Fig. 4: Super-enhancers control muscle fiber-type specification via proximity and chromatin looping. | Nature Communications

Fig. 4: Super-enhancers control muscle fiber-type specification via proximity and chromatin looping.

From: Rewiring of 3D chromatin topology orchestrates transcriptional reprogramming in muscle fiber-type specification and transformation

Fig. 4

a Hockey stick plots based on input-normalized H3K27ac signals in SOL and EDL, highlighting SOL-specific genes (red) and EDL-specific genes (blue). b GO term enrichment for SE-proximal genes. c Tracks of RNA-seq, ATAC-seq, CTCF, histone modifications, and TFBS at the MYH7 locus. SE regions are marked in green. d Expression heatmap of SE-anchored genes. e Epigenetic states and chromatin interactions at MYH1 and MYH4 locus. Virtual 4 C plots show chromatin contact differences; tracks for RNA-seq, ATAC-seq, CTCF, and histone modifications are included. SE regions are marked in green. f 3D chromatin conformation models around the gene locus (bin size: 10 kb). g MYH7 and MYH4 expression changes after SE deletion in differentiated PSCs (n = 3). h Immunofluorescence staining of KLF5, Fast-type MyHC, and Slow-type MyhC in the EDL muscle. (Green: Slow-MyHC; Red: Fast-MyhC; Purple: KLF5; Blue: Nuclei) (n = 3). Fast-MyHC antibody detects total fast skeletal myosin heavy chain isoforms including MYH2, MYH4, and MYH8. The percentages indicate the proportion of KLF5-positive nuclei within slow-type or fast-type fibers. i Aggregate TF footprint plots for KLF5 in SOL and EDL muscles. j Track of KLF5 ChIP-seq in C2C12 myotubes differentiated for 0 day, 2 days, and 5 days. KLF5 peak was filled in green. k qPCR showing the expression change after KLF5 overexpression in differentiated PSCs (n = 3). l ChIP-qPCR showing the enrichment change of H3K27ac in SE-MYH1/4 after KLF5 overexpression in differentiated PSCs (n = 3). The enrichment level is shown as a percentage of input. IgG was used as a negative control. m Dual-reporter assay detecting the enhancer activity of SE-MYH1/4 after KLF5 overexpression in differentiated PSCs (n = 4). n 3C-qPCR showing the interaction frequencies of SE-MYH1/4 with gene promoters after KLF5 overexpression in differentiated PSCs (n = 3). For (g, k–n), significance was tested by a two-tailed unpaired student’s t-test. All values are presented as mean ± SEM.

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