Fig. 8: Maternal obesity (MO) increases DNA methylation in Dio3os promoter in oocytes and offspring brown fat (BAT).
From: Imprinted lncRNA Dio3os preprograms intergenerational brown fat development and obesity resistance
a Diagram displays proximal region (R-A) and promoter (R-B) in Dio3os with enriched CpG island. The region is highly conserved between the human and mouse genomes. b Enrichment of 5-methylcytosine (5mC) quantified by MeDIP-qPCR in Dio3os proximal region R-A and promoter R-B with enriched CpG islands in fetal BAT at E18 and P0. Mock IgG was used as a negative control (n = 7). c Regression of Dio3os DNA methylation with Dio3os expression in fetal BAT. Statistical analyses were performed by Pearson correlation. d, e Diagram of bisulfite pyrosequencing to assay percentage of CpG methylation in the Dio3os promoter (d). CpG methylation percentage in the Dio3os promoter in offspring BAT at E18.5 (n = 8), birth (n = 10), weaning (n = 6) and 4-month-old (n = 8) (e). Whiskers of box plots show mean and individual values from minimum to maximum. f Dynamics of CpG methylation percentage in Dio3os promoter in offspring BAT measured by bisulfite pyrosequencing (n = 8 at E18.5, n = 10 at P0, n = 6 at P21, n = 8 at 4-month-old). g Enrichment of DNA methylation (5mC, % input) in Dio3os proximal CpG rich region R-A and promoter R-B region in oocytes of control and obese dam measured by MeDIP-qPCR (350–400 pooled oocytes, n = 7). Mock IgG was used as a negative control. h, i mRNA expression of Dio3os and Dio3 in dam oocytes (150 pooled oocytes, n = 6). j Mitochondrial DNA (mtDNA) copy in maternal oocytes (25–30 oocytes, n = 6). Amplification of mitochondrial genes was normalized to GAPDH and 18 S rRNA. k Immunoblotting measurement of DIO3, DRP1, DRP1 phosphorylation at S616 and VDAC protein contents in oocytes (250 pooled oocytes, n = 6). β-actin was used as a loading control. l Mitochondria (mito-tracker; green color) and mitochondrial membrane potentials (TMRE; red color) in oocytes. Signal intensity was qualified by Image-J (four oocytes, n = 6 mice). Data are mean ± s.e.m. and each pregnancy (dam) was treated as replicate unit; statistical analysis was conducted with the unpaired Student’s t test with the two-tailed distribution.
