Dnmt3a2 expression and DNA methylation are reduced in Gsk-3 DKO ESCs. A, Western blot of Dnmt3a isoforms in WT and Gsk-3 DKO ESCs. Protein expression of Gsk-3 isoforms in WT and Gsk-3 DKO ESCs is shown in the middle panel, whereas a Western blot for tubulin, which serves as a loading control, is shown in the bottom panel. B, real-time quantitative PCR of Dnmt3a2 showing mRNA expression of Dnmt3a2 in Gsk-3 DKO ESCs relative to WT ESCs (RQ = relative quantitation). Error bars represent S.D. between biological replicates, n = 3 (***, p < 0.001, two-tailed t test). C, real-time quantitative PCR showing H19 and Igf2 mRNA expression in Gsk-3 DKO ESCs relative to WT ESCs. Error bars represent S.D. between biological replicates, n = 3 (*, p < 0.05; ***, p < 0.001, two-tailed t test). D, bisulfite sequencing analysis of the Igf2/H19 DMD. Circles represent 15 CpG dinucleotides analyzed within the Igf2/H19 DMD. Open circles represent unmethylated CpG dinucleotides, whereas filled circles represent methylated CpG dinucleotides (p < 0.01, two-tailed t test).

Reintroduction of Dnmt3a2 into Gsk-3 DKO ESCs rescues DNA methylation. A, Western blot of Dnmt3a2 expression in Gsk-3 DKO ESCs stably expressing Dnmt3a2 from the chicken β-actin (CAG) promoter (Dnmt3a2), DKO ESCs stably expressing the puromycin-resistant empty vector (Vector), and WT ESCs. The WT, Dnmt3a2, and Vector lanes are from the same blot, with intervening lanes removed. B, bisulfite sequencing of the Igf2/H19 DMD in Dnmt3a2 rescued ESCs. Open circles represent unmethylated CpG dinucleotides, whereas filled circles represent methylated CpG dinucleotides.

Activation of components of the insulin signaling pathway reduces DNA methylation. A, Western blots showing that WT ESCs stably transfected with p110* constitutively activate insulin signaling. p-Akt, phospho-Akt; p-Gsk-3, phospho-glycogen synthase kinase-3. B, bisulfite sequencing analysis of the Igf2/H19 DMD in p110* ESCs when compared with WT ESCs (p < 0.01, two-tailed t test). C, bisulfite sequencing analysis of the Igf2r DMR2 in p110* ESCs when compared with WT ESCs. Open circles represent unmethylated CpG dinucleotides, whereas filled circles represent methylated CpG dinucleotides.

Effect on DNA methylation in Gsk-3 DKO ESCs extends to other imprinted genes but is not global. A, real-time quantitative PCR analysis of Air mRNA expression in Gsk-3 DKO ESCs relative to WT ESCs (RQ = relative quantitation). Error bars represent S.D. between biological replicates, n = 3. B, bisulfite sequencing analysis of the Igf2r DMR2 in WT and Gsk-3 DKO ESCs. Circles represent 35 CpG dinucleotides analyzed within the Igf2r DMR2. Open circles represent unmethylated CpG dinucleotides, whereas filled circles represent methylated CpG dinucleotides (p < 0.01, two-tailed t test). C, Southern blot analysis showing methylation of IAP in WT and Gsk-3 DKO ESCs. Genomic DNA from WT ESCs or Gsk-3 DKO ESCs was digested with MspI or HpaII. MspI is not sensitive to DNA methylation and cuts DNA regardless of methylation status. HpaII is an isoschizomer of MspI that does not cut methylated DNA.

Lithium inhibition of Gsk-3 results in hypomethylation. A, WT ESCs were continuously grown in the presence of either 10 mm or 20 mm LiCl for 1 month. DNA methylation at CpG nucleotides within the Igf2/H19 DMD was assessed by bisulfite sequencing (WT versus 20 mm LiCl, p < 0.01, two-tailed t test). B, DNA methylation of CpG nucleotides within the Igf2/H19 DMD in WT NSCs that were continuously grown in the presence of 5 mm LiCl for 1 month (p < 0.01, two-tailed t test). Open circles represent unmethylated CpG dinucleotides, whereas filled circles represent methylated CpG dinucleotides.

N-Myc regulates Dnmt3a2 expression in a Gsk-3-dependent manner. A, schematic representation of the locus encoding Dnmt3a isoforms. Exons are shown as the vertical bars. The Dnmt3a2 promoter we used to drive the luciferase promoter is shown as the gray box. The arrow denotes the putative N-Myc binding site that we mutated in our reporter construct. B, Dnmt3a2 promoter activity was assessed with a luciferase reporter. Reporters containing WT 1.9-kb Dnmt3a2 promoter (WT) or promoter with two point mutations (bases underlined) in putative Myc binding site (CACGTG → CAGCTG; mutation (mut)) were transfected into WT and Gsk-3 DKO ESCs. Bars represent -fold change over promoterless luciferase vector (pGLF). Error bars represent S.D. between replicate transfections, n = 3 (***, p < 0.001, two-tailed t test). C, real-time quantitative PCR analysis of N-myc mRNA expression in Gsk-3 DKO ESCs relative to WT ESCs (RQ = relative quantitation). Error bars represent S.D. between biological replicates, n = 3 (***, p < 0.001, two-tailed t test). D, Western blots showing N-Myc, Gsk-3α, Gsk-3β, and tubulin in WT and Gsk-3 DKO ESCs. E, real-time quantitative PCR analysis of N-myc mRNA expression in WT ESCs transfected with GFP siRNA and N-myc siRNA. Error bars represent S.D. between biological replicates, n = 3 (***, p < 0.001, two-tailed t test). F, Western blot for Dnmt3a2 in WT cells transfected with GFP siRNA and N-myc siRNA. G, model depicting a pathway that leads from PI3K activation to changes in DNA methylation in the nucleus.