Sparse CBX2 nucleates many Polycomb proteins to promote facultative heterochromatinization of Polycomb target genes.

Facultative heterochromatinization of genomic regulators by Polycomb repressive complex (PRC) 1 and 2 is essential in development and differentiation; however, the underlying molecular mechanisms remain obscure. Using genetic engineering, molecular approaches, and live-cell single-molecule imaging, we quantify the number of proteins within condensates formed through liquid-liquid phase separation (LLPS) and find that in mouse embryonic stem cells (mESCs), approximately 3 CBX2 proteins nucleate many PRC1 and PRC2 subunits to form one non-stoichiometric condensate. We demonstrate that sparse CBX2 prevents Polycomb proteins from migrating to constitutive heterochromatin, demarcates the spatial boundaries of facultative heterochromatin, controls the deposition of H3K27me3, regulates transcription, and impacts cellular differentiation.

Mouse Embryonic Stem Cell Pluripotency Factors Regulate RNA Methylation.

The pluripotency of embryonic stem cells (ESCs) is actively promoted by a diverse set of factors, including leukemia inhibitory factor (LIF), glycogen synthase kinase-3 (Gsk-3) and mitogen-activated protein kinase kinase (MEK) inhibitors, ascorbic acid, and α-ketoglutarate. Strikingly, several of these factors intersect with the post-transcriptional methylation of RNA (m A), which has also been shown to play a role in ESC pluripotency. Therefore, we explored the possibility that these factors converge on this biochemical pathway to promote the retention of ESC pluripotency.

Vitamin C and Transferrin Reduce RNA Methylation in Mouse Embryonic Stem Cells.

Methylation of mRNA on adenosine bases (referred to as m A) is the most common internal modification of mRNA in eukaryotic cells. Recent work has revealed a detailed view of the biological significance of m A-modified mRNA, with a role in mRNA splicing, control of mRNA stability, and mRNA translation efficiency. Importantly, m A is a reversible modification, and the primary enzymes responsible for methylating (Mettl3/Mettl14) and demethylating RNA (FTO/Alkbh5) have been identified.

Regulation of eukaryotic translation initiation factor 6 dynamics through multisite phosphorylation by GSK3.

Eukaryotic translation initiation factor 6 (eIF6) is essential for the synthesis of 60S ribosomal subunits and for regulating the association of 60S and 40S subunits. A mechanistic understanding of how eIF6 modulates translation in response to stress, specifically starvation-induced stress, is lacking. We here show a novel mode of eIF6 regulation by glycogen synthase kinase 3 (GSK3) that is predominantly active in response to serum starvation.

Associations between maternal depression and mother and infant oxytocin receptor gene (OXTR_rs53576) polymorphisms.

Polymorphisms in the oxytocin receptor gene, OXTR_rs53576, have been linked to differences in maternal sensitivity and depressive symptoms. Although some studies suggest the A allele confers risk for mood disorders, individuals homozygous for the G allele may exhibit greater sensitivity to both positive and negative social experiences, including in the mother-infant dyad. Given the bi-directional nature of mother-infant influences on maternal mood, we tested the association between both mothers' and infants' OXTR_rs53576 genotype and maternal depression, as assessed through a self-report inventory.

Glycogen Synthase Kinase-3α Promotes Fatty Acid Uptake and Lipotoxic Cardiomyopathy.

Obesity induces lipotoxic cardiomyopathy, a condition in which lipid accumulation in cardiomyocytes causes cardiac dysfunction. Here, we show that glycogen synthase kinase-3α (GSK-3α) mediates lipid accumulation in the heart. Fatty acids (FAs) upregulate GSK-3α, which phosphorylates PPARα at Ser280 in the ligand-binding domain (LBD).

The late positive potential and subjective arousal ratings evoked by negative images vary as a function of oxytocin receptor genotype SNP rs53576.

In the central nervous system the neuropeptide oxytocin mediates a range of behaviors related primarily to emotionality. One factor that influences oxytocinergic communication in the human brain and correlates with emotional behaviors is the single nucleotide polymorphism rs53576 on the oxytocin receptor gene (OXTR). For example, variations in this OXTR genotype are related to parental, altruistic, and other prosocial behaviors.

Live-cell single-molecule dynamics of PcG proteins imposed by the DIPG H3.3K27M mutation.

Over 80% of diffuse intrinsic pontine gliomas (DIPGs) harbor a point mutation in histone H3.3 where lysine 27 is substituted with methionine (H3.3K27M); however, how the mutation affects kinetics and function of PcG proteins remains elusive.

Glycogen synthase kinase-3 (GSK-3) activity regulates mRNA methylation in mouse embryonic stem cells.

Glycogen synthase kinase-3 (GSK-3) activity regulates multiple signal transduction pathways and is also a key component of the network responsible for maintaining stem cell pluripotency. Genetic deletion of α and Gsk-3β or inhibition of GSK-3 activity via small molecules promotes stem cell pluripotency, yet the mechanism underlying the role for GSK-3 in this process remains ambiguous. Another cellular process that has been shown to affect stem cell pluripotency is mRNA methylation (mA).

Glycogen synthase kinase 3 controls migration of the neural crest lineage in mouse and Xenopus.

Neural crest migration is critical to its physiological function. Mechanisms controlling mammalian neural crest migration are comparatively unknown, due to difficulties accessing this cell population in vivo. Here we report requirements of glycogen synthase kinase 3 (GSK3) in regulating the neural crest in Xenopus and mouse models.

Isoform-specific requirement for GSK3α in sperm for male fertility.

Glycogen synthase kinase 3 (GSK3) is a highly conserved protein kinase regulating key cellular functions. Its two isoforms, GSK3α and GSK3β, are encoded by distinct genes. In most tissues the two isoforms are functionally interchangeable, except in the developing embryo where GSK3β is essential.

Glycogen synthase kinase-3 (Gsk-3) plays a fundamental role in maintaining DNA methylation at imprinted loci in mouse embryonic stem cells.

Glycogen synthase kinase-3 (Gsk-3) is a key regulator of multiple signal transduction pathways. Recently we described a novel role for Gsk-3 in the regulation of DNA methylation at imprinted loci in mouse embryonic stem cells (ESCs), suggesting that epigenetic changes regulated by Gsk-3 are likely an unrecognized facet of Gsk-3 signaling. Here we extend our initial observation to the entire mouse genome by enriching for methylated DNA with the MethylMiner kit and performing next-generation sequencing (MBD-Seq) in wild-type and Gsk-3α(-/-);Gsk-3β(-/-) ESCs.

Targeted disruption of glycogen synthase kinase 3A (GSK3A) in mice affects sperm motility resulting in male infertility.

The signaling enzyme glycogen synthase kinase 3 (GSK3) exists as two isoforms-GSK3A and GSK3B. Protein phosphorylation by GSK3 has important signaling roles in several cells. In our past work, we found that both isoforms of GSK3 are present in mouse sperm and that catalytic GSK3 activity correlates with motility of sperm from several species.

Cbx2 stably associates with mitotic chromosomes via a PRC2- or PRC1-independent mechanism and is needed for recruiting PRC1 complex to mitotic chromosomes.

Polycomb group (PcG) proteins are epigenetic transcriptional factors that repress key developmental regulators and maintain cellular identity through mitosis via a poorly understood mechanism. Using quantitative live-cell imaging in mouse ES cells and tumor cells, we demonstrate that, although Polycomb repressive complex (PRC) 1 proteins (Cbx-family proteins, Ring1b, Mel18, and Phc1) exhibit variable capacities of association with mitotic chromosomes, Cbx2 overwhelmingly binds to mitotic chromosomes. The recruitment of Cbx2 to mitotic chromosomes is independent of PRC1 or PRC2, and Cbx2 is needed to recruit PRC1 complex to mitotic chromosomes.

Gene Expression Profiling in Mouse Embryonic Stem Cells Reveals Glycogen Synthase Kinase-3-Dependent Targets of Phosphatidylinositol 3-Kinase and Wnt/β-Catenin Signaling Pathways.

Glycogen synthase kinase-3 (Gsk-3) activity is an important regulator of numerous signal transduction pathways. Gsk-3 activity is the sum of two largely redundant proteins, Gsk-3α and Gsk-3β, and in general, Gsk-3 is a negative regulator of cellular signaling. Genetic deletion of both Gsk-3α and Gsk-3β in mouse embryonic stem cells (ESCs) has previously been shown to lead to the constitutive activation of the Wnt/β-catenin signaling pathway.

A simple and efficient method for transfecting mouse embryonic stem cells using polyethylenimine.

Mouse embryonic stem cells (ESCs) can be transfected by electroporation, liposomal reagents, and viral transduction methods. The cationic polymer polyethylenimine (PEI) has been shown to transfect a variety of differentiated mammalian cell types, including mouse ESCs, but existing methods require the use of additional equipment that is not readily accessible to most labs. Here we describe conditions that permit for the efficient transfection of mouse ESCs with low cytotoxicity and without the need for specialized equipment.

A novel interaction between Glycogen Synthase Kinase-3α (GSK-3α) and the scaffold protein Receptor for Activated C-Kinase 1 (RACK1) regulates the circadian clock.

Glycogen synthase kinase-3α (GSK-3α) and GSK-3β are intracellular kinases with largely redundant functions. However, the deletion of each GSK-3 isoform in the mouse has distinct consequences, suggesting that these related enzymes also have non-overlapping isoform-specific functions. A yeast two-hybrid screen for GSK-3α interacting partners revealed an interaction with the Receptor for Activated C-Kinase 1 (RACK1).

The role for oxidative stress in aberrant DNA methylation in Alzheimer's disease.

Alzheimer's disease (AD) is a common, progressive neurodegenerative disorder without highly effective therapies. The etiology of AD is heterogeneous with amyloid-beta plaques, neurofibrillary tangles, oxidative stress, and aberrant DNA methylation all implicated in the disease pathogenesis. DNA methylation is a well-established process for regulating gene expression and has been found to regulate a growing number of important genes involved in AD development and progression.

Phosphatidylinositol 3-kinase (PI3K) signaling via glycogen synthase kinase-3 (Gsk-3) regulates DNA methylation of imprinted loci.

Glycogen synthase kinase-3 (Gsk-3) isoforms, Gsk-3α and Gsk-3β, are constitutively active, largely inhibitory kinases involved in signal transduction. Underscoring their biological significance, altered Gsk-3 activity has been implicated in diabetes, Alzheimer disease, schizophrenia, and bipolar disorder. Here, we demonstrate that deletion of both Gsk-3α and Gsk-3β in mouse embryonic stem cells results in reduced expression of the de novo DNA methyltransferase Dnmt3a2, causing misexpression of the imprinted genes Igf2, H19, and Igf2r and hypomethylation of their corresponding imprinted control regions.

Gsk3β is required in the epithelium for palatal elevation in mice.

In Wnt/β-catenin signaling pathway, Gsk3β functions to facilitate β-catenin degradation. Inactivation of Gsk3β in mice causes a cleft palate formation, suggesting an involvement of Wnt/β-catenin signaling during palatogenesis. In this study, we have investigated the expression pattern, tissue-specific requirement and function of Gsk3β during mouse palatogenesis.

Functions of B56-containing PP2As in major developmental and cancer signaling pathways.

Members of the B'/B56/PR61 family regulatory subunits of PP2A determine the subcellular localization, substrate specificity, and catalytic activity of PP2A in a wide range of biological processes. Here, we summarize the structure and intracellular localization of B56-containing PP2As and review functions of B56-containing PP2As in several major developmental/cancer signaling pathways.

A noncatalytic domain of glycogen synthase kinase-3 (GSK-3) is essential for activity.

Glycogen synthase kinase-3 (GSK-3) isoforms, GSK-3alpha and GSK-3beta, are serine/threonine kinases involved in numerous cellular processes and diverse diseases, including Alzheimer disease, cancer, and diabetes. GSK-3 isoforms function redundantly in some settings, while, in others, they exhibit distinct activities. Despite intensive investigation into the physiological roles of GSK-3 isoforms, the basis for their differential activities remains unresolved.

PP2A:B56epsilon is required for eye induction and eye field separation.

Eye induction and eye field separation are the earliest events during vertebrate eye development. Both of these processes occur much earlier than the formation of optic vesicles. The insulin-like growth factor (IGF) pathway appears to be essential for eye induction, yet it remains unclear how IGF downstream pathways are involved in eye induction.

JLK inhibitors: isocoumarin compounds as putative probes to selectively target the gamma-secretase pathway.

Alzheimer's disease is characterized by the extracellular deposition of the amyloid beta-peptide that derives from its precursor betaAPP by sequential actions of beta- and gamma- secretases, respectively. Recent studies aimed at identifying these enzymes have been reported as it is thougth that their inhibition should hopefully lead to reduce Abeta load in the AD brains. beta-secretase seems to be due to BACE1, a novel membrane-bound aspartyl protease.

Vesicle-associated protein-A is differentially expressed during intestinal smooth muscle cell differentiation.

Gastrointestinal (GI) smooth muscle diseases represent a major health concern affecting in excess of 2 million people each year. Little is currently known regarding the molecular mechanisms controlling either normal or pathogenic GI smooth muscle development. In an effort to identify the specific gene products responsible for modulating GI smooth muscle cell (SMC) differentiation, we performed differential display on distinct intestinal SMC (ISMC) phenotypes.