1. Volume 18, Issue 2, Pages 229-242 (February 2016)
Regulation of Skeletal Muscle Stem Cell Quiescence by Suv4-20h1-Dependent Facultative Heterochromatin Formation 
Verawan Boonsanay, Ting Zhang, Angelina Georgieva, Sawa Kostin, Hui Qi, Xuejun Yuan, Yonggang Zhou, Thomas Braun 
Cell Stem Cell 
Volume 18, Issue 2, Pages (February 2016)
DOI: /j.stem
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2. Cell Stem Cell 2016 18, 229-242DOI: (10.1016/j.stem.2015.11.002)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1 MuSCs Maintain a Facultative Heterochromatic Configuration
(A) Immunofluorescence staining of the chromatin modification H3K9me3, H3K27me3, H4K20me1, H4K20me2, and H4K20me3 in MuSCs (Pax7+) attached to freshly isolated myofibers from EDL muscles. Scale bar represents 20 μm.
(B) Quantification of different epigenetic modifications and Pax7+ double-positive cells.
(C) Western blot analysis of chromatin modifications in quiescent (Quiescent SC) and proliferating satellite cells (Proliferating SC) after 2 days in culture. Histone H3 is used as internal control.
(D) RT-qPCR expression analysis of enzymes regulating H4K20 methylation in quiescent MuSCs and proliferating MuSCs. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < (n = 3).
(E) RT-qPCR expression analysis of enzymes regulating H3K27 methylation in quiescent and proliferating MuSCs. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p <  (n = 3).
See also Figure S1.
Cell Stem Cell  , DOI: ( /j.stem )
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4. Figure 2
Inactivation of Suv4-20h1 in MuSCs Reduces fHC Formation, Disrupts MuSC Quiescence, and Impairs Long-Term Muscle Regeneration
(A) Representative EM images demonstrating reduction of condensed heterochromatin and increase of cytoplasm in MuSC of Suv4-20h1mKO mice. Scale bar represents 2 μm. Percentages of MuSC numbers with distinct heterochromatin contents (hetero) in WT and mutant muscles are shown on the right. ∗p < 0.05, ∗∗∗p < (n = 3).
(B and C) Immunofluorescence analysis of H4K20me2 (red) (B) and H3K27me3 (red) (C) in MuSCs (Pax7+, green) attached to freshly isolated myofibers of Suv4-20h1mKO mutant and WT mice. Scale bar represents 20 μm. Quantifications are shown on the right. ∗∗p < 0.01, ∗∗∗p < (n = 6).
(D) Immunofluorescence staining of activated (Pax7+/MyoD+) MuSCs of WT and Suv4-20h1mKO mutant mice. Scale bar represents 20 μm. Quantifications are shown on the right. ∗∗p < 0.01 (n = 6).
(E) H&E staining of TA muscles from WT and Suv4-20h1mKO mice after three times CTX injection. Scale bar represents 20 μm.
(F) Quantification of Pax7+ MuSCs in regenerated TA muscles of Suv4-20h1mKO and WT muscles after three rounds of regeneration (ten sections per mouse). ∗∗p < 0.01 (n = 6).
See also Figures S2 and S3.
Cell Stem Cell  , DOI: ( /j.stem )
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5. Figure 3
Inactivation of Suv4-20h1 in Adult MuSCs Causes Loss of fHC, Stem Cell Quiescence, and Long-Term Muscle Regeneration
(A) Representative EM images demonstrating reduction of condensed heterochromatin in MuSCs of Suv4-20h1SKO mice. Scale bar represents 2 μm. Percentages of MuSC numbers with distinct heterochromatin contents (hetero) in WT and mutant muscles are shown on the right. ∗∗p < 0.01, ∗∗∗p < (n = 3).
(B and C) Quantifications of H4K20me2 (B) and H3K27me3 (C) Pax7 double-positive cells in Suv4-20h1sKO mutant mice. ∗∗∗p < (n = 3).
(D) Quantifications of Pax7+/MyoD+ MuSCs. ∗∗∗p < (n = 3).
(E) Immunofluorescence staining of EdU+/Pax7+ double-positive MuSCs in WT and Suv4-20h1sKO after 14 days of EdU incorporation. Quantifications are shown on the right. ∗p < 0.05 (n = 3). Scale bar represents 20 μm.
(F) Macroscopic view of decreased TA muscle mass in Suv4-20h1sKO mutant compared with WT mice after repeated muscle injury. Quantifications of TA muscle masses with and without CTX injection are shown on the right. ∗p < 0.05 (n = 6).
(G) Quantifications of MuSC numbers per 1 mm2 in regenerating and non-regenerating TA muscles of Suv4-20h1sKO and WT muscles after three rounds of regeneration. ∗∗p < 0.01 (n = 6).
(H) Quantification of Pax7+/MyoG+ cells of WT and Suv4-20h1sKO indicating accelerated differentiation in Suv4-20h1sKO mutant mice. ∗p < 0.05 (n = 3).
See also Figure S4.
Cell Stem Cell  , DOI: ( /j.stem )
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6. Figure 4
Suv4-20h1-Mediated fHC Formation Is Required for MyoD Gene Silencing in MuSCs
(A) RT-qPCR analyses of MyoD upregulation in MuSCs of Suv4-20h1mKO compared with WT mice. ∗p < 0.05 (n = 6).
(B) Western blot analysis of increased MyoD protein levels in muscles of Suv4-20h1mKO mice. β-tubulin serves as loading control.
(C–F) ChIP-qPCR assays of Suv4-20h1 (C), H4K20me2 (D), histone H3 (E), and H3K4me3 (F) at distinct MyoD regulatory regions of quiescent MuSCs from Suv4-20h1mKO and WT mice. Relative enrichment of Suv4-20h1 and histone H3 was normalized against input DNA, while enrichment of H4K20me2 and H3K4me3 was normalized to histone H3. ∗p < 0.05 compared with IgG control (n = 3).
(G) Representative MyoD DNA FISH images of the localization of the MyoD locus (red) in the center of Suv4-20h1mKO MuSC nuclei compared to a peripheral localization in nuclei of WT MuSCs. Quantification of the distance (μm) of MyoD loci relative to the nuclear lamina within different groups of MuSCs (y axis) in WT and Suv4-20h1mKO mice is shown on the right; 150 cells from three different pairs of WT and mutant mice were analyzed by DNA FISH.
Scale bar represents 5 μm. See also Figure S5.
Cell Stem Cell  , DOI: ( /j.stem )
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7. Figure 5
Reduction of MyoD Expression Partially Rescues Condensed Heterochromatin Formation in Suv4-20h1-Deficient MuSCs
(A and B) Representative EM micrographs of heterochromatin condensations in different mouse mutants. Scale bar represents 2 μm. Quantifications of the heterochromatin content are shown below. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < (n = 3).
(C and D) Immunofluorescence based quantification of H3K27me3+/Pax7+ MuSC in different mouse mutants.
See also Figure S6.
Cell Stem Cell  , DOI: ( /j.stem )
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8. Figure 6
Reduction of MyoD Expression Partially Restores MuSC Quiescence and Rescues Long-Term Muscle Regeneration in Suv4-20h1-Deficient Muscle
(A) Immunofluorescence based quantification of Pax7+/MyoD+ MuSCs in TA muscles of WT and different mutant mice (ten sections per mouse, ∗p < 0.05, ∗∗p < 0.01; n = 5).
(B) Macroscopic images of regenerating TA muscles of different mouse mutants after three rounds of regeneration. Quantifications of weights of regenerated muscles are shown on the right. ∗∗p < 0.01 (n = 5).
(C) H&E staining of regenerating TA muscles after three rounds of regeneration. Scale bar represents 20 μm.
(D) Quantification of MuSC numbers per 100 myonuclei by EM in regenerating muscles from different mouse mutant strains. ∗∗∗p < (n = 5).
(E) Quantitative assessment of MuSC numbers identified by Pax7 immunofluorescence in regenerating muscles of different mutant strains. ∗p < 0.05, ∗∗p < 0.01 (n = 3).
See also Figure S7.
Cell Stem Cell  , DOI: ( /j.stem )
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9. Figure 7
MuSCs of mdx Mice Show Reduced Suv4-20h1 Expression and fHC Content
(A) Quantification of MyoD+/Pax7+ MuSCs in TA muscles of mdx mice. ∗∗p < 0.01 (n = 3).
(B) RT-qPCR analyses of Suv4-20h1 and MyoD expression in FACS-isolated Pax7+ MuSC from WT and mdx mice. ∗p < 0.05, ∗∗∗p < (n = 3).
(C and D) Quantifications of H4K20me2+/Pax7+ (C) and H3K27me3+/Pax7+ (D) MuSCs in TA muscles. H4K20me2-positive MuSCs were separated into three groups according to staining intensity. ∗p < 0.05, ∗∗p < 0.01, (n = 3).
(E) Representative EM images demonstrating reduction of condensed heterochromatin in MuSCs of mdx compared to WT mice. Scale bar represents 2 μm. Quantitative assessment of the heterochromatin content in MuSCs of WT and mdx mice are shown on the right. ∗∗p < 0.01, ∗∗∗∗p < (n = 3).
(F) Simplified model of the role of Suv4-20h1 in the regulation of fHC formation and MuSC quiescence. Suv4-20h1-dependent fHC formation arrests the MyoD gene locus in the nuclear periphery where transcriptional activity is low.
See also Figure S6.
Cell Stem Cell  , DOI: ( /j.stem )
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