Fig 2: Average DNA methylation levels in DMRs of imprinted genes in 10.5dpc fetus as revealed by qAMP. Diabetic (n = 12 from 4 litters) and control (n = 12 from 4 litters) fetus were collected at 10.5dpc of gestation. DNA was digested with Hha1(Hh), HpaII(Hp) or McrBC (M) and amplified using real-time PCR. The locations of flanked restriction sites were displayed for each DMR. (A) Shown are the average methylation levels of paternally methylated gene H19 DMR; (B, C) represented the DNA methylation status in DMRs of Snrpn and Peg3. Data were presented as mean ± SE for each enzyme employed. White bar, non-diabetic group; black bar, diabetic group.

Fig 3: Exploring molecular determinants of transcriptional bursting.(A to C) Kinetic properties of transcriptional bursting of genes either with or without a TATA box. (D) Schematic representation of calculating reads per million (RPM) at the promoter and gene body from ChIP-seq data. In addition, similar calculations were also performed for enhancers (see Materials and Methods). (E) Heat maps of Spearman’s rank correlation between promoter-, gene body–, or enhancer-associated factors and either normalized intrinsic noise (N. int. noise), burst size, or burst frequency (burst freq.). (F) Effect of the Pol II pause release inhibitor, DRB, and flavopiridol treatment on the kinetic properties of transcriptional bursting. ?normalized intrinsic noise, ?burst size, and ?burst frequency are residuals of normalized intrinsic noise, burst size, and frequency of inhibitor-treated cells from that of control cells, respectively. Error bars indicate 95% confidence interval. (G) Effect of Suz12 K/O on normalized intrinsic noise. Suz12 K/O cell lines derived from Dnmt3l, Dnmt3b, Peg3, and Ctcf KI cell lines were established. Upper panel represents the result of Western blotting. In the lower part of the panel, the ?normalized intrinsic noise, ?burst size, and ?burst frequency compared with the control (cont1) are shown. Error bars indicate 95% confidence interval. Asterisks indicate significance at P < 0.05.

Fig 4: Quantitative DNA methylation in DMRs of imprinted genes in 10.5dpc placentas as revealed by qAMP. Diabetic (n = 26 from 5 litters) and nondiabetic (n = 25 from 5 litters) placentas at mid-gestation were recovered. (A) The paternally methylated H19; (B, C) Percentage methylation values at single and groups of restriction sites in the maternally methylated DMRs of Snrpn and Peg3. The lines represent the DMRs which were analyzed and the letter indicates the recognition sites of the enzymes. M, McrBc; Hh, HhaI; Hp, HpaII. (D) Confirmation of gene methylation level was determined by qAMP to expected values. HhaI and McrBc sites in chr9:106724005–106724149 are unmethylated in the genome. A primer pair flanking these sites was utilized to amplify DNA (NIH 3 T3 mouse genomic DNA and CpG methylated NIH 3 T3 mouse genomic DNA, NEB) mixed at the ratios: 100:0, 75:25, 55:45, 50:50, 45:55, 0:100. The qAMP values were close to expected values. Data are presented as mean ± SE for each enzyme employed. White bar, non-diabetic group; black bar, diabetic group; *P < 0.05, **P < 0.01.

Fig 5: Analysis of the mRNA and protein expression levels of imprinted genes in 10.5dpc placentas and fetus. (A, B) Placentas (n = 30 from 6 litters for each group) and fetus (n = 12 from 4 litters for each group) were collected at 10.5dpc of gestation. Total RNA was purified and reverse transferred into cDNA and then amplified using qRT-PCR. (A) Relative expression levels of H19, Snrpn and Peg3 in placentas. (B) Relative expression levels of H19, Snrpn and Peg3 in fetus. (C) The protein expression of Peg3 in placentas (n = 6) was investigated by western blot analysis and (D) the relative intensity of Peg3/beta-actin was evaluated by gel level analysis. (E) Placentas (n = 6) from diabetic and non-diabetic groups were stained by anti-Peg3 at 1:500 for histological analysis. Scale bar, 100 µm. Data are presented as mean ± SD. White bar, non-diabetic group; black bar, diabetic group; *P < 0.05, **P < 0.01.