During early mammalian embryogenesis, the genome undergoes global epigenetic reprogramming, losing most of its methylation before re-establishing it de novo at implantation. However, faithful maintenance of methylation at imprinted genes during this process is vital for embryonic development, but the DNA methyltransferase responsible for this maintenance has remained unknown. In this issue of Genes & Development, Hirasawa and colleagues (pp. 1607-1616) show that Dnmt1, and not Dnmt3a or Dnmt3b, maintains methylation at genomic imprints during preimplantation development.

Colorectal cancer is the second most common cause of cancer related death in the developed world. To date, no blood or stool biomarkers with both high sensitivity and specificity for potentially curable early stage disease have been validated for clinical use. SELDI and MALDI profiling are being used increasingly to search for biomarkers in both blood and urine. Both techniques provide information predominantly on the low molecular weight proteome (<15 kDa). There have been several reports that colorectal cancer is associated with changes in the serum proteome that are detectable by SELDI and we hypothesised that proteomic changes would also be detectable in urine.

Partial T-cell immunodeficiencies constitute a heterogeneous cluster of disorders characterized by an incomplete reduction in T-cell number or activity. The immune deficiency component of these diseases is less severe than that of the severe T-cell immunodeficiencies and therefore some ability to respond to infectious organisms is retained. Unlike severe T-cell immunodeficiencies, however, partial immunodeficiencies are commonly associated with hyper-immune dysregulation, including autoimmunity, inflammatory diseases and elevated IgE production. This causative association is counter-intuitive--immune deficiencies are caused by loss-of-function changes to the T-cell component, whereas the coincident autoimmune symptoms are the consequence of gain-of-function changes. This Review details the genetic basis of partial T -cell immunodeficiencies and draws on recent advances in mouse models to propose mechanisms by which a reduction in T-cell numbers or function may disturb the population-dependent balance between activation and tolerance.

The p110 isoforms of phosphoinositide 3-kinase (PI3K) are acutely regulated by extracellular stimuli. The class IA PI3K catalytic subunits (p110alpha, p110beta, and p110delta) occur in complex with a Src homology 2 (SH2) domain-containing p85 regulatory subunit, which has been shown to link p110alpha and p110delta to Tyr kinase signaling pathways. The p84/p101 regulatory subunits of the p110gamma class IB PI3K lack SH2 domains and instead couple p110gamma to G protein-coupled receptors (GPCRs). Here, we show, using small-molecule inhibitors with selectivity for p110beta and cells derived from a p110beta-deficient mouse line, that p110beta is not a major effector of Tyr kinase signaling but couples to GPCRs. In macrophages, both p110beta and p110gamma contributed to Akt activation induced by the GPCR agonist complement 5a, but not by the Tyr kinase ligand colony-stimulating factor-1. In fibroblasts, which express p110beta but not p110gamma, p110beta mediated Akt activation by the GPCR ligands stromal cell-derived factor, sphingosine-1-phosphate, and lysophosphatidic acid but not by the Tyr kinase ligands PDGF, insulin, and insulin-like growth factor 1. Introduction of p110gamma in these cells reduced the contribution of p110beta to GPCR signaling. Taken together, these data show that p110beta and p110gamma can couple redundantly to the same GPCR agonists. p110beta, which shows a much broader tissue distribution than the leukocyte-restricted p110gamma, could thus provide a conduit for GPCR-linked PI3K signaling in the many cell types where p110gamma expression is low or absent.

Eukaryotic cells are thought to move across supporting surfaces through a combination of coordinated processes: polarisation; extension of dynamic protrusions from a leading edge; adhesion-associated stabilisation of some protrusions; centripetal pulling against those leading adhesions; and de-adhesion at the rear. Gradients of extracellular ligands can be detected by cells and then used to guide them either towards the source (in the case of a chemoattractant) or away from the source (in the case of a chemorepellent)--such migration is termed chemotaxis. Recent work suggests that chemotaxis probably emerges from the ability of cells to spatially encode extracellular gradients of ligands, a process for which phosphoinositide 3'-kinase (PI3K) signals alone are insufficient, and to use that vectorial information to bias movement by enhancing the survival, and not the formation, of the protrusions that experience the greatest stimulation.

Regulatory T (Treg) cells safeguard against autoimmunity and immune pathology. Because determinants of the Treg cell fate are not completely understood, we have delineated signaling events that control the de novo expression of Foxp3 in naive peripheral CD4 T cells and in thymocytes. We report that premature termination of TCR signaling and inibition of phosphatidyl inositol 3-kinase (PI3K) p110alpha, p110delta, protein kinase B (Akt), or mammalian target of rapamycin (mTOR) conferred Foxp3 expression and Treg-like gene expression profiles. Conversely, continued TCR signaling and constitutive PI3K/Akt/mTOR activity antagonised Foxp3 induction. At the chromatin level, di- and trimethylation of lysine 4 of histone H3 (H3K4me2 and -3) near the Foxp3 transcription start site (TSS) and within the 5' untranslated region (UTR) preceded active Foxp3 expression and, like Foxp3 inducibility, was lost upon continued TCR stimulation. These data demonstrate that the PI3K/Akt/mTOR signaling network regulates Foxp3 expression.

Attempts to identify causal interactions in multivariable biological time series (e.g., gene data, protein data, physiological data) can be undermined by the confounding influence of environmental (exogenous) inputs. Compounding this problem, we are commonly only able to record a subset of all related variables in a system. These recorded variables are likely to be influenced by unrecorded (latent) variables. To address this problem, we introduce a novel variant of a widely used statistical measure of causality--Granger causality--that is inspired by the definition of partial correlation. Our 'partial Granger causality' measure is extensively tested with toy models, both linear and nonlinear, and is applied to experimental data: in vivo multielectrode array (MEA) local field potentials (LFPs) recorded from the inferotemporal cortex of sheep. Our results demonstrate that partial Granger causality can reveal the underlying interactions among elements in a network in the presence of exogenous inputs and latent variables in many cases where the existing conditional Granger causality fails.

We present a statistical approach to the identification of correlated activity in multineuron spike data, based on the value of the correlation determinant. This approach is not compromised by the lack of independence often encountered in this kind of data. We illustrate our method by applying it both to simulated data and to data recorded from neurons in a forebrain region (intermediate medial mesopallium, IMM) of the behaving domestic chick and simultaneously from the corresponding contralateral region. There is no direct anatomical connection between the two sites, and the validity of this technique is strongly supported by the observation that when the test indicates significantly correlated activity for neurons within either hemisphere, this correlation is greatly reduced, and ultimately obliterated, by serial incorporation of activity from neurons in the opposite hemisphere. Since the value of individual correlation coefficients allied to the Bonferroni correction is often used as a diagnostic tool, we also present comparisons of that approach with our correlation determinant approach.

Commitment of hematopoietic progenitor cells to B-lymphoid cell fate has been suggested to coincide with the development of PAX5-expressing B220(+)CD19(+) pro-B cells. We have used a transgenic reporter mouse, expressing human CD25 under the control of the B-lineage-restricted Igll1 (lambda5) promoter to investigate the lineage potential of early progenitor cells in the bone marrow. This strategy allowed us to identify a reporter expressing LIN(-)B220(-)CD19(-)CD127(+)FLT3(+)SCA1(low)KIT(low) population that displays a lack of myeloid and a 90% reduction in in vitro T-cell potential compared with its reporter-negative counterpart. Gene expression analysis demonstrated that these lineage-restricted cells express B-lineage-associated genes to levels comparable with that observed in pro-B cells. These data suggest that B-lineage commitment can occur before the expression of B220 and CD19.

While the extravasation cascade of lymphocytes is well characterized, data on their intraepithelial positioning and morphology are scant. However, the latter process is presumably crucial for many immune functions. Integrin alpha(E)(CD103)beta(7) has previously been implicated in epithelial retention of some T cells through binding to E-cadherin. Our current data suggest that alpha(E)(CD103)beta(7) also determines shape and motility of some lymphocytes. Time-lapse microscopy showed that wild-type alpha(E)(CD103)beta(7) conferred the ability to form cell protrusions/filopodia and to move in an amoeboid fashion on E-cadherin, an activity that was abrogated by alpha(E)(CD103)beta(7)-directed antibodies or cytochalasin D. The alpha(E)-dependent motility was further increased (P < .001) when point-mutated alpha(E)(CD103) locked in a constitutively active conformation was expressed. Moreover, different yellow fluorescent protein-coupled alpha(E)(CD103) species demonstrated that the number and length of filopodia extended toward purified E-cadherin, cocultured keratinocytes, cryostat-cut skin sections, or epidermal sheets depended on functional alpha(E)(CD103). The in vivo relevance of these findings was demonstrated by wild-type dendritic epidermal T cells (DETCs), which showed significantly more dendrites and spanned larger epidermal areas as compared with DETCs of alpha(E)(CD103)-deficient mice (P < .001). Thus, integrin alpha(E)(CD103)beta(7) is not only involved in epithelial retention, but also in shaping and proper intraepithelial morphogenesis of some leukocytes.

Insulin stimulates glucose uptake by adipocytes through increasing translocation of the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane. Fusion of GLUT4-containing vesicles at the cell surface is thought to involve phospholipase D activity, generating the signalling lipid phosphatidic acid, although the mechanism of action is not yet clear.

One often compares cells to computers, and signalling proteins to transistors. Location and wiring of those molecular transistors is paramount in defining the function of the subcellular chips. The bacterial chemotactic sensing apparatus is a large, stable assembly consisting of thousands of receptors, signal transducing kinases and linking proteins, and is responsible for the motile response of the bacterium to environmental signals, whether chemical, mechanical, or thermal. Because of its rich functional repertoire despite its relative simplicity, this chemosome has attracted much attention from both experimentalists and theoreticians, and the bacterial chemotaxis response becoming a benchmark in Systems Biology. Structural and functional models of the chemotactic device have been developed, often based on particular assumptions regarding the topology of the receptor lattice. In this issue of Molecular Microbiology, Briegel et al. provide a detailed view of the receptor arrangement, unravelling the wiring of the molecular signal processors.

miRs (microRNAs) post-transcriptionally regulate gene expression mainly by repressing translation or by inducing mRNA degradation. Dicer, an enzyme responsible for miR biogenesis, is required for T-cell function, suggesting regulatory roles for miRs in lymphocytes. However, specific roles for individual miRs are only just beginning to emerge. miR-155 is encoded within an exon of the non-coding RNA known as bic (B-cell integration cluster) and high levels of bic expression are induced upon antigen receptor stimulation of B- and T-cells, as well as TLR (Toll-like receptor) stimulation of macrophages and dendritic cells. High levels of bic/miR-155 are found in B-cell lymphomas and solid tumours, indicating that this locus may also be linked to cancer. Indeed, transgenic mice overexpressing miR-155 develop B-cell malignancies. To define the in vivo role of bic/miR-155 (bic), we have studied bic-deficient mice. These mice are immunodeficient and fail to generate high levels of class-switched antibody upon immunization with thymus-dependent and thymus-independent antigens. This defect is intrinsic to B-cells and manifested at the level of differentiation of switched plasmablasts into mature antibody secreting plasma cells. In addition, bic-deficient T-cells show skewed differentiation into the Th2 lineage under a variety of in vitro culture conditions. Microarray analysis of bic-deficient B- and T-cells under different conditions has revealed a wide spectrum of targets regulated by an miR-155 and suggested mechanisms for the regulation of lymphocyte differentiation by a single miR.

Dopamine is a potentially toxic neurotransmitter that has long been speculated to contribute to the pathogenesis of Parkinson's disease (PD). Recent work has demonstrated the importance of proper storage of dopamine in vesicles to maintain dopamine homeostasis, thus protecting neurons from the detrimental effects of dopamine accumulation and breakdown in the cytosol. These studies suggest that factors which affect dopamine storage might increase the susceptibility of dopamine neurons to further environmental or genetic insults, exacerbating the neuronal degeneration that characterizes PD. This review seeks to revisit the pathogenicity of cytosolic dopamine and further address the critical role of neurotransmitter storage in dopamine-mediated neurotoxicity.

Wallerian degeneration slow (Wld(S)) mice express a chimeric protein that delays axonal degeneration. The N-terminal domain (N70), which is essential for axonal protection in vivo, binds valosin-containing protein (VCP) and targets both Wld(S) and VCP to discrete nuclear foci. We characterized the formation, composition and localization of these potentially important foci. Missense mutations show that the N-terminal sixteen residues (N16) of Wld(S) are essential for both VCP binding and targeting Wld(S) to nuclear foci. Removing N16 abolishes foci, and VCP binding sequences from ataxin-3 or HrdI restore them. In vitro, these puncta co-localize with proteasome subunits. In vivo, Wld(S) assumes a range of nuclear distribution patterns, including puncta, and its neuronal expression and intranuclear distribution is region-specific and varies between spontaneous and transgenic Wld(S) models. We conclude that VCP influences Wld(S) intracellular distribution, and thus potentially its function, by binding within the N70 domain required for axon protection.

Monoallelic gene expression exposes an organism to the risks associated with the unmasking of recessive mutations. A recent study by Gimelbrant and colleagues, supported by results from two methodologically different studies, demonstrated that random monoallelic expression is surprisingly widespread among autosomal genes. This raises important questions about why, when and how cells choose and tolerate monoallelism and whether functional hemizygosity might provide an unappreciated advantage.

The equatorial subsegment (EqSS) was originally identified by atomic force microscopy as a discrete region within the equatorial segment of Artiodactyl spermatozoa. In this investigation, we show that the EqSS is enriched in tyrosine phosphorylated proteins and present preliminary evidence for its presence in mouse and rat spermatozoa. The anti-phosphotyrosine monoclonal antibody (McAb) 4G10 bound strongly and discretely to the EqSS of permeabilized boar, ram, and bull spermatozoa. It also bound to a small patch on the posterior acrosomal region of permeabilized mouse and rat spermatozoa, suggesting that the EqSS is not restricted to the order Artiodactyla. An anti-HSPA1A (formerly Hsp70) antibody recognized the EqSS in boar spermatozoa. Immunogold labeling with McAb 4G10 localized the tyrosine phosphorylated proteins to the outer acrosomal membrane. This was verified by freeze-fracture electron microscopy, which identified the EqSS in three overlying membranes, the plasma membrane, outer acrosomal membrane, and inner acrosomal membrane. In all five species, tyrosine phosphorylated proteins became restricted to the EqSS during sperm maturation in the epididymis. The major tyrosine phosphorylated proteins in the EqSS of boar and ram spermatozoa were identified by mass spectrometry as orthologs of human SPACA1 (formerly SAMP32). Immunofluorescence with a specific polyclonal antibody localized SPACA1 to the equatorial segment in boar spermatozoa. We speculate that the EqSS is an organizing center for assembly of multimolecular complexes that initiate fusion competence in this area of the plasma membrane following the acrosome reaction.

The CD45 tyrosine phosphatase is required for T cell development and function by virtue of its role as a positive regulator of src family kinase activity. In addition, recent data have highlighted that CD45 also acts as a negative regulator of Lck function by dephosphorylation of critical tyrosine residues. Lck functionality and TCR responsiveness are elevated in transgenic mice expressing the CD45RO isoform at 'intermediate' (10-40% of wild type) levels, indicating that the expression level of CD45 is critical in determining the sensitivity of T cells to TCR stimulation. However, it is unclear whether such a phenotype is specific for the CD45RO isoform, typically expressed by activated T cells. In the present work, the roles of three isoforms of CD45, RO, RB and RABC, in thymocyte development, T cell responses and TCR signalling pathways were directly compared. The data demonstrate that expression of CD45RB or CD45RABC at intermediate levels also results in CD4(+) T cell hyper-reactivity, as previously published for CD45RO. These data emphasize the dual functions of CD45 as both a positive and a negative regulators of TCR signalling irrespective of specific isoform expression.

Increases in cytosolic free Ca2+ ([Ca2+]i) represent a ubiquitous signalling mechanism that controls a variety of cellular processes, including proliferation, metabolism and gene transcription, yet under certain conditions increases in intracellular Ca2+ are cytotoxic. Thus, in using Ca2+ as a messenger, cells walk a tightrope in which [Ca2+]i is strictly maintained within defined boundaries. To adhere to these boundaries and to sustain their modified phenotype, many cancer cells remodel the expression or activity of their Ca2+ signalling apparatus. Here, we review the role of Ca2+ in promoting cell proliferation and cell death, how these processes are remodelled in cancer and the opportunities this might provide for therapeutic intervention.

The LIM homeobox containing genes of the LIM-3 group, Lhx3 and Lhx4, are critical for normal development. Both genes are involved in the formation of the pituitary and the motoneuron system and loss of either gene causes perinatal lethality. Previous studies had shown that Lhx3 is overexpressed in hyperplastic placentas of mouse interspecies hybrids. To determine the role of LHX3 in the mouse placenta, we performed expression and function analyses. Our results show that Lhx3 exhibits specific spatial and temporal expression in the mouse placenta. However, deletion of Lhx3 does not produce a placental phenotype. To test whether this is due to functional substitution by Lhx4, we performed a phenotype analysis of Lhx3-/-; Lhx4-/- double-mutant placentas. A subset of Lhx3-/-; Lhx4-/- placentas exhibited abnormal structure of the labyrinth. However, absence of both LIM-3 genes did not interfere with placental transport nor consistently with expression of target genes such as Gnrhr. Thus, LHX3 and LHX4 appear to be dispensable for placental development and function.

Rapamycin is an inhibitor of mTOR, a key component of the mTORC1 complex that controls the growth and survival of cells in response to growth factors, nutrients, energy balance and stresses. The downstream targets of mTORC1 include ribosome biogenesis, transcription, translation and macroautophagy. Recently it was proposed that rapamycin and its derivatives enhance the clearance (and/or reduce the accumulation) of mutant intracellular proteins causing proteinopathies such as tau, alpha-synuclein, ataxin-3, and full-length or fragments of huntingtin containing a polyglutamine (polyQ) expansion, by upregulating macroautophagy. We tested this proposal directly using macroautophagy-deficient fibroblasts. We found that rapamycin inhibits the aggregation of a fragment of huntingtin (exon 1) containing 97 polyQs similarly in macroautophagy-proficient (Atg5(+/+)) and macroautophagy-deficient (Atg5(-/-)) cells. These data demonstrate that autophagy is not the only mechanism by which rapamycin can alleviate the accumulation of misfolded proteins. Our data suggest that rapamycin inhibits mutant huntingtin fragment accumulation due to inhibition of protein synthesis. A model illustrates how a modest reduction in polyQ synthesis can lead to a long-lasting reduction in polyQ aggregation. We propose that several mechanisms exist by which rapamycin reduces the accumulation and potential toxicity of misfolded proteins in diseases caused by protein misfolding and aggregation.

Epigenetic modifications influence gene expression pattern and provide a unique signature of a cell differentiation status. Without external stimuli or signalling events, this cell identity remains stable and unlikely to change over many cell divisions. The epigenetic signature of a particular cell fate therefore needs to be replicated faithfully in daughter cells; otherwise a cell lineage cannot be maintained. However, the mechanism of transmission of cellular memory from mother to daughter cells remains unclear. It has been suggested that the inheritance of an active or silent gene state involves different kinds of epigenetic mechanisms, e.g. DNA methylation, histone modifications, replacement of histone variants, Polycomb group (PcG) and Trithorax group (TrxG) proteins. Emerging evidence supports the role of histone variant H3.3 in maintaining an active gene status and in remodelling nucleosomal composition. Here we discuss some recent findings on the propagation of epigenetic memory and propose a model for the inheritance of an active gene state through the interaction of H3.3 with other epigenetic components.