Extended Data Fig. 8: Epigenetic inheritance of hypermethylation at the Gck promoter throughout HG offspring development.

a, BS analysis of H19 imprinted control region methylation in sperm, oocytes, male and female pronuclei isolated from aphidicolin-treated PN3–4 zygotes. Fully methylated H19 in sperm and male pronuclei as well as unmethylated H19 in oocytes and female pronuclei indicate high purity of germ cells and pronuclei without somatic cell contamination. b, BS analysis of Line 1 5’ region methylation in the sperm and male pronuclei. Hypermethylation at 5’ Line 1 elements suggests compromised Tet3-mediated demethylation in the HG group. c–d, BS analysis of the Gck promoter methylation in sperm, MII oocytes (c) and female pronuclei (d) of the indicated groups. Relatively high levels of methylation in sperm and low levels of methylation in MII oocytes and female pronuclei at the Gck promoter were observed. e, Schematic diagram of the strategy to distinguish Gck methylation patterns on the paternal allele from maternal allele. sgRNA targeting sequence and PAM sequence are underlined and labeled in red, respectively. Through CRISPR/Cas9 mediated editing, a homozygous mutant male with a 35 bp-deletion before the Gck promoter was generated. PCR primers for bisulfite methylation analysis are also indicated. The forward primers used are allele-specific. f, Bisulfite sequencing analysis of promoter methylation of the paternal (left) and maternal (right) Gck in aphidicolin-treated PN5 zygotes using the strategy described in e. a–f, Open and filled circles represent unmethylated and methylated CpG sites, respectively. g–j, Pyrosequencing analysis of the Gck promoter methylation in pancreatic islets from offspring at 3 weeks (male: g, female: h) and 16 weeks (male: i, female: j) of age. n of the groups is indicated. k, Timeline of the average methylation level of the Gck corresponding sites in Figure 3c–e and Extended Data Fig. 8g–j. Only the percentages of 5mC were included in the methylation level in male pronuclei. l, Reduced Gck mRNA expression in female offspring pancreatic islets. n = 9 mice per group. m, Relative mRNA expression of genes with hyper-DMRs involved in insulin secretion. RT-qPCR analysis was performed for pancreatic islets from 3-week-old male offspring. Ctrl, HG: n = 11 and 12 mice, respectively. n–p, Using glucokinase activator (GKA) Dorzagliatin to reverse the effect of Gck downregulation in HG offspring. n, Glucose tolerance test (GTT) results of male offspring at 24 weeks of age after HFD feeding. Ctrl, HG, and HG + GKA: n = 8 mice per group. P values for comparisons of HG + GKA and HG are presented using *, HG and Ctrl using †. o, The stimulation index of insulin release from isolated pancreatic islets of male offspring at 24 weeks of age. Ctrl, HG and HG + GKA: n = 6, 9 and 7 mice, respectively. p, Total insulin released based on the corresponding data in o. g–p, Data are presented as the bar charts and line charts (g–l, n–p, mean ± s.e.m.) and box-and-whisker plots (m, the median (middle lines), interquartile range (boxes) and minimum to maximum values (whiskers)); two-tailed P values were calculated by one-way repeated-measurements ANOVA followed by the post hoc unpaired t-test (n) or the unpaired Student’s t-test (g–m, o–p). *P < 0.05, **P < 0.01 and ***P < 0.001. Statistical details are in Supplementary Table 5.

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