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-3β 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-3β-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.
Highlights
Glycogen synthase kinase-3 (GSK-3) activity regulates multiple signal transduction pathways and is a key component of the network responsible for maintaining stem cell pluripotency
Because the stereotypical role for GSK-3 is to negatively regulate target substrates, our hypothesis was that FTO protein levels are kept low by GSK-3 activity, and we should observe an increase in FTO protein levels in Gsk-3 DKO embryonic stem cells (ESCs)
We initiated our study by examining the levels of FTO protein in WT and Gsk-3 DKO ESCs grown under standard conditions
Summary
Reduced GSK-3 activity and decreased levels of m6A are both associated with promotion of pluripotency. It had been shown previously that Gsk-3 DKO ESCs would remain pluripotent when grown in the absence of LIF for as long as 14 days [12]. Under these same conditions, WT ESCs progressively differentiate. WT ESCs were grown in the absence of LIF for 14 days and treated with increasing doses of the GSK-3 inhibitors SB-415,286 or lithium chloride for 48 h. Examination of FTO protein by Western blotting revealed a dose-dependent increase in FTO protein (Fig. 2, A and B) These results strongly support the notion that GSK-3 inhibition, even for a short time, is sufficient to result in elevated levels of FTO protein in ESCs
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