An ERK5-KLF2 signalling module regulates early embryonic gene expression and telomere rejuvenation in stem cells.

The ERK5 MAP kinase signalling pathway drives transcription of naïve pluripotency genes in mouse Embryonic Stem Cells (mESCs). However, how ERK5 impacts on other aspects of mESC biology has not been investigated. Here, we employ quantitative proteomic profiling to identify proteins whose expression is regulated by the ERK5 pathway in mESCs. This reveals a function for ERK5 signalling in regulating dynamically expressed early embryonic 2-cell stage (2C) genes including the mESC rejuvenation factor ZSCAN4. ERK5 signalling and ZSCAN4 induction in mESCs increases telomere length, a key rejuvenative process required for prolonged culture. Mechanistically, ERK5 promotes ZSCAN4 and 2C gene expression via transcription of the KLF2 pluripotency transcription factor. Surprisingly, ERK5 also directly phosphorylates KLF2 to drive ubiquitin-dependent degradation, encoding negative feedback regulation of 2C gene expression. In summary, our data identify a regulatory module whereby ERK5 kinase and transcriptional activities bi-directionally control KLF2 levels to pattern 2C gene transcription and a key mESC rejuvenation process.

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

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 lineage-specified 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.

Brd4-Brd2 isoform switching coordinates pluripotent exit and Smad2-dependent lineage specification.

Pluripotent stem cells (PSCs) hold great clinical potential, as they possess the capacity to differentiate into fully specialised tissues such as pancreas, liver, neurons and cardiac muscle. However, the molecular mechanisms that coordinate pluripotent exit with lineage specification remain poorly understood. To address this question, we perform a small molecule screen to systematically identify novel regulators of the Smad2 signalling network, a key determinant of PSC fate. We reveal an essential function for BET family bromodomain proteins in Smad2 activation, distinct from the role of Brd4 in pluripotency maintenance. Mechanistically, BET proteins specifically engage Nodal gene regulatory elements (NREs) to promote Nodal signalling and Smad2 developmental responses. In pluripotent cells, Brd2-Brd4 occupy NREs, but only Brd4 is required for pluripotency gene expression. Brd4 downregulation facilitates pluripotent exit and drives enhanced Brd2 NRE occupancy, thereby unveiling a specific function for Brd2 in differentiative Nodal-Smad2 signalling. Therefore, distinct BET functionalities and Brd4-Brd2 isoform switching at NREs coordinate pluripotent exit with lineage specification.

Protein Kinases in Pluripotency-Beyond the Usual Suspects.

Post-translational modification of proteins by phosphorylation plays a key role in regulating all aspects of eukaryotic biology. Embryonic stem cell (ESC) pluripotency, defined as the ability to differentiate into all cell types in the adult body, is no exception. Maintenance and dissolution of pluripotency are tightly controlled by phosphorylation. As a result, key signalling pathways that regulate pluripotency have been identified and their functions well characterised. Amongst the best studied are the fibroblast growth factor (FGF)-ERK1/2 pathway, PI3K-AKT, the leukemia inhibitory factor (LIF)-JAK-STAT3 axis, Wnt-GSK3 signalling, and the transforming growth factor (TGF)ß family. However, these kinase pathways constitute only a small proportion of the protein kinase complement of pluripotent cells, and there is accumulating evidence that diverse phosphorylation systems modulate ESC pluripotency. Here, we review recent progress in understanding the overarching role of phosphorylation in mediating communication from the cellular environment, metabolism, and cell cycle to the core pluripotency machinery.

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

Embryonic stem cells (ESCs) can self-renew or differentiate into any cell type, a phenomenon known as pluripotency. Distinct pluripotent states, termed naive and primed pluripotency, have been described. However, the mechanisms that control naive-primed pluripotent transition are poorly understood. Here, we perform a targeted screen for kinase inhibitors, which modulate the naive-primed pluripotent transition. We find that XMD compounds, which selectively inhibit Erk5 kinase and BET bromodomain family proteins, drive ESCs toward primed pluripotency. Using compound selectivity engineering and CRISPR/Cas9 genome editing, we reveal distinct functions for Erk5 and Brd4 in pluripotency regulation. We show that Erk5 signaling maintains ESCs in the naive state and suppresses progression toward primed pluripotency and neuroectoderm differentiation. Additionally, we identify a specialized role for Erk5 in defining ESC lineage selection, whereby Erk5 inhibits a cardiomyocyte-specific differentiation program. Our data therefore reveal multiple critical functions for Erk5 in controlling ESC identity.

p73 plays a role in erythroid differentiation through GATA1 induction.

The TP73 gene gives rise to transactivation domain-p73 isoforms (TAp73) as well as DeltaNp73 variants with a truncated N terminus. Although TAp73alpha and -beta proteins are capable of inducing cell cycle arrest, apoptosis, and differentiation, DeltaNp73 acts in many cell types as a dominant-negative repressor of p53 and TAp73. It has been proposed that p73 is involved in myeloid differentiation, and its altered expression is involved in leukemic degeneration. However, there is little evidence as to which p73 variants (TA or DeltaN) are expressed during differentiation and whether specific p73 isoforms have the capacity to induce, or hinder, this differentiation in leukemia cells. In this study we identify GATA1 as a direct transcriptional target of TAp73alpha. Furthermore, TAp73alpha induces GATA1 activity, and it is required for erythroid differentiation. Additionally, we describe a functional cooperation between TAp73 and DeltaNp73 in the context of erythroid differentiation in human myeloid cells, K562 and UT-7. Moreover, the impaired expression of GATA1 and other erythroid genes in the liver of p73KO embryos, together with the moderated anemia observed in p73KO young mice, suggests a physiological role for TP73 in erythropoiesis.