Phosphatidylinositol 3-Kinase (PI3K) Signaling via Glycogen Synthase Kinase-3 (Gsk-3) Regulates DNA Methylation of Imprinted Loci

Anthony P Popkie,Leigh C Zeidner,Ashley M Albrecht,Anthony D'Ippolito,Sigrid Eckardt,David E Newsom,Joanna Groden,Bradley W Doble,Bruce Aronow,K John Mclaughlin,Peter White,Christopher J Phiel

doi:10.1074/jbc.m110.170704

Abstract

Glycogen synthase kinase-3 (Gsk-3) isoforms, Gsk-3α and Gsk-3β, are constitutively active, largely inhibitory kinases involved in signal transduction. Underscoring their biological significance, altered Gsk-3 activity has been implicated in diabetes, Alzheimer disease, schizophrenia, and bipolar disorder. Here, we demonstrate that deletion of both Gsk-3α and Gsk-3β in mouse embryonic stem cells results in reduced expression of the de novo DNA methyltransferase Dnmt3a2, causing misexpression of the imprinted genes Igf2, H19, and Igf2r and hypomethylation of their corresponding imprinted control regions. Treatment of wild-type embryonic stem cells and neural stem cells with the Gsk-3 inhibitor, lithium, phenocopies the DNA hypomethylation at these imprinted loci. We show that inhibition of Gsk-3 by phosphatidylinositol 3-kinase (PI3K)-mediated activation of Akt also results in reduced DNA methylation at these imprinted loci. Finally, we find that N-Myc is a potent Gsk-3-dependent regulator of Dnmt3a2 expression. In summary, we have identified a signal transduction pathway that is capable of altering the DNA methylation of imprinted loci.

Highlights

Glycogen synthase kinase-3 (Gsk-3) isoforms, Gsk-3␣ and Gsk-3␤, are constitutively active, largely inhibitory kinases involved in signal transduction
Bisulfite sequencing of the H19/Igf2 differentially methylated domain (DMD) and Igf2r differentially methylated region 2 (DMR2) demonstrated a substantial reduction of DNA methylation levels in p110* stable cells, similar to that seen in Gsk-3 double knock-out (DKO) embryonic stem cells (ESCs) (Fig. 5, B and C)
We provide evidence that several imprinted genes are hypomethylated as a consequence of the loss of Gsk-3 activity, it remains to be determined whether DNA methylation is reduced at all known imprinted loci in Gsk-3 DKO ESCs or whether the effect on DNA methylation extends to non-imprinted genes

Summary

Introduction

Glycogen synthase kinase-3 (Gsk-3) isoforms, Gsk-3␣ and Gsk-3␤, are constitutively active, largely inhibitory kinases involved in signal transduction. We demonstrate that deletion of both Gsk-3␣ and Gsk-3␤ in mouse embryonic stem cells results in reduced expression of the de novo DNA methyltransferase Dnmt3a2, causing misexpression of the imprinted genes Igf2, H19, and Igf2r and hypomethylation of their corresponding imprinted control regions.

Results

Conclusion