2i Maintains a Naive Ground State in ESCs through Two Distinct Epigenetic Mechanisms

Abstract

SummaryMouse embryonic stem cells (ESCs) are maintained in serum with leukemia inhibitory factor (LIF) to maintain self-renewal and pluripotency. Recently, a 2i culture method was reported using a combination of MEK inhibition (MEKi) and GSK3 inhibition (GSK3i) with LIF to maintain ESCs in a naive ground state. How 2i maintains a ground state of ESCs remains elusive. Here we show that MEKi and GSK3i maintain the ESC ground state by downregulating global DNA methylation through two distinct mechanisms. MEK1 phosphorylates JMJD2C for ubiquitin-mediated protein degradation. Therefore, MEKi increased JMJD2C protein levels but decreased DNMT3 expression. JMJD2C promotes TET1 activity to increase 5-hydroxymethylcytosine (5hmC) levels. GSK3i suppressed DNMT3 expression, thereby decreasing DNA methylation without affecting 5hmC levels. Furthermore, 2i increased PRDM14 expression to inhibit DNMT3A/B protein expression by promoting G9a-mediated DNMT3A/B protein degradation. Collectively, 2i allows ESCs to maintain a naive ground state through JMJD2C-dependent TET1 activation and PRDM14/G9a-mediated DNMT3A/B protein degradation.

KLF4 protein stability regulated by interaction with pluripotency transcription factors overrides transcriptional control

“…Naïve pluripotent ES cells cultured in LIF/2i are thought to maintain a more balanced and stable state than ES cells maintained in LIF/serum through higher and more uniform expression of the major pluripotency transcription factors in the cell population (Tosolini and Jouneau 2016;Sim et al 2017). Our results indicate that for KLF4 protein, this higher and more uniform expression in LIF/2i is achieved through posttranslational protein stabilization.…”

Zfp281 orchestrates interconversion of pluripotent states by engaging Ehmt1 and Zic2

“…We, however, showed that Zfp281 drives exit from naïve pluripotency independent of Dnmt3a, Dnmt3b, Tet1, and Tet2. Although the specific contributions of 5-methylcytosine, H3K9me2, and other Ehmt substrates (Sim et al, 2017) to pluripotent cell plasticity remain to be determined, our findings suggest that resolution of naïve pluripotency in vitro is masked or mechanistically distinct in heterogeneous Serum/Lif ESC cultures. Similarly, Zic2 has previously been reported to act as a repressor in metastable serum/Lif ESCs (Luo et al, 2015), but we detect only minor transcriptional defects in naïve Zic2 KO ESCs.…”

“…Nevertheless, 38% of target genes were downregulated. Changes in the absence of Ehmt1 are therefore likely consequential to both direct and indirect effects and may also include the contribution of non-histone Ehmt1 substrates (Sim et al, 2017) to transcription. Based on mRNA levels, Zic2 32 h cells were not separated from matching control cells ( Fig 6A-D).…”

Physical properties and actin organization in embryonic stem cells depend on differentiation stage

“…Embryonic stem cells are known to exhibit dramatic changes in morphology and mechanics during the first few days after fertilization. Cultured ESCs can be maintained in a ground state of totipotency or pluripotency by maintaining certain culture conditions and their exact stage can be detected by associated expression levels of transcription factors (9,10). Cells cultured in serumfree media with the addition of two small molecule inhibitors of GSK3 and MEK along with leukemia inhibitory factor (LIF) resemble the 3.5 day post fertilization (11)(12)(13).…”

“…Cells cultured in serumfree media with the addition of two small molecule inhibitors of GSK3 and MEK along with leukemia inhibitory factor (LIF) resemble the 3.5 day post fertilization (11)(12)(13). These cells exhibit a greater level of pluripotency than cells grown in serum media supplemented with LIF (9,13,14) and exhibit a remarkably different morphology when grown on a substrate. To our knowledge, no study has quantitatively investigated the structural and physical properties of cells cultured in these two widely used culture conditions.…”