FGF Signaling Inhibition in ESCs Drives Rapid Genome-wide Demethylation to the Epigenetic Ground State of Pluripotency

Abstract

SummaryGenome-wide erasure of DNA methylation takes place in primordial germ cells (PGCs) and early embryos and is linked with pluripotency. Inhibition of Erk1/2 and Gsk3β signaling in mouse embryonic stem cells (ESCs) by small-molecule inhibitors (called 2i) has recently been shown to induce hypomethylation. We show by whole-genome bisulphite sequencing that 2i induces rapid and genome-wide demethylation on a scale and pattern similar to that in migratory PGCs and early embryos. Major satellites, intracisternal A particles (IAPs), and imprinted genes remain relatively resistant to erasure. Demethylation involves oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), impaired maintenance of 5mC and 5hmC, and repression of the de novo methyltransferases (Dnmt3a and Dnmt3b) and Dnmt3L. We identify a Prdm14- and Nanog-binding cis-acting regulatory region in Dnmt3b that is highly responsive to signaling. These insights provide a framework for understanding how signaling pathways regulate reprogramming to an epigenetic ground state of pluripotency.

Esrrb extinction triggers dismantling of naïve pluripotency and marks commitment to differentiation

“…In contrast, the Esrrb enhancer (Festuccia et al , 2012; Moorthy et al , 2017), which is active in ESCs but inactive in primed pluripotent cells, becomes completely methylated as cells commit to differentiate. Our results build on previous reports that accumulation of DNA methylation at the regulatory regions of pluripotency genes accompanies loss of naïve pluripotency (Ficz et al , 2013; Habibi et al , 2013; Hackett et al , 2013; Leitch et al , 2013). Moreover, our results illustrate the progressive nature by which accumulation of DNA methylation occurs, as exemplified by the intermediate levels of methylation detected at the Esrrb enhancer in Esrrb‐GFP medium ESC.…”

“…Locus‐specific DNA methylation at pluripotency gene regulatory regions characterizes loss of naïve pluripotency (Ficz et al , 2013; Habibi et al , 2013; Hackett et al , 2013; Leitch et al , 2013). Therefore, to test whether CpG methylation drives the loss of pluripotency associated with Esrrb extinction, CpG DNA methylation was analyzed in fractionated SSEA1 + E‐GFPd1 ESCs.…”

Stable Methylation at Promoters Distinguishes Epiblast Stem Cells from Embryonic Stem Cells and the In Vivo Epiblasts

“…This is also the case for reverted ESCs, which become more demethylated when grown in the same serum-free conditions (data not shown). Several studies have highlighted a complex crosstalk between FGF signaling, Prdm14, Dnmt3b, and Tet that could play an inductive role in this process [19,64,65]. In vivo, FGF signaling activity as revealed by phosphorylated Erk1/2 is low in the pregastrulation epiblast cells but becomes activated upon derivation in the presence of FGF2-containing medium [62,66].…”

The molecular underpinnings of totipotency

“…The PGC-associated gene Prdm14 maintains potency in part by repressing DNA methylation enzymes [56]. ES cell cultures maintained in 2i/ LIF, which contain a totipotent subpopulation of cells, are hypomethylated [57][58][59] and exhibit reduced levels of the repressive chromatin mark H3K27me3 [59]. Similarly, in 2C-like ES cells, MuERV-L elements are hypomethylated and exhibit an increase in histone marks associated with active transcription, such as H3K4 methylation and H3 and H4 acetylation [31].…”