Fig. 6: Long-range interactions connect WHR-associated rs878639 to EIF6 expression.
From: Skeletal muscle enhancer interactions identify genes controlling whole-body metabolism
a Regional visualization of WHR GWAS data7 at the region around UQCC1 with highlight of rs878639 and linked SNPs. Position of all enhancers (green), palmitate or TNFα regulated enhancers (red) are indicated below. Identified Promoter Capture Hi-C interactions for the UQCC1 + 100 kb (pink) and the UQCC1 + 26 kb, UQCC1 + 16 kb, and UQCC1 + 13 kb enhancer region (blue) are illustrated. b Quantification of H3K27ac counts pr. million at the UQCC1 + 100 kb and UQCC1 + 26 kb, UQCC1 + 16 kb, and UQCC1 + 13 kb enhancers from control, palmitate-treated or TNFα-treated cells. Values are represented as mean ± S.D. (n = 4 biological replicates). c, Quantification of MMP24, EIF6 and GDF5 RNA-seq counts pr. million from control, palmitate-treated or TNFα-treated cells. Values are represented as mean ± S.D. (n = 4 biological replicates). For b, c; Stars indicate enhancers or genes that are significantly regulated in the ChIP-seq or RNA-seq analyses (****FDR < 0.0001, ***FDR < 0.001, **FDR < 0.01, *FDR < 0.05). d, e eQTL analysis in skeletal muscle between rs878639 and EIF6 (d) or MMP24 (e) expression. Data are presented as box plots where the horizontal line represent the median, vertical middle bars represent the first and third quartiles, and black dots denote outliers beyond 1.5 times the interquartile range (Tukey plot). f–h Skeletal muscle expression of Eif6 is positively correlated with running distance (f), VO2basal (g) and VO2max (h) in BXD mice strains. Statistics were performed using Spearmans rank correlation analysis. i, Eif6 mRNA levels in siScr or siEif6#2 transfected C2C12 myoblasts. Expression data was normalized to housekeeping Gapdh expression levels. Values are represented as mean ± S.D. (n = 7) J, Real-time measurements of oxygen consumption rates (OCR) by Seahorse Extracellular Flux Analyzer in siScr or siEif6#2 transfected C2C12 cells. OCR was measured under basal conditions and after injection of oligomycin, FCCP, and antimycin A combined with rotenone at indicated time points. Values are represented as mean ± S.D. (n = 8). k–l OCR area under the curve (AUC) values (k) or mean OCR for the time points during FCCP-induced maximal respiration (l) for siScr or siEif6#2 transfected C2C12 myoblasts. Values are represented as mean ± S.D. (n = 8) m, Quantification of Western blots of the mitochondrial oxidative complexes V, III, II and I in siScr or siEif6#2 transfected C2C12 cells. Values are represented as mean ± S.D. (n = 12), statistical tests were performed by a two-tailed t-test (n.s., p > 0.05).
