITGA7+/VCAM1+/CD11b-/CD45-/CD31-/Ly6a-) were isolated by Fluorescence Activated Cell Sorting (FACS) from 3 WT (C57BL/6) male mice (6 weeks old). MuSCs were sorted directly into 2%FBS in PBS and Cut&Run was performed using the commercially available Cut&Run Kit (Epicypher, 14-1048) according to the manufacturer’s guidelines with the H3K27me3 primary antibody (Cell Signalling, Cell Signalling, C36B11). Cut&Run for REST/NRSF was performed on low passage cultured myoblasts. Briefly, 100 000 cells were isolated by FACS and cells were cultured for 1 week in growth media. Cells were then trypsinized, washed and Cut&Run was performed using the commercially available Cut&Run Kit (Epicypher, 14-1048) according to the manufacturer’s guidelines with the anti-REST antibody (Milllipore, 17-641). All Cut&Run libraries were prepared with Cut&Run Library Prep Kit (Epicypher, 14-1001) according to the manufacturer’s guidelines and the samples were sequenced on NovaSeq6000 Sprime Paired End (PE) 150 bp.