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Thymocytes are tested for productive rearrangement of the tcrb locus by expression of a pre-TCR in a process termed β-selection, which requires both Notch1 and CXCR4 signaling. It has been shown that activation of the GTPase Ras allows thymocytes to proliferate and differentiate in the absence of a Pre-TCR; the direct targets of Ras at this checkpoint have not been identified, however. Mice with a mutant allele of p110γ unable to bind active Ras revealed that CXCR4-mediated PI3K activation is Ras dependent. The Ras-p110γ interaction was necessary for efficient β-selection-promoted proliferation but was dispensable for the survival or differentiation of thymocytes. Uncoupling Ras from p110γ provides unambiguous identification of a Ras interaction required for thymic β-selection.

The pro-apoptotic BH3 only protein BIM(EL) is phosphorylated by ERK1/2 and this targets it for proteasome-dependent degradation. A recent study has shown that ERK5, an ERK1/2-related MAPK, is activated during mitosis and phosphorylates BIM(EL) to promote cell survival. Here we show that treatment of cells with nocodazole or paclitaxel does cause phosphorylation of BIM(EL), which is independent of ERK1/2. However, this was not due to ERK5-catalysed phosphorylation, since it was not reversed by the MEK5 inhibitor BIX02189 and proceeded normally in ERK5-/- fibroblasts. Indeed, although ERK5 is phosphorylated at multiple sites in the C-terminal transactivation region during mitosis, these do not include the activation-loop and ERK5 kinase activity does not increase. Mitotic phosphorylation of BIM(EL) occurred at proline-directed phospho-acceptor sites and was abolished by selective inhibition of CDK1. Furthermore, cyclin B1 was able to interact with BIM and cyclin B1/CDK1 complexes could phosphorylate BIM in vitro. Finally, we show that CDK1-dependent phosphorylation of BIM(EL) drives its polyubiquitylation and proteasome-dependent degradation to protect cells during mitotic arrest. These results provide new insights into the regulation of BIM(EL) and may be relevant to the therapeutic use of agents such as paclitaxel.

Imprinted genes are the prototypical epigenetically regulated genes. On the basis of findings in adult lung stem cells, Zacharek et al. (2011) suggest in this issue of Cell Stem Cell that epigenetic silencing of imprinted genes is a common requirement for maintaining self-renewal in adult stem cell populations.

Blood platelets undergo a carefully regulated change in shape to serve as the primary mediators of hemostasis and thrombosis. These processes manifest through platelet spreading and aggregation and are dependent on platelet actin cytoskeletal changes orchestrated by the Rho GTPase family member Rac1. To elucidate how Rac1 is regulated in platelets, we captured Rac1-interacting proteins from platelets and identified Rac1-associated proteins by mass spectrometry.

Calcium (Ca(2+)) is a critical regulator of cardiac myocyte function. Principally, Ca(2+) is the link between the electrical signals that pervade the heart and contraction of the myocytes to propel blood. In addition, Ca(2+) controls numerous other myocyte activities, including gene transcription. Cardiac Ca(2+) signaling essentially relies on a few critical molecular players--ryanodine receptors, voltage-operated Ca(2+) channels, and Ca(2+) pumps/transporters. These moieties are responsible for generating Ca(2+) signals upon cellular depolarization, recovery of Ca(2+) signals following cellular contraction, and setting basal conditions. Whereas these are the central players underlying cardiac Ca(2+) fluxes, networks of signaling mechanisms and accessory proteins impart complex regulation on cardiac Ca(2+) signals. Subtle changes in components of the cardiac Ca(2+) signaling machinery, albeit through mutation, disease, or chronic alteration of hemodynamic demand, can have profound consequences for the function and phenotype of myocytes. Here, we discuss mechanisms underlying Ca(2+) signaling in ventricular and atrial myocytes. In particular, we describe the roles and regulation of key participants involved in Ca(2+) signal generation and reversal.

DNA hypomethylation is assumed to be a feature of the mammalian placenta; however, its role in regulating placental gene expression is not well defined. In this study, MeDIP and Sequenom MassARRAY were used to identify hypomethylated gene promoters in the human placenta. Among the genes identified, the hypomethylation of an alternative promoter for KCNH5 was found to be restricted to the placenta and chorion. Complete methylation of this promoter correlates with a silenced KCNH5 transcript in embryonic tissues, including the amnion. Unusually, this hypomethylated promoter and the alternative first exon are derived from a SINE (AluY) retrotransposon. Examination of additional retrotransposon-derived gene promoters in the placenta confirmed that retrotransposon hypomethylation permits the placenta-specific expression of these genes. Furthermore, the lineage-specific methylation displayed by KCNH5, INSL4, and ERVWE1 revealed that dichotomous methylation establishes differential retrotransposon silencing between the extra-embryonic and embryonic lineages. The hypomethylation of the retrotransposons that regulate these genes, each of which arose during recent primate evolution, is consistent with these genes having functional roles that are unique to the invasive haemochorial placentas of humans and recent primates.

The noncoding Tsix RNA is an antisense repressor of Xist and regulates X inactivation in mice. Tsix is essential for preventing the inactivation of the maternally inherited X chromosome in extraembryonic lineages where imprinted X-chromosome inactivation (XCI) occurs. Here we establish an inducible Tsix expression system for investigating Tsix function in development. We show that Tsix has a clear functional window in extraembryonic development. Within this window, Tsix can repress Xist, which is accompanied by DNA methylation of the Xist promoter. As a consequence of Xist repression, reactivation of the inactive X chromosome (Xi) is widely observed. In the parietal endoderm, Tsix represses Xist and causes reactivation of an Xi-linked GFP transgene throughout development, whereas Tsix progressively loses its Xist-repressing function from embryonic day 9.5 (E9.5) onward in trophoblast giant cells and spongiotrophoblast, suggesting that Tsix function depends on a lineage-specific environment. Our data also demonstrate that the maintenance of imprinted XCI requires Xist expression in specific extraembryonic tissues throughout development. This finding shows that reversible XCI is not exclusive to pluripotent cells, and that in some lineages cell differentiation is not accompanied by a stabilization of the Xi.

Dynamic changes in gene expression punctuate lymphocyte development and are a characteristic of lymphocyte activation. A prevailing view has been that these changes are driven by DNA transcription factors, which are the dominant force in gene expression. Accumulating evidence is challenging this DNA centric view and has highlighted the prevalence and dynamic nature of RNA handling mechanisms. Alternative splicing and differential polyadenylation appear to be more widespread than first thought. Changes in mRNA decay rates also affect the abundance of transcripts and this mechanism may contribute significantly to gene expression. Additional RNA handling mechanisms that control the intracellular localization of mRNA and association with translating ribosomes are also important. Thus, gene expression is regulated through the coordination of transcriptional and post-transcriptional mechanisms. Developing a more "RNA centric" view of gene expression will allow a more systematic understanding of how gene expression and cell function are integrated.

Dysregulated release of neutrophil azurophilic granules causes increased tissue damage and amplified inflammation during autoimmune disease. Antineutrophil cytoplasmic antibodies (ANCAs) are implicated in the pathogenesis of small vessel vasculitis and promote adhesion and exocytosis in neutrophils. ANCAs activate specific signal transduction pathways in neutrophils that have the potential to be modulated therapeutically to prevent neutrophil activation by ANCAs. We have investigated a role for diacylglycerol kinase (DGK) and its downstream product phosphatidic acid (PA) in ANCA-induced neutrophil exocytosis. Neutrophils incubated with the DGK inhibitor R59022, before treatment with ANCAs, exhibited a reduced capacity to release their azurophilic granules, demonstrated by a component release assay and flow cytometry. PA restored azurophilic granule release in DGK-inhibited neutrophils. Confocal microscopy revealed that R59022 did not inhibit translocation of granules, indicating a role for DGK during the process of granule fusion at the plasma membrane. In investigating possible mechanisms by which PA promotes neutrophil exocytosis, we demonstrated that exocytosis can only be restored in R59022-treated cells through simultaneous modulation of membrane fusion and increasing cytosolic calcium. PA and its associated pathways may represent viable drug targets to reduce tissue injury associated with ANCA-associated vasculitic diseases and other neutrophilic inflammatory disorders.

The immunological synapse (IS) serves a dual role for sustained T cell receptor (TCR) signaling and for TCR downregulation. TC21 (Rras2) is a RRas subfamily GTPase that constitutively associates with the TCR and is implicated in tonic TCR signaling by activating phosphatidylinositol 3-kinase. In this study, we demonstrate that TC21 both cotranslocates with the TCR to the IS and is necessary for TCR internalization from the IS through a mechanism dependent on RhoG, a small GTPase previously associated with phagocytosis. Indeed, we found that the TCR triggers T cells to phagocytose 1-6 μm beads through a TC21- and RhoG-dependent pathway. We further show that TC21 and RhoG are necessary for the TCR-promoted uptake of major histocompatibility complex (MHC) from antigen-presenting cells. Therefore, TC21 and RhoG dependence underlie the existence of a common phagocytic mechanism that drives TCR internalization from the IS together with its peptide-MHC ligand.

BMP is thought to induce hESC differentiation toward multiple lineages including mesoderm and trophoblast. The BMP-induced trophoblast phenotype is a long-standing paradox in stem cell biology. Here we readdressed BMP function in hESCs and mouse epiblast-derived cells. We found that BMP4 cooperates with FGF2 (via ERK) to induce mesoderm and to inhibit endoderm differentiation. These conditions induced cells with high levels of BRACHYURY (BRA) that coexpressed CDX2. BRA was necessary for and preceded CDX2 expression; both genes were essential for expression not only of mesodermal genes but also of trophoblast-associated genes. Maximal expression of the latter was seen in the absence of FGF but these cells coexpressed mesodermal genes and moreover they differed in cell surface and epigenetic properties from placental trophoblast. We conclude that BMP induces human and mouse pluripotent stem cells primarily to form mesoderm, rather than trophoblast, acting through BRA and CDX2.

Typically, only a fraction of the ≥600 ribosomal RNA (rRNA) gene copies in human cells are transcriptionally active. Expressed rRNA genes coalesce in specialized nuclear compartments - the nucleoli - and are believed to replicate during the first half of S phase. Paradoxically, attempts to visualize replicating rDNA during early S phase have failed. Here, I show that, in human (HeLa) cells, early-replicating rDNA is detectable at the nucleolar periphery and, more rarely, even outside nucleoli. Early-replicated rDNA relocates to the nucleolar interior and reassociates with the transcription factor UBF, implying that it predominantly represents expressed rDNA units. Contrary to the established model for active gene loci, replication initiates randomly throughout the early-replicating rDNA. By contrast, mostly silent rDNA copies replicate inside the nucleoli during mid and late S phase. At this stage, replication origins are fired preferentially within the non-transcribed intergenic spacers (NTSs), and ongoing rDNA transcription is required to maintain this specific initiation pattern. I propose that the unexpected spatial dynamics of the early-replicating rDNA repeats serve to ensure streamlined efficient replication of the most heavily transcribed genomic loci while simultaneously reducing the risk of chromosome breaks and rDNA hyper-recombination.

Autophagy is a cellular response to starvation which generates autophagosomes to carry cellular organelles and long-lived proteins to lysosomes for degradation. Degradation through autophagy can provide an innate defence against virus infection, or conversely autophagosomes can promote infection by facilitating assembly of replicase proteins. We demonstrate that the avian coronavirus, Infectious Bronchitis Virus (IBV) activates autophagy. A screen of individual IBV non-structural proteins (nsps) showed that autophagy was activated by IBV nsp6. This property was shared with nsp6 of mammalian coronaviruses Mouse Hepatitis Virus, and Severe Acute Respiratory Syndrome Virus, and the equivalent nsp5-7 of the arterivirus Porcine Reproductive and Respiratory Syndrome Virus. These multiple-spanning transmembrane proteins located to the endoplasmic reticulum (ER) where they generated Atg5 and LC3II-positive vesicles, and vesicle formation was dependent on Atg5 and class III PI3 kinase. The vesicles recruited double FYVE-domain containing protein (DFCP) indicating localised concentration of phosphatidylinositol 3 phosphate, and therefore shared many features with omegasomes formed from the ER in response to starvation. Omegasomes induced by viral nsp6 matured into autophagosomes that delivered LC3 to lysosomes and therefore recruited and recycled the proteins needed for autophagosome nucleation, expansion, cellular trafficking and delivery of cargo to lysosomes. The coronavirus nsp6 proteins activated omegasome and autophagosome formation independently of starvation, but activation did not involve direct inhibition of mTOR signalling, activation of sirtuin1 or induction of ER stress.

Genomic imprinting is an important and enigmatic form of gene regulation in mammals in which one copy of a gene is silenced in a manner determined by its parental history. Imprinted genes range from those with constitutive monoallelic silencing to those, typically more remote from imprinting control regions, that display developmentally regulated, tissue-specific or partial monoallelic expression. This diversity may make these genes, and the processes they control, more or less sensitive to factors that modify or disrupt epigenetic marks. Imprinted genes have important functions in development and physiology, including major endocrine/neuroendocrine axes. Owing to is central role in coordinating growth, metabolism and reproduction, as well as evidence from genetic and knockout studies, the hypothalamus may be a focus for imprinted gene action. Are there unifying principles that explain why a gene should be imprinted? Conflict between parental genomes over limiting maternal resources, but also co-adaptation between mothers and offspring, have been invoked to explain the evolution of imprinting. Recent reports suggest there may be many more genes imprinted in the hypothalamus than hitherto expected, and it will be important for these new candidates to be validated and to determine whether they conform to current notions of how imprinting is regulated. In fully evaluating the role of imprinted genes in the hypothalamus, much work needs to be done to identify the specific neuronal populations in which particular genes are expressed, establish whether there are pathways in common and whether imprinted genes are involved in long-term programming of hypothalamic functions.

Considering the many differences between mice and humans, it is perhaps surprising how well mice model late-onset human neurodegenerative disease. Models of Alzheimer's disease, frontotemporal dementia, Parkinson's disease and Huntington's disease show some striking similarities to the corresponding human pathologies in terms of axonal transport disruption, protein aggregation, synapse loss and some behavioural phenotypes. However, there are also major differences. To extrapolate from mouse models to human disease, we need to understand how these differences relate to intrinsic limitations of the mouse system and to the effects of transgene overexpression. In the present paper, we use examples from an amyloid-overexpression model and a mutant-tau-knockin model to illustrate what we learn from each type of approach and what the limitations are. Finally, we discuss the further contributions that knockin and similar approaches can make to understanding pathogenesis and how best to model disorders of aging in a short-lived mammal.

The conserved Polycomb repressive complex 2 (PRC2) generates trimethylation of histone 3 lysine 27 (H3K27me3), a modification associated with stable epigenetic silencing. Much is known about PRC2-induced silencing but key questions remain concerning its nucleation and stability. Vernalization, the perception and memory of winter in plants, is a classic epigenetic process that, in Arabidopsis, involves PRC2-based silencing of the floral repressor FLC. The slow dynamics of vernalization, taking place over weeks in the cold, generate a level of stable silencing of FLC in the subsequent warm that depends quantitatively on the length of the prior cold. These features make vernalization an ideal experimental system to investigate both the maintenance of epigenetic states and the switching between them. Here, using mathematical modelling, chromatin immunoprecipitation and an FLC:GUS reporter assay, we show that the quantitative nature of vernalization is generated by H3K27me3-mediated FLC silencing in the warm in a subpopulation of cells whose number depends on the length of the prior cold. During the cold, H3K27me3 levels progressively increase at a tightly localized nucleation region within FLC. At the end of the cold, numerical simulations predict that such a nucleation region is capable of switching the bistable epigenetic state of an individual locus, with the probability of overall FLC coverage by silencing H3K27me3 marks depending on the length of cold exposure. Thus, the model predicts a bistable pattern of FLC gene expression in individual cells, a prediction we verify using the FLC:GUS reporter system. Our proposed switching mechanism, involving the local nucleation of an opposing histone modification, is likely to be widely relevant in epigenetic reprogramming.

Follicular helper (T(FH)) cells provide crucial signals to germinal center B cells undergoing somatic hypermutation and selection that results in affinity maturation. Tight control of T(FH) numbers maintains self tolerance. We describe a population of Foxp3(+)Blimp-1(+)CD4(+) T cells constituting 10-25% of the CXCR5(high)PD-1(high)CD4(+) T cells found in the germinal center after immunization with protein antigens. These follicular regulatory T (T(FR)) cells share phenotypic characteristics with T(FH) and conventional Foxp3(+) regulatory T (T(reg)) cells yet are distinct from both. Similar to T(FH) cells, T(FR) cell development depends on Bcl-6, SLAM-associated protein (SAP), CD28 and B cells; however, T(FR) cells originate from thymic-derived Foxp3(+) precursors, not naive or T(FH) cells. T(FR) cells are suppressive in vitro and limit T(FH) cell and germinal center B cell numbers in vivo. In the absence of T(FR) cells, an outgrowth of non-antigen-specific B cells in germinal centers leads to fewer antigen-specific cells. Thus, the T(FH) differentiation pathway is co-opted by T(reg) cells to control the germinal center response.

The exploding number of computational models produced by Systems Biologists over the last years is an invitation to structure and exploit this new wealth of information. Researchers would like to trace models relevant to specific scientific questions, to explore their biological content, to align and combine them, and to match them with experimental data. To automate these processes, it is essential to consider semantic annotations, which describe their biological meaning. As a prerequisite for a wide range of computational methods, we propose general and flexible similarity measures for Systems Biology models computed from semantic annotations. By using these measures and a large extensible ontology, we implement a platform that can retrieve, cluster, and align Systems Biology models and experimental data sets. At present, its major application is the search for relevant models in the BioModels Database, starting from initial models, data sets, or lists of biological concepts. Beyond similarity searches, the representation of models by semantic feature vectors may pave the way for visualisation, exploration, and statistical analysis of large collections of models and corresponding data.

Major eukaryotic genomic elements, including the ribosomal DNA (rDNA), are composed of repeated sequences with well-defined copy numbers that must be maintained by regulated recombination. Although mechanisms that instigate rDNA recombination have been identified, none are directional and they therefore cannot explain precise repeat number control. Here, we show that yeast lacking histone chaperone Asf1 undergo reproducible rDNA repeat expansions. These expansions do not require the replication fork blocking protein Fob1 and are therefore independent of known rDNA expansion mechanisms. We propose the existence of a regulated rDNA repeat gain pathway that becomes constitutively active in asf1Δ mutants. Cells lacking ASF1 accumulate rDNA repeats with high fidelity in a processive manner across multiple cell divisions. The mechanism of repeat gain is dependent on highly repetitive sequence but, surprisingly, is independent of the homologous recombination proteins Rad52, Rad51 and Rad59. The expansion mechanism is compromised by mutations that decrease the processivity of DNA replication, which leads to progressive loss of rDNA repeats. Our data suggest that a novel mode of break-induced replication occurs in repetitive DNA that is dependent on high homology but does not require the canonical homologous recombination machinery.

The utilization of neural stem cells and their progeny in applications such as disease modelling, drug screening or safety assessment will require the development of robust methods for consistent, high quality uniform cell production. Previously, we described the generation of adherent, homogeneous, non-immortalized mouse and human neural stem cells derived from both brain tissue and pluripotent embryonic stem cells (Conti et al., 2005; Sun et al., 2008). In this study, we report the isolation or derivation of stable neurogenic human NS (hNS) lines from different regions of the 8-9 gestational week fetal human central nervous system (CNS) using new serum-free media formulations including animal component-free conditions. We generated more than 20 adherent hNS lines from whole brain, cortex, lobe, midbrain, hindbrain and spinal cord. We also compared the adherent hNS to some aspects of the human CNS-stem cells grown as neurospheres (hCNS-SCns), which were derived from prospectively isolated CD133(+)CD24(-/lo) cells from 16 to 20 gestational week fetal brain. We found, by RT-PCR and Taqman low-density array, that some of the regionally isolated lines maintained their regional identity along the anteroposterior axis. These NS cells exhibit the signature marker profile of neurogenic radial glia and maintain neurogenic and multipotential differentiation ability after extensive long-term expansion. Similarly, hCNS-SC can be expanded either as neurospheres or in extended adherent monolayer with a morphology and marker expression profile consistent with radial glia NS cells. We demonstrate that these lines can be efficiently genetically modified with standard nucleofection protocols for both protein overexpression and siRNA knockdown of exogenously expressed and endogenous genes exemplified with GFP and Nestin. To investigate the functional maturation of neuronal progeny derived from hNS we (a) performed Agilent whole genome microarray gene expression analysis from cultures undergoing neuronal differentiation for up to 32 days and found increased expression over time for a number of drugable target genes including neurotransmitter receptors and ion channels and (b) conducted a neuropharmacology study utilizing Fura-2 Ca(2+) imaging which revealed a clear shift from an initial glial reaction to carbachol to mature neuron-specific responses to glutamate and potassium after prolonged neuronal differentiation. Fully automated culture and scale-up of select hNS was achieved; cells supplied by the robot maintained the molecular profile of multipotent NS cells and performed faithfully in neuronal differentiation experiments. Here, we present validation and utility of a human neural lineage-restricted stem cell-based assay platform, including scale-up and automation, genetic engineering and functional characterization of differentiated progeny.

Co-localization of networks of genes in the nucleus is thought to play an important role in determining gene expression patterns. Based upon experimental data, we built a dynamical model to test whether pure diffusion could account for the observed co-localization of genes within a defined subnuclear region. A simple standard Brownian motion model in two and three dimensions shows that preferential co-localization is possible for co-regulated genes without any direct interaction, and suggests the occurrence may be due to a limitation in the number of available transcription factors. Experimental data of chromatin movements demonstrates that fractional rather than standard Brownian motion is more appropriate to model gene mobilizations, and we tested our dynamical model against recent static experimental data, using a sub-diffusion process by which the genes tend to colocalize more easily. Moreover, in order to compare our model with recently obtained experimental data, we studied the association level between genes and factors, and presented data supporting the validation of this dynamic model. As further applications of our model, we applied it to test against more biological observations. We found that increasing transcription factor number, rather than factory number and nucleus size, might be the reason for decreasing gene co-localization. In the scenario of frequency- or amplitude-modulation of transcription factors, our model predicted that frequency-modulation may increase the co-localization between its targeted genes.

Foxp3(+) regulatory T cells play a pivotal role in maintaining self-tolerance and immune homeostasis. In the absence of regulatory T cells, generalized immune activation and multiorgan T cell-driven pathology occurs. Although the phenomenon of immunologic control by Foxp3(+) regulatory T cells is well recognized, the comparative effect over different arms of the immune system has not been thoroughly investigated. Here, we generated a cohort of mice with a continuum of regulatory T-cell frequencies ranging from physiologic levels to complete deficiency. This titration of regulatory T-cell depletion was used to determine how different effector subsets are controlled. We found that in vivo Foxp3(+) regulatory T-cell frequency had a proportionate relationship with generalized T-cell activation and Th1 magnitude, but it had a surprising disproportionate relationship with Th2 magnitude. The asymmetric regulation was associated with efficient suppression of Th2 cells through additional regulations on the apoptosis rate in Th2 cells and not Th1 cells and could be replicated by CTLA4-Ig or anti-IL-2 Ab. These results indicate that the Th2 arm of the immune system is under tighter control by regulatory T cells than the Th1 arm, suggesting that Th2-driven diseases may be more responsive to regulatory T-cell manipulation.

Elucidating how and to what extent CpG islands (CGIs) are methylated in germ cells is essential to understand genomic imprinting and epigenetic reprogramming. Here we present, to our knowledge, the first integrated epigenomic analysis of mammalian oocytes, identifying over a thousand CGIs methylated in mature oocytes. We show that these CGIs depend on DNMT3A and DNMT3L but are not distinct at the sequence level, including in CpG periodicity. They are preferentially located within active transcription units and are relatively depleted in H3K4me3, supporting a general transcription-dependent mechanism of methylation. Very few methylated CGIs are fully protected from post-fertilization reprogramming but, notably, the majority show incomplete demethylation in embryonic day (E) 3.5 blastocysts. Our study shows that CGI methylation in gametes is not entirely related to genomic imprinting but is a strong factor in determining methylation status in preimplantation embryos, suggesting a need to reassess mechanisms of post-fertilization demethylation.