Fig. 5: Esrrg pre-mRNA splicing reshapes muscle metabolic pattern.
From: Kdm2a inhibition in skeletal muscle improves metabolic flexibility in obesity
a, ChIP–seq of isolated Gas tissue from HFD-fed Kdm2afl/fl and Kdm2aSKO mice. Venn diagram showing co-upregulated genes revealed by RNA-seq and ChIP–seq (left), and heatmap showing the differentially expressed genes (right) (n = 3 per group). FC, fold change. b, RT–qPCR (n = 6 per group) and c, western blot analysis (n = 4 per group) of Esrrg expression levels in Gas tissue from HFD-fed Kdm2afl/fl and Kdm2aSKO mice. d, ChIP–seq analysis of the Esrrg locus showing the peak locations relative to the intron (n = 3 per group). e, ChIP–qPCR analysis showing intron fragments marked by H3K36me2 in Gas tissue from HFD Kdm2afl/fl and Kdm2aSKO mice (n = 6 per group). f, RT–qPCR analysis of transcribed Esrrg intron fragments in Gas tissue from HFD-fed Kdm2afl/fl and Kdm2aSKO mice (n = 6 per group). g, Relative RNA levels of transcribed Esrrg exons 4–5 and intron 5 between Gas tissue from HFD-fed Kdm2afl/fl and Kdm2aSKO mice (n = 6 per group). h, Relative RNA levels of transcribed Esrrg introns between Gas tissue from HFD-fed Kdm2afl/fl and Kdm2aSKO mice (n = 12 per group). i, Relative expression of different isoforms of Esrrg between Gas tissue from HFD-fed Kdm2afl/fl and Kdm2aSKO mice (n = 12 per group). j, The transcribed ratio of intron 1 to exons 2–3 in Gas tissue from HFD-fed Kdm2afl/fl and Kdm2aSKO mice (n = 12 per group). k, Transfection of C2C12 myotubes with Mrg15-Flag expressing plasmids. Immunoprecipitated Mrg15-Flag was probed for H3K36me2 using an anti-H3K36me2 antibody. The quantification is in the right panel (n = 4 per group). l, In HFD Kdm2afl/fl and Kdm2aSKO mice Gas tissue, immunoprecipitated H3K36me2 was probed for Mrg15 using an anti-Mrg15 antibody. The quantification is in the right panel (n = 4 per group). m, Western blot measurement of Mrg15, H3K36me2, Kdm2a and Esrrg expression levels in C2C12 myotubes (n = 4 per group). n–o, Male mouse model of skeletal muscle-specific Esrrg knockdown, (n) detected Pgc1a, Myh4 and Myh7 protein levels and (o) the representative TEM images of Gas from Kdm2afl/fl-shCTR, Kdm2aSKO-shCTR and Kdm2aSKO-shEsrrg mice (n = 4 per group). Mitochondria are indicated by arrows. Scale bars, 10 μm. Data are expressed as mean ± s.e.m. Statistical significance was assessed by two-sided Student’s t-test (b, c, e–n).
