Dissecting dual roles of MyoD during lineage conversion to mature myocytes and myogenic stem cells

Abstract

The generation of myotubes from fibroblasts upon forced MyoD expression is a classic example of transcription factor-induced reprogramming. We recently discovered that additional modulation of signaling pathways with small molecules facilitates reprogramming to more primitive induced myogenic progenitor cells (iMPCs). Here, we dissected the transcriptional and epigenetic dynamics of mouse fibroblasts undergoing reprogramming to either myotubes or iMPCs using a MyoD-inducible transgenic model. Induction of MyoD in fibroblasts combined with small molecules generated Pax7⁺ iMPCs with high similarity to primary muscle stem cells. Analysis of intermediate stages of iMPC induction revealed that extinction of the fibroblast program preceded induction of the stem cell program. Moreover, key stem cell genes gained chromatin accessibility prior to their transcriptional activation, and these regions exhibited a marked loss of DNA methylation dependent on the Tet enzymes. In contrast, myotube generation was associated with few methylation changes, incomplete and unstable reprogramming, and an insensitivity to Tet depletion. Finally, we showed that MyoD's ability to bind to unique bHLH targets was crucial for generating iMPCs but dispensable for generating myotubes. Collectively, our analyses elucidate the role of MyoD in myogenic reprogramming and derive general principles by which transcription factors and signaling pathways cooperate to rewire cell identity.

Keywords: DNA methylation; MyoD; dedifferentiation; epigenetic reprogramming; induced myogenic progenitor cells (iMPCs); satellite cells; transdifferentiation.

Figure 1.

A versatile MyoD-inducible system for the study of myogenic reprogramming. (A) Development of a doxycycline (Dox)-inducible MyoD transgenic system for studying myogenic reprogramming to myocytes and iMPCs. (B) Representative bright-field images of MEFs exposed to MyoD, FRC, or MyoD + FRC for 10 d. Scale bar, 100 µm. White arrowheads highlight myotubes; black arrowheads highlight iMPC colonies. (C) Quantitative RT-PCR analysis for myogenic markers after 10 d of exposure of MEFs to MyoD, FRC, or MyoD + FRC. Values normalized to untreated MEFs. Error bars indicate mean ± SD (n = 3). Two-tailed unpaired Student's t-test: (***) P < 0.001, (****) P < 0.0001. (D) FACS analysis for Pax7-nGFP⁺ cells using MEFs exposed to MyoD, FRC, or MyoD + FRC for 10 d. (E) Immunofluorescence images showing expression of MyoD, Desmin (Des), and Pax7-nGFP in MEFs exposed for 10 d to MyoD or MyoD + FRC. Scale bar, 50 µm. Arrowheads indicate Pax7-nGFP⁺ cells. (F) Assay to determine temporal requirement of MyoD expression (Dox) to generate Pax7-nGFP⁺ iMPCs at day 10. Error bars indicate mean ± SD (n = 2). (G) Time-course FACS analysis for Pax7-nGFP⁺ cells using Rosa26-M2rtTA; Col1a1-tetO-MyoD (rtTA/MyoD) heterozygous (Het) and homozygous (Homo) MEFs exposed to Dox and FRC. (H,I) Requirement of small molecules for iMPC establishment (H) and maintenance (I). FACS analysis for Pax7-nGFP⁺ cells using transgenic MEFs exposed to the indicated small molecules for 10 d (H) or three passages (I). Error bars indicate mean ± SD (n = 3).

Figure 2.

Pax7⁺ iMPCs share key characteristics with satellite cells. (A) Experimental outline to isolate Pax7-nGFP⁺ fresh SCs (fSCs), cultured SCs (cSCs), and iMPCs. (Dox WD) Dox withdrawal. (B) PCA of RNA-seq and ATAC-seq data for the indicated samples (n = 2 per sample). (C) Venn diagram showing the overlap of up-regulated and down-regulated differentially expressed genes (DEGs) when comparing MEFs with either iMPCs, fSCs, or cSCs (n = 2 per sample). (D) RNA-seq gene tracks of representative myogenic genes highlighted in C. (E) Immunofluorescence images showing expression of MyoD, Des, and Pax7-nGFP in established iMPCs maintained in FRC without exogenous MyoD (Dox). Scale bar, 10 µm. (F) Quantification of Pax7⁺/MyoD⁻ and Pax7⁺/MyoD⁺ iMPC subsets shown in E. Error bars indicate mean ± SD (n = 4). (G) UMAP embedding of single-cell RNA-seq data for indicated samples. (H) Expression of representative genes using the same embedding as in G. (I) Expression levels of indicated genes in Pax7-nGFP⁺ iMPCs with (“positive”) or without (“negative”) endogenous Pax7 mRNA signal. (J) Heat map showing differentially expressed marker genes (RNA-seq) between Pax7-nGFP^high (n = 2) and Pax7-nGFP^low (n = 2) iMPCs. (K) Effect of DAPT (Notch inhibitor) on iMPC maintenance.

Figure 3.

PRC2-dependent, gradual silencing of the fibroblast program precedes late induction of the stem cell program. (A) Experimental outline to study transcriptional dynamics of dedifferentiation. Pax7-nGFP⁺ fSC, cSC, and iMPC (d10) samples are identical to those in Figure 2. (B) PCA of RNA-seq data for the indicated samples (n = 2 per sample). (C) Hierarchical clustering dendrogram of RNA-seq data for the indicated samples. (D) Heat maps of gene expression (z-scores of log₂ transformed RPKM relative to all samples) for genes differentially expressed (fold change [FC] > 2, FDR < 0.01) in iMPCs (GFP⁺) and at least fSCs or cSCs compared with MEFs (n = 2 for each). (E) k-means clustering of temporal bulk gene expression profiles with representative GO terms and genes shown below. Gene expression was normalized and scaled from minimum to maximum among MEFs, reprogramming intermediates (d2–d8) and iMPCs. (F) UMAP embedding of single-cell RNA-seq data of MEFs, Pax7-nGFP⁺ iMPCs, and intermediates (Dox + FRC d2, d4, and d8). Dotted oval indicates Myog⁺ population common to days 4 and 8 and iMPCs. (G) Expression of representative genes using the same UMAP embedding as in F. (H) Expression of MEF signature genes (MEF pseudobulk) and iMPC signature genes (iMPC pseudobulk) using the same UMAP embedding as shown in F. (I) Expression of “transient UP (I)” gene signature using the same UMAP embedding. transient UP (I) genes (n = 95) were extracted from bulk RNA-seq data shown in E. (J) Quantification of Pax7-nGFP⁺ cells using MEFs exposed to MyoD + FRC and indicated siRNAs for 10 d. Targeted genes are highlighted with red dots in D. Error bars indicate mean ± SD (n = 3). Two-tailed unpaired Student's t-test: (***) P < 0.001, (****) P < 0.0001. (K) Effect of DZNep (Ezh2 inhibitor) on iMPC maintenance. (L) Quantitative RT-PCR analysis for indicated fibroblast and myogenic markers after 2 d of exposure to MyoD + FRC with or without DZNep. Values normalized to MEFs. Error bars indicate mean ± SD (n = 3). Two-tailed unpaired Student's t-test: (****) P < 0.0001, (n.s.) not significant.

Figure 4.

Chromatin opening at stem cell loci precedes their transcriptional activation. (A) PCA of ATAC-seq data for the indicated samples (n = 2 per sample). (B) Heat maps showing differentially accessible regions (DARs) that open or close (FC > 2, FDR < 0.01) in iMPCs (nGFP⁺) and at least fSCs or cSCs relative to MEFs (n = 2 for each). (C) Transcription factor motif analysis using DARs that open or close in iMPCs (Pax7-nGFP⁺) compared with MEFs. (D) Box plots of the levels of ATAC-seq signal (z-scores of log₂ transformed RPKM relative to all samples) among subsets of MyoD-enriched sites with the strongest (top 10%, n = 4482) and the weakest (bottom 10%, n = 3884) MyoD occupancy calculated from public ChIP-seq data; two-tailed unpaired Student's t-test. (E) Expression levels of indicated genes during dedifferentiation (RNA-seq). Values for Six1 and Tcf12 are relative to MEFs, while values for Jund and Fosl2 are relative to iMPCs. Error bars indicate mean ± SD (n = 2). (F) FACS analysis of Pax7-nGFP⁺ cells using MyoD-inducible MEFs exposed to indicated siRNAs. Targeted genes are highlighted with red dots in B. Error bars indicate mean ± SD (n = 3). Two-tailed unpaired Student's t-test: (***) P < 0.001. (G) Joint clustering analysis of RNA-seq and ATAC-seq dynamics for promoter regions (TSS ± 3 kb). RNA-seq expression (RPKM) and ATAC-seq signal (read density over TSS ± 3 kb) are normalized to the range between minimal and maximal levels for each gene. Representative gene tracks for each category are shown below. (H) Detection of Pax7-nGFP⁺ iMPCs using MEFs upon lentiviral overexpression of different MRFs. Error bars indicate mean ± SD (n = 3). (I) Detection of Pax7-nGFP⁺ cells in iMPCs (p3) derived from MRF-overexpressing MEFs. Error bars indicate mean ± SD (n = 3). (J) Experimental outline to determine the roles of Myf5 and Myod1 in iMPC establishment and maintenance. (R26 reporter) Cre-dependent Rosa26-GFP (R26NG) reporter. (K) Quantification of GFP⁺ cells in Cre-treated cdHomo and cdHet iMPCs maintained in FRC for three passages (p1–p4). Error bars indicate mean ± SD (n = 2). (L) Immunofluorescence images showing expression of MyoD, Des, and Rosa26-GFP after exposure of cdHomo and cdHet iMPCs (scale bar, 50 µm) to differentiation medium (DM; 2% horse serum) for 4 d following expansion in FRC for 6 d. Arrowheads for cdHomo iMPCs depict GFP⁻/Des⁺ myotubes. Arrowheads for cdHet iMPCs depict GFP⁺/Des⁺ myotubes.

Figure 5.

Active DNA demethylation is required, while de novo methylation is dispensable for reprogramming to a Pax7⁺ state. (A) Number of differentially methylated regions (DMRs) that are gained or lost when comparing MEFs with the indicated time points using RRBS analysis (FDR < 0.01, CpGs > 8, and difference > 0.2). (B) Heat maps showing DMRs that are gained or lost between MEFs and iMPCs, with inclusion of fSC and cSC samples. (C) Genome browser tracks of representative DMRs shown in B. (D) Violin plots showing DNA methylation (DNAme) levels for CpGs located within DARs (differentially accessible regions) that open (n = 6756) or close (n = 3516) between MEFs and iMPCs. (E) Transcription factor motif enrichment for regions that gain accessibility and lose methylation (Pax7-nGFP⁺ d10 iMPCs vs. MEFs). (F) Scatter plot showing correlation between global DNAme (1-kb tiles, n = 345,904) and known MyoD targets (n = 10,733) when comparing MEFs (n = 2) with Pax7-nGFP⁺ iMPCs d10 (n = 2). (G) Violin plots for DNAme at CpGs located within cSC-specific enhancers (n = 639). (H) Genome browser track showing chromatin accessibility and DNAme changes at the Mamstr locus during dedifferentiation and in established iMPCs, fSCs, and cSCs. (I) Quantitative RT-PCR analysis for Pax7 in conditional Dnmt3a knockout (Dnmt3a fl/fl) or Dnmt3b knockout (Dnmt3b fl/fl) MEFs exposed to Cre and MyoD + FRC for 10 d. Values normalized to untreated control (no Cre, MyoD, or FRC). Error bars indicate mean ± SD (n = 3). Two-tailed unpaired Student's t-test: (n.s.) not significant. (J) Detection of Pax7-nGFP⁺ cells using MyoD-inducible MEFs treated with the indicated siRNAs. Error bars indicate mean ± SD (n = 3). Two-tailed unpaired Student's t-test: (***) P < 0.001, (****) P < 0.0001, (n.s.) not significant. (K) Detection of Pax7-nGFP⁺ cells using MyoD-inducible MEFs transduced with two different shRNA vectors targeting Tet2 or a scrambled control. Error bars indicate mean ± SD (n = 3). Two-tailed unpaired Student's t-test: (****) P < 0.0001. (L) Effect of DMOG (Tet inhibitor) on iMPC maintenance. (M) Detection of Pax7-nGFP⁺ cells using MyoD-inducible MEFs overexpressing a Tet2 WT or Tet2 HD (catalytic mutant) lentiviral construct. Error bars indicate mean ± SD (n = 3). Two-tailed unpaired Student's t-test: (**) P < 0.01, (***) P < 0.001. (N, top) Dot blot analysis for global 5hmC levels in MEFs and dedifferentiation intermediates. (N, bottom) Values are normalized to methylene blue and MEFs (n = 1).

Figure 6.

Dedifferentiation and transdifferentiation follow different trajectories due to cooperative effects of MyoD and FRC. (A) Experimental outline. (B) Venn diagram depicting the overlap of DEGs up-regulated in MEFs exposed to FRC, MyoD, or MyoD + FRC (n = 2 for each) for 8 d relative to untreated MEFs. (C) Venn diagram depicting the overlap of DARs that open in MEFs exposed to FRC, MyoD, or MyoD + FRC (n = 2 for each) for 8 d relative to untreated MEFs. Enriched GO terms (bold) and selected genes are highlighted. (D) Diffusion pseudotime (DPT) representation of single-cell RNA-seq data, comparing transdifferentiating (MyoD) and dedifferentiating (MyoD + FRC) cells. Data from MyoD + FRC cells are equivalent to those in Figure 3. (E) Expression of representative genes in the DPT representation shown in D. (F) Immunofluorescence analysis of MyoD, Des, and Pax7-nGFP expression in MyoD-inducible MEFs exposed to Dox ± FRC for 7 d before withdrawing Dox for another 3 d. Scale bar, 50 µm. (G) Quantification of MyoD⁺, Pax7⁺, and MyoD⁺/Pax7⁺ cells and multinucleated myotubes shown in F. Error bars indicate mean ± SD (n = 3). Two-tailed unpaired Student's t-test: (**) P < 0.01, (***) P < 0.001. (H) Heat map showing DNAme levels for DMRs that are lost in iMPCs compared with MEFs (n = 757) with selected genes highlighted. (I) Violin plots showing DNAme levels at CpGs located within DARs that open in iMPCs relative to MEFs (n = 6756). (J) Gene tracks of the Six1 locus in MyoD, FRC, and MyoD + FRC cells with annotation of fSC/cSC enhancers below. (K) Analysis of DNAme and previously validated MyoD targets between MyoD (n = 2) and MyoD + FRC cells at day 8 (n = 2). (L) Bisulfite sequencing of putative MyoD enhancer in MEFs exposed to FRC, MyoD, or MyoD + FRC for 10 d.

Figure 7.

DNA and cofactor binding activity of MyoD uncouple its potential for dedifferentiation versus transdifferentiation. (A) Depiction of MyoD/Neurod2 mutants used in this experiment. (B) Representative bright-field images of MEFs expressing either MyoD, Neurod2, MN, WCS, or MNWCS for 10 d with or without FRC. Scale bar, 100 µm. Myotubes are highlighted by white arrowheads, and iMPC colonies are highlighted by black arrowheads. Immunofluorescence images (top of each bright-field image) show expression of Myh1. Scale bar, 100 µm. (C) Quantitative RT-PCR analysis for indicated genes in MEFs expressing MyoD, Neurod2, MN, WCS, or MNWCS for 10 d in the presence of FRC. Values are normalized to the MyoD + FRC condition. Error bars indicate mean ± SD (n = 3). (D) Detection of Pax7-nGFP⁺ cells using MEFs expressing MyoD, MN, WCS, or MNWCS for 10 d in the presence of FRC. (E) Venn diagram showing the overlap of down-regulated and up-regulated DEGs (FC > 2, FDR < 0.01) between MyoD + FRC and MN + FRC cells (day 6) relative to MEFs (n = 2 per sample). Enriched GO terms (bold) and selected genes are highlighted. (F) Heat maps showing DARs that close or open (FC > 2, FDR < 0.01) relative to MEFs in MyoD + FRC and MN + FRC cells at day 6 (n = 2 per sample). (G) Transcription factor motif enrichment analysis based on DARs that open in MyoD + FRC and MN + FRC cells relative to MEFs at day 6. (H) Analysis of DNAme and published MyoD targets (n = 10,733) between MyoD + FRC (n = 2) and MN + FRC (n = 2) cells at day 6. (I) Quantification of Pax7-nGFP⁺ cells using MyoD-inducible MEFs exposed to the indicated siRNAs. Error bars indicate mean ± SD (n = 3). Two-tailed unpaired Student's t-test: (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001. (J) Effect of AG14631 (Parp1 inhibitor) on iMPC induction. (K) Effect of AG14631 on transdifferentiation. Scale bars, 50 µm.