Fig 2: ModelAttenuated epigenetic suppression of muscle stem cell necroptosis is required for efficient regeneration of dystrophic muscles. CHD4/NuRD suppresses Ripk3 expression and thereby necroptosis in MuSCs of healthy muscles (bottom). Chronic dystrophy continuously evocates a demand for regeneration and persistent activation of MuSCs. Necroptosis secures removal of defective MuSCs that otherwise inhibit MuSC expansion in a non-cell-autonomous manner if not eliminated. Necroptosis in MuSCs is mediated by attenuated recruitment of CHD4/NuRD to the Ripk3 promoter in dystrophic muscles and leads to a lasting increase in necroptosis even in the absence of pro-necroptotic signals (“memory” effect) (top).

Fig 3: Chd4 Is Required for Muscle Regeneration and Suppresses Necroptosis of MuSCs(A) Macroscopic images of uninjured and CTX-injured TA muscles from control and Chd4mKO mice.(B and C) H&E staining of sections of uninjured muscle (B) and injured muscle (C) derived from muscles shown in (A). Scale bar (A–C), 100 μm.(D) Quantification of PAX7+ cells from control and Chd4mKO muscles using PAX7 antibodies (n = 3 for each group).(E–J) Immunofluorescence staining of TA muscle sections 2 weeks after CTX injury from control and mdx mice (n = 3 for each group) using antibodies against PAX7 and pMLKL to detect necroptosis (E–G) and PAX7 and cleaved CASP3 (H–J) to detect apoptosis (n = 5 for each group). Scale bar, 25 μm. (F, G, I, and J) Absolute and relative quantification of data represented in (E) and (H) (n = 3–5 for each group).Statistical analysis: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗∗p < 0.001, two-way ANOVA followed by Bonferroni post-test with alpha = 5%. All analyses indicated across the experiments were biological replicates unless otherwise stated.

Fig 4: CHD4 binds and regulates essential pancreatic β cell genes.(A) Volcano plot showing the distribution of differentially expressed genes (DEGs) in Chd4 βKO mice. (B) Heatmap showing a subset of differentially expressed β cell genes between control and Chd4 βKO mice. Heat map is mean centered using z-score scale. (C) UpSet plot showing all the genes that overlap in various combinations throughout the five datasets, Chd4 control and βKO ATAC-seq, Chd4 and Nkx2.2 βKO RNA-seq and Nkx2.2 ChIP-seq datasets. 62 genes overlapped in all five datasets. (D) IGV tracks showing genome locations and sequencing read peaks of Chd4 control and βKO ATAC-seq, Chd4 control and βKO RNA-seq and Nkx2.2 ChIP-seq datasets overlapped with CHD4 binding sites through qPCR primer locations for Slc2a2 and Kcnj5. (E) Quantification of qPCR using fold enrichment over IgG of MafA, Slc2a2 and Kcnj5 representing binding sites of CHD4 at these genes (two-tailed Student’s t-test). **P≤0.01.

Fig 5: Chd4 βKO mice are hyperglycemic with disrupted islet architecture and dysfunctional β cells.Unless otherwise noted, mouse data consists of control (Chd4fl/fl : white bars) and mutant/Chd4 βKO (Chd4fl/fl; Ins1Cre/+: red bars) genotypes.(A) Measurements of fasted blood glucose for 6-week control (n =20) and mutant (n=23) mice. (B) Islet hormone quantification at 6-weeks for Insulin (INS), Glucagon (GCG) and Somatostatin (SOM) for control (n=5) and mutant (n=3) mice. (C) Representative immunofluorescent images for 6-week control and mutant mice showing DAPI (blue), Insulin (INS, Magenta), and Glucagon (GCG, green). Scale bars: 100 μm. (D) Glucose Tolerance Test (GTT) of 6-week control (black line, n=13) and mutant (red line, n=13) mice (multiple two-tailed Student’s t-test). Area under the curve (AUC) measurement for GTT. All points normalized to 0 minutes time point prior to AUC calculation. (E) Quantification of 6-week control (n=5) and mutant (n=3) total islet area. (F) Insulin content from dissected mouse pancreas of 6-week Control (n=3) and Mutant (n=3). (G) Glucose Stimulated Insulin Secretion (GSIS) assay of 6-week control and mutant pancreas slices. Conditions were 2mM glucose, control (n=4) and mutant (n=4), 11mM glucose, Control (n=4) and Mutant (n=4), 20mM glucose, control (n=4) and mutant (n=4), 20mM glucose + KCl, control (n=4) and mutant (n=4). Fold Change is each condition normalized to 2mM basal secretion levels. For all figures, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001; two-tailed Student’s t-test unless otherwise specified.