ABSTRACT
Glycogen synthase kinase-3 (GSK-3) activity regulates multiple signal transduction pathways and is also a key component of the network responsible for maintaining stem cell pluripotency. Genetic deletion of Gsk-3α and Gsk-3ß or inhibition of GSK-3 activity via small molecules promotes stem cell pluripotency, yet the mechanism underlying the role for GSK-3 in this process remains ambiguous. Another cellular process that has been shown to affect stem cell pluripotency is mRNA methylation (m6A). Here, we describe an intersection between these components, the regulation of m6A by GSK-3. We find that protein levels for the RNA demethylase, FTO (fat mass and obesity-associated protein), are elevated in Gsk-3α;Gsk-3ß-deficient mouse embryonic stem cells (ESCs). FTO is normally phosphorylated by GSK-3, and MS identified the sites on FTO that are phosphorylated in a GSK-3-dependent fashion. GSK-3 phosphorylation of FTO leads to polyubiquitination, but in Gsk-3 knockout ESCs, that process is impaired, resulting in elevated levels of FTO protein. As a consequence of altered FTO protein levels, mRNAs in Gsk-3 knockout ESCs have 50% less m6A than WT ESCs, and m6A-Seq analysis reveals the specific mRNAs that have reduced m6A modifications. Taken together, we provide the first evidence for how m6A demethylation is regulated in mammalian cells and identify a putative novel mechanism by which GSK-3 activity regulates stem cell pluripotency.
Subject(s)
Adenosine/analogs & derivatives , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/metabolism , Glycogen Synthase Kinase 3/physiology , Mouse Embryonic Stem Cells/metabolism , RNA, Messenger/metabolism , Adenosine/metabolism , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics , Animals , Cells, Cultured , Methylation , Mice , Mice, Knockout , Mouse Embryonic Stem Cells/cytology , Phosphorylation , RNA, Messenger/genetics , UbiquitinationABSTRACT
Glycogen synthase kinase-3 (Gsk-3) is a key regulator of multiple signal transduction pathways. Recently we described a novel role for Gsk-3 in the regulation of DNA methylation at imprinted loci in mouse embryonic stem cells (ESCs), suggesting that epigenetic changes regulated by Gsk-3 are likely an unrecognized facet of Gsk-3 signaling. Here we extend our initial observation to the entire mouse genome by enriching for methylated DNA with the MethylMiner kit and performing next-generation sequencing (MBD-Seq) in wild-type and Gsk-3α(-/-);Gsk-3ß(-/-) ESCs. Consistent with our previous data, we found that 77% of known imprinted loci have reduced DNA methylation in Gsk-3-deficient ESCs. More specifically, we unambiguously identified changes in DNA methylation within regions that have been confirmed to function as imprinting control regions. In many cases, the reduced DNA methylation at imprinted loci in Gsk-3α(-/-);Gsk-3ß(-/-) ESCs was accompanied by changes in gene expression as well. Furthermore, many of the Gsk-3-dependent, differentially methylated regions (DMRs) are identical to the DMRs recently identified in uniparental ESCs. Our data demonstrate the importance of Gsk-3 activity in the maintenance of DNA methylation at a majority of the imprinted loci in ESCs and emphasize the importance of Gsk-3-mediated signal transduction in the epigenome.
Subject(s)
DNA Methylation , Embryonic Stem Cells/metabolism , Genetic Loci , Genomic Imprinting , Glycogen Synthase Kinase 3/metabolism , Animals , Glycogen Synthase Kinase 3/genetics , Glycogen Synthase Kinase 3 beta , High-Throughput Nucleotide Sequencing , Mice , Mice, Knockout , Signal TransductionABSTRACT
Kinship analysis allows the determination of sibship based on the individuals' genetic profile. In a recent empirical study, amplified fragment length polymorphism (AFLP) analysis was proposed as a test to determine kinship between Phormia regina individuals useful in inferring postmortem transport of a corpse. In order to validate this technique, mitochondrial DNA gene cytochrome oxidase II was sequenced for all individuals used in the previous study. Then, the relatedness coefficient based on AFLP profiles was determined for the pairs of individuals that had different haplotypes, and thus could not be full siblings, to determine a conservative false positive error rate of this proposed test. A majority, 96%, of pair wise comparisons of individuals with different haplotypes had relatedness coefficients <0.41 supporting the conclusion that AFLP analysis for full sibship is a valid and robust technique and thus useful for the detection of postmortem movement of a corpse.
