Glycogen Synthase Kinase-3 Loss-Of-Function Studies In Mus musculus and Murine Embryonic Stem Cells

Glycogen synthase kinase-3 (Gsk-3) isoforms, Gsk-3alpha and Gsk-3beta, are constitutively active, largely inhibitory serine/threonine kinases that are involved in diverse cellular pathways ranging from Wnt signaling and insulin signaling to the maintenance of pluripotency in embryonic stem cells and neural progenitors. Underscoring their biological significance, altered Gsk-3 activity has been implicated in diabetes, cancer, Alzheimer's disease, schizophrenia, and bipolar disorder. Gsk-3 isoforms show gross functional redundancy in many cellular contexts including cultured embryonic stem cells (ESC) and neural progenitors. However, the consequences of genetic deletion are not equivalent for each isoform in vivo, while Gsk-3alpha null mice are viable, Gsk-3beta null mice are embryonic lethal. The presence of two distinct Gsk-3 isoforms is evolutionarily conserved throughout vertebrate species, and the distinct phenotypes of Gsk-3alpha and Gsk-3beta null mice suggest unique functions for each isoform. This work was designed to test the hypothesis that Gsk-3alpha and Gsk-3beta have unique functions in the regulation of downstream transcriptional targets. In order to explore these potentially distinct functions, we carried out genome-wide gene expression analysis in Gsk-3alpha-/-, Gsk-3beta -/-, and Gsk-3beta-/- Gsk-3beta-/- double knockout (DKO) ESCs. We found that Gsk-3alpha and Gsk-3beta are essentially redundant in the context of the regulation of gene expression in cultured ESCs. However, we found a large number of genes are misregulated in the absence of both isoforms, many of which have not been previously linked with Gsk-3. In addition, we have also generated Gsk-3alpha and Gsk-3beta conditional knockout (CKO) mice in order to understand the distinct functions of Gsk-3 isoforms in vivo. We have shown that these conditional alleles are functional, and that Gsk-3alpha-/- and Gsk-3beta -/- mice derived from our CKO lines concur with previously reported phenotypes in conventional knockout mice. In the course of our studies, we have also discovered a novel role for Gsk-3 isoforms in the regulation of DNA methylation at imprinted loci. We found that deletion of both Gsk-3alpha and Gsk-3beta in mouse embryonic stem cells (ESCs), resulted in reduced expression of the de novo DNA methyltransferase Dnmt3a2, causing misregulated expression of imprinted genes and hypomethylation of corresponding imprinted loci. Treatment of wild-type ESCs and neural stem cells with the Gsk-3 inhibitor, lithium, phenocopies the DNA hypomethylation defect at imprinted loci. We show that phosphorylation and inhibition of Gsk-3 isoforms via activation of phosphoinositide 3-kinase (PI3K) also results in reduced DNA methylation. Finally, we find 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 DNA methylation at imprinted loci.