Fig 1.
PCM1 selectively labels myonuclei in isolated single fibers.
(A) Max intensity Z projection of single fiber from transgenic mouse expressing the H2B-GFP (Green) construct under the control of ACTA1 stained against PCM1 (magenta). Counterstained with DAPI to visualize DNA (Blue). Arrow: nucleus negative for both PCM1 and H2B-GFP. Scale bar 50 μm (B) High resolution image of single fiber showing GFP positive and negative nuclei. Scale bar 10 μm.
Fig 2.
PCM1 can be used to isolate the myonuclei population from whole skeletal muscle tissue.
(A) Nuclei from whole muscle lysate stained with PCM1 antibody (green) and Hoechst to visualize DNA (blue). Scale bar 20 μm. (B) Representative scatterplot showing identification of single nuclei from skeletal muscle tissue by flow cytometry. (C-D) Representative scatterplot of nuclei labeled with PCM1 and IgG, respectively. (E) Representative scatterplot of co-localization between nuclei expressing H2B-GFP and are positive for PCM1 in tibialis anterior (TA) from mouse expressing the H2B-GFP construct under the control of ACTA1, analyzed by flow cytometry. F) Co-localization between PCM1 and GFP positive nuclei in TA in the transgenic mouse model (n = 3), error bars SEM. (G) Workflow used to isolate the myo-specific nuclei from skeletal muscle. (H-J) Representative histograms of nuclear distribution and magnetic sorting efficiency for the three muscles TA, EDL and soleus (SOL) from mice by flow cytometry. (K) Quantification of nuclear distribution in full tissue (n = 3). (L) Quantification of sorting efficiency (n = 3).
Fig 3.
The epigenome of whole tissue is different from the myonuclei specific.
(A-B) Comparison of H3K27ac enrichment at gene promoter regions from whole muscle tissue and PCM1 isolated myonuclei for soleus and EDL, respectively. (C-D) Gene ontology enrichment analysis of promoter regions with H3K27ac enrichment specific for whole muscle tissue (grey). See S1A and S1B Table for full list. (E-F) Heatmaps showing H3K27ac enrichment at the promoter regions in (A-B) -whole tissue and sorted myonuclei, in soleus and EDL, respectively. (G) ChIP-Seq profiles of H3K27ac enrichment in whole tissue and sorted myonuclei for loci used to define cellular populations. Whole muscle H3K27ac data from [42]. Gene ontology identified with GREAT [143] using single closest gene.
Fig 4.
The differences in the epigenome reflects the physiological differences.
(A-B) Venn diagrams showing numbers of peaks with similar or different enrichment for the histone marks H3K27ac and H3K4me3 in myonuclei. (C) ChIP-Seq profiles of the myo-specific H3K4me3 and H3K27ac enrichment at the genomic loci containing the genes coding for the contractile proteins defining the muscle types: the main myosin types at the MyHC locus at chromosome 11 (Myh1,-2,-4) coding for the fast isoforms as well as the embryonically expressed Myh3 and the non-coding RNA Linc-Myh, the slow Myh7 gene at chromosome 14, and the Troponin genes, the slow Tnnt1 and the fast Tnnt3 at chromosome 7. (D) Relative H3K27ac enrichment for peaks overlapping the promoter regions for the four-principle myosin heavy chains (Myh1,-2,-4 and -7) and protein level for the corresponding proteins assessed by immunohistochemistry (MyHC1, MyHC-2A, -2B and -2X) from [72]. (E) Fold change differences in soleus/EDL in the H3K27ac enrichment in peaks overlapping the promoter region for the genes coding for phenotype defining contractile proteins. Gene names in black, protein names in grey. No significant difference in the enrichment for Mybpc1.
Fig 5.
Genes involved in the calcium signaling displayed differences in the epigenome.
(A) ChIP-Seq profiles of the myo-specific H3K4me3 and H3K27ac enrichment at the genomic loci containing the genes coding for calcium signaling proteins: ATPase Sarcoplasmic/Endoplasmic Reticulum Ca2+ Transporting (Atp2a1,SERCA1) at chromosome 7, Atp2a2 (SERCA2) at chromosome 5, Calsequestrin 1 (Casq1) at chromosome 1, Calsequestrin 2 (Casq2) at chromosome 3 and Parvalbumin (Pvalb) at chromosome 15, in soleus and EDL, respectively.
Fig 6.
Genes related to metabolism displayed differences in the epigenome.
(A-B) Plot of enriched gene ontologies associated with the myo-specific differently enriched H3K27ac peaks in soleus and EDL, respectively. The ontologies are shown after redundancy reduction with ReViGO (148) by functional similarity. Terms are clustered in the semantic space by their similarity, without intrinsic meaning to the semantic space units. Selected terms for each cluster are shown, see S3A and S3B Table for full list. Ontologies were identified with GREAT [143] using closest promoter. (C-D) ChIP-Seq profiles of the myo-specific H3K4me3 and H3K27ac enrichment in soleus and EDL, for two loci with different enrichment, encoding Fatty acid translocase (Cd36/Fat) at chromosome 5 and Lactate dehydrogenase A (LDHA) at chromosome 7, respectively. (E-F) Schematic representation showing the genes encoding the enzymes involved in the fatty-acid oxidation and glycolysis, respectively. Genes with a differently enriched H3K27ac promoter colored in red and blue for soleus and EDL, respectively. Grey denoted no difference in enrichment. Differently enriched alternative promoters and enhancer regions are shown as nodes with relative distance to the primary promoter region. Color code denotes the FDR significance level of the differential enrichment between the two muscle types.
Fig 7.
Soleus and EDL are enriched for different regulatory networks.
Transcriptional regulatory network for soleus (A) and EDL (B). Top row constitutes of transcription factors with motif enriched in DE H3K27ac peaks, the middle of associated genes with a DE promoter (< 100 kb). The last row shows biological functions assigned to the genes. Edges between transcription factor and genes represent identification of predicted binding sites for the respective transcription factor and the gene it is connected to. Edges between genes and biological function denote the functional properties assigned to the gene. Transcription factor motifs were identified with AME [151] using high-quality motifs from human and mouse FDR < 1x10-5. For factors with similar motifs, the factor with the most significantly enriched promoter in the respective muscle type was used. Biological functions represent functional annotation clusters associated with the differential enriched genes (Clusters identified with David [144, 145] using an enrichment score >2.2 for the clusters and a p-value and fold change for the ontologies at < 5x10-2 and >3, respectively). Network visualized with Cytoscape. For the sake of clarity, only selected genes, with their most significant edge to each transcription factor, and terms are shown. See S5 Table for the full names of transcription factors and motifs and S6 Table for functional annotation of clusters and genes.