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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jun 11, 2024 |
| Title |
ATAC-Seq of single myofibers and muscle stem cells (MuSCs) |
| Organism |
Mus musculus |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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| Summary |
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.
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| Overall design |
5000 MuSCs (ITGA7+/VCAM1+/CD11b-/CD45-/CD31-/Ly6a-) were isolated by Fluorescence Activated Cell Sorting (FACS) from 4 REST-cKO and 4 WT male mice (4-6 weeks old) and sorted directly into ATAC-Seq lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% NP-40, 0.01% Digitonin). Samples were processed as previously described OMNI-ATAC-Seq protocol (Corces, M.R. et al. Nature Methods, 2017). Single myofiber ATAC-Seq (smfATAC-Seq) was performed on uninjured Extensor Digitorum Longus (EDL) myofibers isolated from 2 REST-cKO and 2 WT male mice (4-6 weeks old) (same mice used for smfRNA-Seq). Single myofibers were isolated and smfATAC-Seq was performed as previously described (Sahinyan K. et al. eLife, 2022 , Sahinyan K. et al. bioProtocol, 2022). All the ATAC-Seq libraries underwent 150 bp paired end sequencing on NovaSeq6000 Sprime Paired End (PE) 150 bp.
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| Contributor(s) |
Soleimani VD, Simon M |
| Citation |
https://doi.org/10.21203/rs.3.rs-4396883/v1
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| Submission date |
May 06, 2024 |
| Last update date |
Jun 12, 2024 |
| Contact name |
Vahab D. Soleimani |
| E-mail(s) |
vahab.soleimani@mcgill.ca
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| Organization name |
Jewish General Hospital
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| Department |
Lady Davis Institute for Medical Research
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| Street address |
3755 Chem. de la Côte-Sainte-Catherine
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| City |
Montreal |
| State/province |
Quebec |
| ZIP/Postal code |
H3T 1E2 |
| Country |
Canada |
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| Platforms (1) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
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| Samples (12)
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| Relations |
| BioProject |
PRJNA1108299 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE266720_RAW.tar |
1.6 Gb |
(http)(custom) |
TAR (of BW) |
SRA Run Selector |
| Raw data are available in SRA |
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