High basal γH2AX levels sustain self-renewal of mouse embryonic and induced pluripotent stem cells.

Phosphorylation of histone H2AX (γH2AX) is known to be the earliest indicator of DNA double-strand breaks. Recently, it has been shown that mouse embryonic stem cells (mESCs) have very high basal levels of γH2AX, even when they have not been exposed to genotoxic agents. As the specialized role of high basal γH2AX levels in pluripotent stem cells is still debated, we investigated whether H2AX phosphorylation is important in maintaining self-renewal of these cells. Here, we report that not only mESCs but also mouse-induced pluripotent stem cells (miPSCs), have high basal levels of γH2AX. We show that basal γH2AX levels decrease upon ESC and iPSC differentiation and increase when the cells are treated with self-renewal-enhancing small molecules. We observe that self-renewal activity is highly compromised in H2AX-/- cells and that it can be restored in these cells through reconstitution with a wild-type, but not a phospho-mutated, H2AX construct. Taken together, our findings suggest a novel function of H2AX that expands the knowledge of this histone variant beyond its role in DNA damage and into a new specialized biological function in mouse pluripotent stem cells.

Controlling embryonic stem cell proliferation and pluripotency: the role of PI3K- and GSK-3-dependent signalling.

ESCs (embryonic stem cells) are derived from the inner cell mass of pre-implantation embryos and are pluripotent, meaning they can differentiate into all of the cells that make up the adult organism. This property of pluripotency makes ESCs attractive as a model system for studying early development and for the generation of specific cell types for use in regenerative medicine and drug screening. In order to harness their potential, the molecular mechanisms regulating ESC pluripotency, proliferation and differentiation (i.e. cell fate) need to be understood so that pluripotency can be maintained during expansion, while differentiation to specific lineages can be induced accurately when required. The present review focuses on the potential roles that PI3K (phosphoinositide 3-kinase) and GSK-3 (glycogen synthase kinase 3)-dependent signalling play in the co-ordination and integration of mouse ESC pluripotency and proliferation and contrast this with our understanding of their functions in human ESCs.

Characterization of the phosphoinositide 3-kinase-dependent transcriptome in murine embryonic stem cells: identification of novel regulators of pluripotency.

Phosphoinositide 3-kinase (PI3K)-dependent signaling has been implicated in the regulation of embryonic stem (ES) cell fate. To gain further insight into the mechanisms regulated by PI3Ks in murine ES cells, we have performed expression profiling using Affymetrix GeneChips to characterize the transcriptional changes that arise as a result of inhibition of PI3K-dependent signaling. Using filtering of greater than 1.5-fold change in expression and an analysis of variance significance level of p < .05, we have defined a dataset comprising 646 probe sets that detect changes in transcript expression (469 down and 177 up) on inhibition of PI3Ks. Changes in expression of selected genes have been validated by quantitative reverse transcription polymerase chain reaction. Gene ontology analyses reveal significant over-representation of transcriptional regulators within our dataset. In addition, several known regulators of ES cell pluripotency, for example, Nanog, Esrrb, Tbx3, and Tcl-1, are among the downregulated genes. To evaluate the functional involvement of selected genes in regulation of ES cell self-renewal, we have used short interfering RNA-mediated knockdown. These studies identify genes not previously associated with control of ES cell fate that are involved in regulating ES cell pluripotency, including the protein tyrosine phosphatase Shp-1 and the Zscan4 family of zinc finger proteins. Further gain-of-function analyses demonstrate the importance of Zscan4c in regulation of ES cell pluripotency.

Digital movie analysis for quantification of beating frequencies, chronotropic effects, and beating areas in cardiomyocyte cultures.

Software, named Cardio Analyser, was developed for digital movie analysis of beating frequencies, drug-induced chronotropic effects, and quantification of beating areas of contracting cardiomyocyte cultures. A major novelty of the software is the introduction of automated noise filtering and automated movie analysis of beating frequencies and areas of contracting cardiomyocyte cultures. The software was based on the observation that the intensity of light transmitted through a contractive tissue changes periodically in a way that correlates with the contractions. We provided proof of principle for the method by derivation of relevant data from movies of multicellular cardiomyocyte cultures derived from embryonic stem cells. Moreover, we compared the data to equivalent results obtained by extracellular electric field potential recordings. The comparison demonstrated higher sensitivity to chronotropic effects of the beta-adrenoceptor agonist isoprenaline, and hence implied that more embryonic stem cells underwent differentiation into beta-adrenoceptor-responding cardiomyocytes, in the experimental setup applied for movie analysis than in the setup used for extracellular electric field potential recordings. Our study indicates that the movie analysis method may have potential to be optimized for screening in early drug discovery, aiming to identify cardiac drug candidates or to alert for adverse effects on heart functionality or embryonic heart development.

Zscan4 is regulated by PI3-kinase and DNA-damaging agents and directly interacts with the transcriptional repressors LSD1 and CtBP2 in mouse embryonic stem cells.

The Zscan4 family of genes, encoding SCAN-domain and zinc finger-containing proteins, has been implicated in the control of early mammalian embryogenesis as well as the regulation of pluripotency and maintenance of genome integrity in mouse embryonic stem cells. However, many features of this enigmatic family of genes are poorly understood. Here we show that undifferentiated mouse embryonic stem cell (ESC) lines simultaneously express multiple members of the Zscan4 gene family, with Zscan4c, Zscan4f and Zscan4-ps2 consistently being the most abundant. Despite this, between only 0.1 and 0.7% of undifferentiated mouse pluripotent stem cells express Zscan4 protein at a given time, consistent with a very restricted pattern of Zscan4 transcripts reported previously. Herein we demonstrate that Zscan4 expression is regulated by the p110α catalytic isoform of phosphoinositide 3-kinases and is induced following exposure to a sub-class of DNA-damage-inducing agents, including Zeocin and Cisplatin. Furthermore, we observe that Zscan4 protein expression peaks during the G2 phase of the cell cycle, suggesting that it may play a critical role at this checkpoint. Studies with GAL4-fusion proteins suggest a role for Zscan4 in transcriptional regulation, further supported by the fact that protein interaction analyses demonstrate that Zscan4 interacts with both LSD1 and CtBP2 in ESC nuclei. This study advances and extends our understanding of Zscan4 expression, regulation and mechanism of action. Based on our data we propose that Zscan4 may regulate gene transcription in mouse ES cells through interaction with LSD1 and CtBP2.

Glycogen synthase kinase-3 inhibition enhances translation of pluripotency-associated transcription factors to contribute to maintenance of mouse embryonic stem cell self-renewal.

Maintenance of embryonic stem cell (ESC) self-renewal and pluripotency are controlled by extrinsic factors, molecular signaling pathways and transcriptional regulators. While many of the key players have been studied in depth, how the molecular signals interact with transcription factors of the pluripotency network to regulate their action remains less well understood. Inhibition of glycogen synthase kinase 3 (Gsk-3) has been implicated in the maintenance of mouse ESC pluripotency, although there is contradictory data on its role, with enhancement of cell survival and metabolism, stabilisation of c-Myc and activation of Wnt signalling proposed as potential mechanisms. We have discovered that suppression of Gsk-3 activity leads to enhanced protein levels of key transcriptional regulators of the pluripotency network, notably Nanog, Tbx3 and c-Myc. Protein stability was unchanged following Gsk-3 inhibition, although interestingly, Nanog and Tbx3 proteins were found to have half-lives of 1-3 h, while that of Oct4 protein was longer, at 6 h. We demonstrate that the effects on protein levels seen following inhibition of Gsk-3 are due to both enhanced de novo synthesis of Nanog protein and increases in the proportion of Nanog and Tbx3 RNAs bound to polysomes, findings consistent with Gsk-3 regulating translation of these factors. These effects were not due to changes in regulators of general translation initiation machinery nor mediated via the 5' or 3' UTR sequences of Nanog alone. The data we present provide both new conceptual insight into the mechanisms regulated by Gsk-3 that may contribute to ESC self-renewal and, importantly, establish control of protein translation as an additional mechanism involved in modulation of ESC pluripotency.