Tissue generation and repair requires a stepwise process of cell fate restriction to ensure that adult stem cells differentiate in a timely and appropriate manner. A crucial role has been implicated for Polycomb-group (PcG) proteins and the H3K27me3 repressive histone mark in coordinating the transcriptional programmes necessary for this process, but the targets and developmental timing for this repression remain unclear. To address these questions, we generated novel genome-wide maps of H3K27me3 and H3K4me3 in freshly isolated muscle stem cells. These data, together with the analysis of two conditional Ezh2-null mouse strains, identified a critical proliferation phase in which Ezh2 activity is essential. Mice lacking Ezh2 in satellite cells exhibited decreased muscle growth, severely impaired regeneration and reduced stem cell number, due to a profound failure of the proliferative progenitor population to expand. Surprisingly, deletion of Ezh2 after the onset of terminal differentiation did not impede muscle repair or homeostasis. Using these knockout models and the RNA-Seq and ChIP-Seq datasets, we show that Ezh2 does not regulate the muscle differentiation process in vivo. These results emphasise the lineage and cell-type-specific functions of Ezh2 and Polycomb repressive complex 2.

Little is known about the evolutionary relationship between vertebrate adrenergic receptors and invertebrate octopamine and tyramine receptors. The complexity of the adrenergic signalling system is believed to be an innovation of the vertebrate lineage but the presence of noradrenaline has been reported in some invertebrate species. The cephalochordate, amphioxus (Branchiostoma floridae), is an ideal model organism for studying the evolution of vertebrate GPCRs, given its unique position at the base of the chordate lineage. Here, we describe the pharmacological characterisation and second messenger coupling abilities of AmphiAmR4, which clusters with α₂-adrenergic receptors in a phylogenetic tree but also shares a high sequence similarity to invertebrate octopamine/tyramine receptors in both BLAST and Hidden Markov Model analyses. Thus, it was of particular interest to determine if AmphiAmR4 displayed similar functional properties to the vertebrate α₂-adrenergic receptors or to invertebrate octopamine or tyramine receptors. When stably expressed in Chinese hamster ovary (CHO) cells, noradrenaline couples the receptor to both the activation of adenylyl cyclase and to the activation of the MAPKinase pathway. Pharmacological studies with a wide range of agonists and antagonists suggest that AmphiAmR4 functions as an α₂-adrenergic-like receptor when expressed in CHO cells.

mTORC1 (mammalian target of rapamycin complex 1) controls transcriptional programs that determine CD8+ cytolytic T cell (CTL) fate. In some cell systems, mTORC1 couples phosphatidylinositol-3 kinase (PI3K) and Akt to the control of glucose uptake and glycolysis. However, PI3K-Akt-independent mechanisms control glucose metabolism in CD8+ T cells, and the role of mTORC1 has not been explored. The present study now demonstrates that mTORC1 activity in CD8+ T cells is not dependent on PI3K or Akt but is critical to sustain glucose uptake and glycolysis in CD8+ T cells. We also show that PI3K- and Akt-independent pathways mediated by mTORC1 regulate the expression of HIF1 (hypoxia-inducible factor 1) transcription factor complex. This mTORC1-HIF1 pathway is required to sustain glucose metabolism and glycolysis in effector CTLs and strikingly functions to couple mTORC1 to a diverse transcriptional program that controls expression of glucose transporters, multiple rate-limiting glycolytic enzymes, cytolytic effector molecules, and essential chemokine and adhesion receptors that regulate T cell trafficking. These data reveal a fundamental mechanism linking nutrient and oxygen sensing to transcriptional control of CD8+ T cell differentiation.

For all organisms promoting protein homeostasis is a high priority in order to optimize cellular functions and resources. However, there is accumulating evidence that aging leads to a collapse in protein homeostasis and widespread non-disease protein aggregation. This review examines these findings and discusses the potential causes and consequences of this physiological aggregation with age in particular in relation to disease protein aggregation and toxicity. Importantly, recent evidence points to unexpected differences in protein-quality-control and susceptibility to protein aggregation between neurons and other cell types. In addition, new insight into the cell-non-autonomous coordination of protein homeostasis by neurons will be presented.

ARAP3, a GTPase activating protein for Rho and Arf family GTPases, is one of many phosphoinositide 3-OH kinase (PI3K) effectors. In this study, we investigate the regulatory input of PI3K upstream of ARAP3 by analyzing neutrophils from an ARAP3 pleckstrin homology (PH) domain point mutation knock-in mouse (R302, 303A), in which ARAP3 is uncoupled from activation by PI3K. ARAP3 PH domain point mutant neutrophils are characterized by disturbed responses linked to stimulation by either integrin ligands or immobilized immune complexes. These cells exhibit increased β2 integrin inside-out signaling (binding affinity and avidity), and our work suggests the disturbed responses to immobilized immune complexes are secondary to this. In vitro, neutrophil chemotaxis is affected in the mutant. In vivo, ARAP3 PH domain point mutant bone marrow chimeras exhibit reduced neutrophil recruitment to the peritoneum on induction of sterile peritonitis and also reduced inflammation in a model for rheumatoid arthritis. The current work suggests a dramatic regulatory input of PI3K into the regulation of β2 integrin activity, and processes dependent on this, by signaling through its effector ARAP3.

Experimental techniques for the investigation of three-dimensional (3D) genome organization are being developed at a fast pace. Currently, the associated computational methods are mostly specific to the individual experimental approach. Here we present a general statistical framework that is widely applicable to the analysis of genomic contact maps, irrespective of the data acquisition and normalization processes. Within this framework DNA-DNA contact data are represented as a complex network, for which a broad number of directly applicable methods already exist. In such a network representation, DNA segments and contacts between them are denoted as nodes and edges, respectively. Furthermore, we present a robust method for generating randomized contact networks that explicitly take into account the inherent 3D nature of the genome and serve as realistic null-models for unbiased statistical analyses. By integrating a variety of large-scale genome-wide datasets we demonstrate that meiotic crossover sites display enriched genomic contacts and that cohesin-bound genes are significantly colocalized in the yeast nucleus. We anticipate that the complex network framework in conjunction with the randomization of DNA-DNA contact networks will become a widely used tool in the study of nuclear architecture.

Fundamental to genomic imprinting in mammals is the acquisition of epigenetic marks that differ in male and female gametes at 'imprinting control regions' (ICRs). These marks mediate the allelic expression of imprinted genes in the offspring. Much has been learnt about the nature of imprint marks, the times during gametogenesis at which they are laid down and some of the factors responsible especially for DNA methylation. Recent work has revealed that transcription and histone modifications are critically involved in DNA methylation acquisition, and these findings allow us to propose rational models for methylation establishment. A completely novel perspective on gametic DNA methylation has emerged from epigenomic profiling. Far more differentially methylated loci have been identified in gametes than known imprinted genes, which leads us to revise the notion that methylation of ICRs is a specifically targeted process. Instead, it seems to obey default processes in germ cells, giving rise to distinct patterns of DNA methylation in sperm and oocytes. This new insight, together with the identification of proteins that preserve DNA methylation after fertilization, emphasizes the key role played by mechanisms that selectively retain differential methylation at imprinted loci during early development. Addressing these mechanisms will be essential to understanding the specificity and evolution of genomic imprinting.

In mammalian development, epigenetic modifications, including DNA methylation patterns, play a crucial role in defining cell fate but also represent epigenetic barriers that restrict developmental potential. At two points in the life cycle, DNA methylation marks are reprogrammed on a global scale, concomitant with restoration of developmental potency. DNA methylation patterns are subsequently re-established with the commitment towards a distinct cell fate. This reprogramming of DNA methylation takes place firstly on fertilization in the zygote, and secondly in primordial germ cells (PGCs), which are the direct progenitors of sperm or oocyte. In each reprogramming window, a unique set of mechanisms regulates DNA methylation erasure and re-establishment. Recent advances have uncovered roles for the TET3 hydroxylase and passive demethylation, together with base excision repair (BER) and the elongator complex, in methylation erasure from the zygote. Deamination by AID, BER and passive demethylation have been implicated in reprogramming in PGCs, but the process in its entirety is still poorly understood. In this review, we discuss the dynamics of DNA methylation reprogramming in PGCs and the zygote, the mechanisms involved and the biological significance of these events. Advances in our understanding of such natural epigenetic reprogramming are beginning to aid enhancement of experimental reprogramming in which the role of potential mechanisms can be investigated in vitro. Conversely, insights into in vitro reprogramming techniques may aid our understanding of epigenetic reprogramming in the germline and supply important clues in reprogramming for therapies in regenerative medicine.

Inositol 1,4,5'-triphosphate receptor II (IP(3)RII) calcium channel expression is increased in both hypertrophic failing human myocardium and experimentally induced models of the disease. The ectopic calcium released from these receptors induces pro-hypertrophic gene expression and may promote arrhythmias. Here, we show that IP(3)RII expression was constitutively restrained by the muscle-specific miRNA, miR-133a. During the hypertrophic response to pressure overload or neurohormonal stimuli, miR-133a down-regulation permitted IP(3)RII levels to increase, instigating pro-hypertrophic calcium signaling and concomitant pathological remodeling. Using a combination of in vivo and in vitro approaches, we demonstrated that IP(3)-induced calcium release (IICR) initiated the hypertrophy-associated decrease in miR-133a. In this manner, hypertrophic stimuli that engage IICR set a feed-forward mechanism in motion whereby IICR decreased miR-133a expression, further augmenting IP(3)RII levels and therefore pro-hypertrophic calcium release. Consequently, IICR can be considered as both an initiating event and a driving force for pathological remodeling.

Glycation by endogenous dicarbonyl metabolites such as glyoxal is an important spontaneous post-translational (PTM) modification of peptides and proteins associated with structural and functional impairment. The aim of this study was to investigate types and site of PTM of glyoxal-derived advanced glycation end-products-in the neuropeptide substance P by ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR), mass spectrometry, and tandem mass spectrometry (MS/MS) experiments. The main site of PTM by glyoxal was the side chain guanidine moiety of the arginine residue. Binding site identification has been achieved by electron capture dissociation, double-resonance electron capture dissociation, and collision-activated dissociation, with assignment of the modified amino acid residue with mass error <1 ppm.

We created SynSysNet, available online at http://bioinformatics.charite.de/synsysnet, to provide a platform that creates a comprehensive 4D network of synaptic interactions. Neuronal synapses are fundamental structures linking nerve cells in the brain and they are responsible for neuronal communication and information processing. These processes are dynamically regulated by a network of proteins. New developments in interaction proteomics and yeast two-hybrid methods allow unbiased detection of interactors. The consolidation of data from different resources and methods is important to understand the relation to human behaviour and disease and to identify new therapeutic approaches. To this end, we established SynSysNet from a set of ∼1000 synapse specific proteins, their structures and small-molecule interactions. For two-thirds of these, 3D structures are provided (from Protein Data Bank and homology modelling). Drug-target interactions for 750 approved drugs and 50 000 compounds, as well as 5000 experimentally validated protein-protein interactions, are included. The resulting interaction network and user-selected parts can be viewed interactively and exported in XGMML. Approximately 200 involved pathways can be explored regarding drug-target interactions. Homology-modelled structures are downloadable in Protein Data Bank format, and drugs are available as MOL-files. Protein-protein interactions and drug-target interactions can be viewed as networks; corresponding PubMed IDs or sources are given.

A direct interaction of the Arabidopsis thaliana immunophilin ROF1 with phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-bisphosphate was identified using a phosphatidylinositol-phosphate affinity chromatography of cell suspension extracts, combined with a mass spectrometry (nano LC ESI-MS/MS) analysis. The first FK506 binding domain was shown sufficient to bind to both phosphatidylinositol-phosphate stereoisomers. GFP-tagged ROF1 under the control of a 35S promoter was localised in the cytoplasm and the cell periphery of Nicotiana tabacum leaf explants. Immunofluorescence microscopy of Arabidopsis thaliana root tips verified its cytoplasmic localization and membrane association and showed ROF1 localization in the elongation zone which was expanded to the meristematic zone in plants grown on high salt media. Endogenous ROF1 was shown to accumulate in response to high salt treatment in Arabidopsis thaliana young leaves as well as in seedlings germinated on high salt media (0.15 and 0.2 M NaCl) at both an mRNA and protein level. Plants over-expressing ROF1, (WSROF1OE), exhibited enhanced germination under salinity stress which was significantly reduced in the rof1(-) knock out mutants and abolished in the double mutants of ROF1 and of its interacting homologue ROF2 (WSrof1(-)/2(-)). Our results show that ROF1 plays an important role in the osmotic/salt stress responses of germinating Arabidopsis thaliana seedlings and suggest its involvement in salinity stress responses through a phosphatidylinositol-phosphate related protein quality control pathway.

Mice deficient for the adapter protein Slp65 (also known as Blnk), a key component in precursor-BCR (pre-BCR) signaling, spontaneously develop pre-B cell leukemia. In these leukemias, proliferation is thought to be driven by constitutive Jak3/Stat5 signaling, mostly due to autocrine production of IL-7, together with high surface expression of the pre-BCR. In this study, we investigated whether particular IgH specificities would predispose Slp65-deficient pre-B cells to malignant transformation. Whereas V(H)-D-J(H) junctions were diverse, we found highly restricted Ig V(H) gene usage: 55 out of 60 (~92%) leukemias used a V(H)14/SM7-family gene, mainly V(H)14-1 and V(H)14-2. When combined with surrogate or conventional L chains, these V(H)14 IgH chains did not provide increased proliferative signals or exhibit enhanced poly- or autoreactivity. We therefore conclude that pre-BCR specificity per se did not contribute to oncogenic transformation. Remarkably, in a high proportion of Slp65-deficient leukemias, the nonexpressed IgH allele also harbored a V(H)14-family rearrangement (10 out of 50) or was in the germline configuration (10 out of 50). V(H)14-1 and V(H)14-2 gene regions differed from their neighboring V(H) genes in that they showed active H3K4me3 histone modification marks and germline transcription at the pro-B cell stage in Rag1-deficient mice. Taken together, these findings demonstrate that in Slp65-deficient mice, malignant transformation is largely limited to particular pre-B cells that originate from pro-B cells that had restricted IgH V(H) region accessibility at the time of V(H)-to D-J(H) recombination.

'Dying back' axon degeneration is a prominent feature of many age-related neurodegenerative disorders and is widespread in normal ageing. Although the mechanisms of disease- and age-related losses may differ, both contribute to symptoms. Here, we review recent advances in understanding axon pathology in age-related neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and glaucoma. In particular, we highlight the importance of axonal transport, autophagy, traumatic brain injury, and mitochondrial quality control. We then place these disease mechanisms in the context of changes to axons and dendrites that occur during normal ageing. We discuss what makes ageing such an important risk factor for many neurodegenerative disorders and conclude that the processes of normal ageing and disease combine at the molecular, cellular or systems levels in a range of disorders to produce symptoms. Pathology identical to disease also occurs at the cellular level in most elderly individuals. Thus, normal ageing and age-related disease are inextricably linked and the term 'healthy ageing' downplays the important contributions of cellular pathology. For a full understanding of normal ageing or age-related disease we must study both processes.

The autoimmune regulator (Aire), mediates central tolerance of peripheral self. Its activity in thymic epithelial cells (TECs) directs the ectopic expression of thousands of tissue-restricted antigens (TRAs), causing the deletion of autoreactive thymocytes. The molecular mechanisms orchestrating the breadth of transcriptional regulation by Aire remain unknown. One prominent model capable of explaining both the uniquely high number of Aire-dependent targets and their specificity posits that tissue-specific transcription factors induced by Aire directly activate their canonical targets, exponentially adding to the total number of Aire-dependent TRAs. To test this "Hierarchical Transcription" model, we analysed mice deficient in the pancreatic master transcription factor pancreatic and duodenal homeobox 1 (Pdx1), specifically in TECs (Pdx1(ΔFoxn1) ), for the expression and tolerance of pancreatic TRAs. Surprisingly, we found that lack of Pdx1 in TECs did not reduce the transcription of insulin or somatostatin, or alter glucagon expression. Moreover, in a model of thymic deletion driven by a neo-TRA under the control of the insulin promoter, Pdx1 in TECs was not required to affect thymocyte deletion or the generation of regulatory T (Treg) cells. These findings suggest that the capacity of Aire to regulate expression of a huge array of TRAs relies solely on an unconventional transcriptional mechanism, without intermediary transcription factors.

Embryonic (ES) and epiblast (EpiSC) stem cells are pluripotent but committed to an embryonic lineage fate. Conversely, trophoblast (TS) and extraembryonic endoderm (XEN) stem cells contribute predominantly to tissues of the placenta and yolk sac, respectively. Here we show that each of these four stem cell types is defined by a unique DNA methylation profile. Despite their distinct developmental origin, TS and XEN cells share key epigenomic hallmarks, chiefly characterized by robust DNA methylation of embryo-specific developmental regulators, as well as a subordinate role of 5-hydroxymethylation. We also observe a substantial methylation reinforcement of pre-existing epigenetic repressive marks that specifically occurs in extraembryonic stem cells compared to in vivo tissue, presumably due to continued high Dnmt3b expression levels. These differences establish a major epigenetic barrier between the embryonic and extraembryonic stem cell types. In addition, epigenetic lineage boundaries also separate the two extraembryonic stem cell types by mutual repression of key lineage-specific transcription factors. Thus, global DNA methylation patterns are a defining feature of each stem cell type that underpin lineage commitment and differentiative potency of early embryo-derived stem cells. Our detailed methylation profiles identify a cohort of developmentally regulated sequence elements, such as orphan CpG islands, that will be most valuable to uncover novel transcriptional regulators and pivotal "gatekeeper" genes in pluripotency and lineage differentiation.

We have investigated the contribution of individual phosphoinositide 3-kinase (PI3K) Class I isoforms to the regulation of neutrophil survival using (i) a panel of commercially available small molecule isoform-selective PI3K Class I inhibitors, (ii) novel inhibitors, which target single or multiple Class I isoforms (PI3Kα, PI3Kβ, PI3Kδ, and PI3Kγ), and (iii) transgenic mice lacking functional PI3K isoforms (p110δ(KO)γ(KO) or p110γ(KO)). Our data suggest that there is considerable functional redundancy amongst Class I PI3Ks (both Class IA and Class IB) with regard to GM-CSF-mediated suppression of neutrophil apoptosis. Hence pharmacological inhibition of any 3 or more PI3K isoforms was required to block the GM-CSF survival response in human neutrophils, with inhibition of individual or any two isoforms having little or no effect. Likewise, isolated blood neutrophils derived from double knockout PI3K p110δ(KO)γ(KO) mice underwent normal time-dependent constitutive apoptosis and displayed identical GM-CSF mediated survival to wild type cells, but were sensitized to pharmacological inhibition of the remaining PI3K isoforms. Surprisingly, the pro-survival neutrophil phenotype observed in patients with an acute exacerbation of chronic obstructive pulmonary disease (COPD) was resilient to inactivation of the PI3K pathway.

The class 1 PI3Ks are lipid kinases with key roles in cell surface receptor-triggered signal transduction pathways. Two isoforms of the catalytic subunits, p110γ and p110δ, are enriched in leucocytes in which they promote activation, cellular growth, proliferation, differentiation and survival through the generation of the second messenger PIP3. Genetic inactivation or pharmaceutical inhibition of these PI3K isoforms in mice result in impaired immune responses and reduced susceptibility to autoimmune and inflammatory conditions. We review the PI3K signal transduction pathways and the effects of inhibition of p110γ and/or p110δ on innate and adaptive immunity. Focusing on rheumatoid arthritis and systemic lupus erythematosus we discuss the preclinical evidence and prospects for small molecule inhibitors of p110γ and/or p110δ in autoimmune disease.

Budding yeast centromeres are sequence-defined point centromeres and are, unlike in many other organisms, not embedded in heterochromatin. Here we show that Fun30, a poorly understood SWI/SNF-like chromatin remodeling factor conserved in humans, promotes point centromere function through the formation of correct chromatin architecture at centromeres. Our determination of the genome-wide binding and nucleosome positioning properties of Fun30 shows that this enzyme is consistently enriched over centromeres and that a majority of CENs show Fun30-dependent changes in flanking nucleosome position and/or CEN core micrococcal nuclease accessibility. Fun30 deletion leads to defects in histone variant Htz1 occupancy genome-wide, including at and around most centromeres. FUN30 genetically interacts with CSE4, coding for the centromere-specific variant of histone H3, and counteracts the detrimental effect of transcription through centromeres on chromosome segregation and suppresses transcriptional noise over centromere CEN3. Previous work has shown a requirement for fission yeast and mammalian homologs of Fun30 in heterochromatin assembly. As centromeres in budding yeast are not embedded in heterochromatin, our findings indicate a direct role of Fun30 in centromere chromatin by promoting correct chromatin architecture.

Psoriasis is a chronic inflammatory skin disease triggered by interplay between immune mediators from both innate and adaptive immune systems and skin tissue, in which the IL-23/IL-17 axis is critical. PI3Kδ and PI3Kγ play important roles in various immune cell functions. We found that mice lacking functional PI3Kδ or PI3Kγ are largely protected from imiquimod (IMQ)-induced psoriasis-like dermatitis, correlating with reduced IL-17 levels in the lesions, serum, and the draining lymph nodes. TCRγδ T cells were the major IL-17-producing population in the draining lymph nodes and were significantly diminished in IMQ-treated PI3Kδ knockin and PI3Kγ knockout mice. We also show that PI3Kδ and PI3Kγ inhibitors reduced IFN-γ production by human TCRγδ T cells and IL-17 and IFN-γ production by PBMCs from psoriatic or healthy donors. In addition, inhibition of PI3Kγ, but not PI3Kδ, blocked chemotaxis of CCR6(+)IL-17-producing cells from IMQ-treated mice or healthy human donors. Taken together, these data indicate that PI3Kδ and/or PI3Kγ inhibitors should be considered for treating IL-17-driven diseases, such as psoriasis.

Mononucleosomes, the basic building blocks of chromatin, contain two copies of each core histone. The associated posttranslational modifications regulate essential chromatin-dependent processes, yet whether each histone copy is identically modified in vivo is unclear. We demonstrate that nucleosomes in embryonic stem cells, fibroblasts, and cancer cells exist in both symmetrically and asymmetrically modified populations for histone H3 lysine 27 di/trimethylation (H3K27me2/3) and H4K20me1. Further, we obtained direct physical evidence for bivalent nucleosomes carrying H3K4me3 or H3K36me3 along with H3K27me3, albeit on opposite H3 tails. Bivalency at target genes was resolved upon differentiation of ES cells. Polycomb repressive complex 2-mediated methylation of H3K27 was inhibited when nucleosomes contain symmetrically, but not asymmetrically, placed H3K4me3 or H3K36me3. These findings uncover a potential mechanism for the incorporation of bivalent features into nucleosomes and demonstrate how asymmetry might set the stage to diversify functional nucleosome states.

The function of the gastrointestinal tract relies on a monolayer of epithelial cells, which are essential for the uptake of nutrients. The fragile lining requires protection against insults by a diverse array of antigens. This is accomplished by the mucosa-associated lymphoid tissues of the gastrointestinal tract, which constitute a highly organized immune organ. In this Review, we discuss several recent findings that provide a compelling link between dietary compounds and the organization and maintenance of immune tissues and lymphocytes in the intestine. We highlight some of the molecular players involved, in particular ligand-activated nuclear receptors in lymphoid cells.

The Kinetic Simulation Algorithm Ontology (KiSAO) supplies information about existing algorithms available for the simulation of Systems Biology models, their characteristics, parameters and inter-relationships. KiSAO enables the unambiguous identification of algorithms from simulation descriptions. Information about analogous methods having similar characteristics and about algorithm parameters incorporated into KiSAO is desirable for simulation tools. To retrieve this information programmatically an application programming interface (API) for KiSAO is needed.