Erk5 Is a Key Regulator of Naive-Primed Transition and Embryonic Stem Cell Identity

Williams, Charles A. C.; Fernandez-Alonso, Rosalia; Wang, Jinhua; Toth, Rachel; Gray, Nathanael S.; Findlay, Greg M.

doi:10.1016/j.celrep.2016.07.033

Cited by 39 publications

(51 citation statements)

References 31 publications

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“…Fetal Bovine Serum (FBS; Gibco) and 5% knockout serum replacement (Invitrogen). Embryoid bodies (EBs) were formed by aggregation of 1200 cells in 20 µl of mESC medium without LIF forming hanging drops 56 . At day 4, EBs were transferred onto gelatin-coated plates and maintained for a further 2-6 days.…”

Section: Methodsmentioning

confidence: 99%

Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation

et al. 2020

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Embryonic Stem Cell (ESC) differentiation requires complex cell signalling network dynamics, although the key molecular events remain poorly understood. Here, we use phosphoproteomics to identify an FGF4-mediated phosphorylation switch centred upon the key Ephrin receptor EPHA2 in differentiating ESCs. We show that EPHA2 maintains pluripotency and restrains commitment by antagonising ERK1/2 signalling. Upon ESC differentiation, FGF4 utilises a bimodal strategy to disable EPHA2, which is accompanied by transcriptional induction of EFN ligands. Mechanistically, FGF4-ERK1/2-RSK signalling inhibits EPHA2 via Ser/Thr phosphorylation, whilst FGF4-ERK1/2 disrupts a core pluripotency transcriptional circuit required for Epha2 gene expression. This system also operates in mouse and human embryos, where EPHA receptors are enriched in pluripotent cells whilst surrounding lineagespecified trophectoderm expresses EFNA ligands. Our data provide insight into function and regulation of EPH-EFN signalling in ESCs, and suggest that segregated EPH-EFN expression coordinates cell fate with compartmentalisation during early embryonic development.

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Section: Methodsmentioning

confidence: 99%

Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation

et al. 2020

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“…Protein extracts were separated in 10% SDS-PAGE and electrotransferred to PVDF membranes (Immobilon-P; EMD Millipore). Antibodies used were as follows: MYC #ab32 (Abcam, Cambridge, USA) [ 56 ], #sc764 (Santa Cruz Biotechnology, USA) [ 57 ]; DNMT3B #ab122932 (Abcam, Cambridge, USA) [ 58 ],#nb100-56514 (Novus Biologicals, USA) [ 59 ]; TUBULIN #ab6046 (Abcam, Cambridge, USA) [ 22 ]; β-ACTIN #A5441 (Sigma-Aldrich, USA); GAPDH #sc25778 (Santa Cruz Biotechnology, USA) [ 60 ]; Anti-MOUSE HRP #31430 (Thermo Fisher, USA) [ 61 ]; Anti-RABBIT HRP #31460 (Thermo Fisher, USA) [ 62 ]. Protein quantitation was performed using ImageJ software ( https://imagej.nih.gov/ij/ ) [ 63 ].…”

Section: Methodsmentioning

confidence: 99%

DNMT3B overexpression contributes to aberrant DNA methylation and MYC-driven tumor maintenance in T-ALL and Burkitt’s lymphoma

et al. 2017

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Aberrant DNA methylation is a hallmark of cancer. However, our understanding of how tumor cell-specific DNA methylation patterns are established and maintained is limited. Here, we report that in T-cell acute lymphoblastic leukemia (T-ALL) and Burkitt’s lymphoma the MYC oncogene causes overexpression of DNA methyltransferase (DNMT) 1 and 3B, which contributes to tumor maintenance. By utilizing a tetracycline-regulated MYC transgene in a mouse T-ALL (EμSRα-tTA;tet-o-MYC) and human Burkitt’s lymphoma (P493-6) model, we demonstrated that DNMT1 and DNMT3B expression depend on high MYC levels, and that their transcription decreased upon MYC-inactivation. Chromatin immunoprecipitation indicated that MYC binds to the DNMT1 and DNMT3B promoters, implicating a direct transcriptional regulation. Hence, shRNA-mediated knock-down of endogenous MYC in human T-ALL and Burkitt’s lymphoma cell lines downregulated DNMT3B expression. Knock-down and pharmacologic inhibition of DNMT3B in T-ALL reduced cell proliferation associated with genome-wide changes in DNA methylation, indicating a tumor promoter function during tumor maintenance. We provide novel evidence that MYC directly deregulates the expression of both de novo and maintenance DNMTs, showing that MYC controls DNA methylation in a genome-wide fashion. Our finding that a coordinated interplay between the components of the DNA methylating machinery contributes to MYC-driven tumor maintenance highlights the potential of specific DNMTs for targeted therapies.

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“…The primary kinase targets of these inhibitors are presented in Table 1 . We also demonstrate the applicability of this screen to identify inhibitors which stabilize the primed state 14 .…”

Section: Representative Resultsmentioning

confidence: 86%

“…mESC were treated with a 228 compound kinase inhibitor library at a final concentration of 1 µM. Lysates were prepared and transferred onto nitrocellulose membranes for immuno dot-blot analysis, and Nanog and Dnmt3b levels determined (Figure adapted from Williams et al 14 ). Please click here to view a larger version of this figure.…”

Section: Representative Resultsmentioning

confidence: 99%

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A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening

Williams

Gray

Findlay

2017

JoVE

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Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been described, termed "naïve" and "primed" pluripotency. The mechanisms that control naïve-primed transition are poorly understood. In particular, we remain poorly informed about protein kinases that specify naïve and primed pluripotent states, despite increasing availability of high-quality tool compounds to probe kinase function. Here, we describe a scalable platform to perform targeted small molecule screens for kinase regulators of the naïve-primed pluripotent transition in mouse ESCs. This approach utilizes simple cell culture conditions and standard reagents, materials and equipment to uncover and validate kinase inhibitors with hitherto unappreciated effects on pluripotency. We discuss potential applications for this technology, including screening of other small molecule collections such as increasingly sophisticated kinase inhibitors and emerging libraries of epigenetic tool compounds.

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Erk5 Is a Key Regulator of Naive-Primed Transition and Embryonic Stem Cell Identity

Cited by 39 publications

References 31 publications

Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation

Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation

DNMT3B overexpression contributes to aberrant DNA methylation and MYC-driven tumor maintenance in T-ALL and Burkitt’s lymphoma

A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening

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