ZFP36L1 and ZFP36L2 are RNA-binding proteins (RBPs) that interact with AU-rich elements in the 3' untranslated region of mRNA, which leads to mRNA degradation and translational repression. Here we show that mice that lacked ZFP36L1 and ZFP36L2 during thymopoiesis developed a T cell acute lymphoblastic leukemia (T-ALL) dependent on the oncogenic transcription factor Notch1. Before the onset of T-ALL, thymic development was perturbed, with accumulation of cells that had passed through the beta-selection checkpoint without first expressing the T cell antigen receptor beta-chain (TCRbeta). Notch1 expression was higher in untransformed thymocytes in the absence of ZFP36L1 and ZFP36L2. Both RBPs interacted with evolutionarily conserved AU-rich elements in the 3' untranslated region of Notch1 and suppressed its expression. Our data establish a role for ZFP36L1 and ZFP36L2 during thymocyte development and in the prevention of malignant transformation.

The muscle satellite cell is established as the major stem cell contributing to fiber growth and repair. p38 MAPK signaling is essential for myoblast differentiation and in particular for up-regulation of promyogenic Igf2 expression. p38 exists as four isoforms (alpha, beta, gamma, and delta), of which p38gamma is uniquely abundant in muscle. The aim of this study was to characterize p38 isoform expression and importance (using shRNA knockdown; demonstrated via both reduced protein and kinase activities) during myoblast differentiation. p38alpha and -gamma mRNA levels were most abundant in differentiating C2 cells with low/negligible contributions from p38beta and -delta, respectively. Increased phosphorylation of p38alpha and -gamma occurred during differentiation but via different mechanisms: p38alpha protein levels remained constant, whereas total p38gamma levels increased. Following shRNA knockdown of p38alpha, myoblast differentiation was dramatically inhibited [reduced myosin heavy chain (MHC), myogenin, pAkt protein levels]; significantly, Igf2 mRNA levels and promoter-reporter activities decreased. In contrast, knockdown of p38gamma induced a transient increase in both myogenin and MHC protein levels with no effect on Igf2 mRNA levels or promoter-reporter activity. Knockdown of p38alpha/beta markedly increased but that of p38gamma decreased caspase 3 activity, suggesting opposite actions on apoptosis. p38gamma was initially proposed to have a promyogenic function; however, p38gamma overexpression could not rescue reduced myoblast differentiation following p38alpha/beta inhibition. Therefore, p38alpha is essential for myoblast differentiation, and part of its action is to convert signals that indicate cell density into promyogenic gene expression in the form of the key peptide, IGF-II; p38gamma has a minor, yet opposing antimyogenic, function.

The various membranes of eukaryotic cells differ in composition, but it is at present unclear if this results in differences in physical properties. The sequences of transmembrane domains (TMDs) of integral membrane proteins should reflect the physical properties of the bilayers in which they reside. We used large datasets from both fungi and vertebrates to perform a comprehensive comparison of the TMDs of proteins from different organelles. We find that TMDs are not generic but have organelle-specific properties with a dichotomy in TMD length between the early and late parts of the secretory pathway. In addition, TMDs from post-ER organelles show striking asymmetries in amino acid compositions across the bilayer that is linked to residue size and varies between organelles. The pervasive presence of organelle-specific features among the TMDs of a particular organelle has implications for TMD prediction, regulation of protein activity by location, and sorting of proteins and lipids in the secretory pathway.

An important facet of transcriptional repression by Polycomb repressive complex 1 (PRC1) is the mono-ubiquitination of histone H2A by the combined action of the Posterior sex combs (Psc) and Sex combs extra (Sce) proteins. Here, we report that two ubiquitin-specific proteases, USP7 and USP11, co-purify with human PRC1-type complexes through direct interactions with the Psc orthologues MEL18 and BMI1, and with other PRC1 components. Ablation of either USP7 or USP11 in primary human fibroblasts results in de-repression of the INK4a tumour suppressor accompanied by loss of PRC1 binding at the locus and a senescence-like proliferative arrest. Mechanistically, USP7 and USP11 regulate the ubiquitination status of the Psc and Sce proteins themselves, thereby affecting their turnover and abundance. Our results point to a novel function for USPs in the regulation and function of Polycomb complexes.

Vomeronasal neurons undergo continuous neurogenesis throughout development and adult life. These neurons originate as stem cells in the apical zone of the lumen of the vomeronasal organ (VNO) and are described as nestin-expressing glia-like progenitor cells (Murdoch and Roskams, 2008). They then migrate horizontally along the basal zone where they differentiate into functional VNO neurons (Kaba et al., 1988). We harvested progenitor cells from the adult VNO and, after 3-6 months of invitro culture, these VNO neurons remained in a stable undifferentiated state expressing nestin, beta-tubulin III and vomeronasal type 2 (V2r), but not vomeronasal type 1 (V1r) receptors. Application of histone-deacetylase inhibitors induced development of a neural phenotype that expressed V2r receptors, a down-regulation of nestin expression and no change in any specific genetic markers associated with glial cells. Treatment with valproic acid induced extensive changes in gene expression in the axon guidance pathway. The adult VNO is known to functionally adapt throughout life as a consequence of changes in both a mouse's physiological status and its social environment. These pluripotent cultured neurons may provide valuable insights into how changes in both physiology and environment, exert epigenetic effects on vomeronasal neurons as they undergo continuous neurogenesis and development throughout the life of a mouse.

The Runx genes (Runx1, 2, and 3) regulate cell fate in development and can operate as either oncogenes or tumor suppressors in cancer. The oncogenic potential of ectopic Runx expression has been shown in transgenic mice that develop lymphoma in potent synergy with overexpressed Myc, and in established fibroblasts that display altered morphology and increased tumorigenicity. Candidate oncogenic functions of overexpressed Runx genes include resistance to apoptosis in response to intrinsic and extrinsic stresses. In a search for gene targets responsible for this aspect of Runx phenotype, we have identified three key enzymes in sphingolipid metabolism (Sgpp1, Ugcg, and St3gal5/Siat9) as direct targets for Runx transcriptional regulation in a manner consistent with survival and apoptosis resistance. Consistent with these changes in gene expression, mass spectrometric analysis showed that ectopic Runx reduces intracellular long-chain ceramides in NIH3T3 fibroblasts and elevated extracellular sphingosine 1 phosphate. Runx expression also opposed the activation of c-Jun-NH(2)-kinase and p38(MAPK), key mediators of ceramide-induced death, and suppressed the onset of apoptosis in response to exogenous tumor necrosis factor alpha. The survival advantage conferred by ectopic Runx could be partially recapitulated by exogenous sphingosine 1 phosphate and was accompanied by reduced phosphorylation of p38(MAPK). These results reveal a novel link between transcription factor oncogenes and lipid signaling pathways involved in cancer cell survival and chemoresistance.

Quantitative models of biochemical and cellular systems are used to answer a variety of questions in the biological sciences. The number of published quantitative models is growing steadily thanks to increasing interest in the use of models as well as the development of improved software systems and the availability of better, cheaper computer hardware. To maximise the benefits of this growing body of models, the field needs centralised model repositories that will encourage, facilitate and promote model dissemination and reuse. Ideally, the models stored in these repositories should be extensively tested and encoded in community-supported and standardised formats. In addition, the models and their components should be cross-referenced with other resources in order to allow their unambiguous identification.

Leukocyte migration across the endothelial lining is a critical step in the body's response to infection and inflammation. The homophilic interaction between endothelial PECAM and leukocyte PECAM is essential for this process. The molecular events that are triggered in the endothelial cell by PECAM engagement have been well characterized; however, the function of leukocyte PECAM remains to be elucidated. To study this, we first blocked leukocyte transmigration using anti-PECAM Ab and then specifically activated leukocyte PECAM. This was sufficient to overcome the block and promote transmigration, suggesting an active signaling role for leukocyte PECAM. Consistent with this, we found that ligation of leukocyte PECAM induces phosphorylation of two tyrosine residues on its cytoplasmic tail. By performing RNA interference-rescue experiments, we demonstrate that these phosphorylation events are indispensable for transendothelial migration. Finally, we show that leukocyte PECAM translocates to a detergent-resistant membrane (DRM) during transmigration. PECAM localized in DRMs displays reduced phosphorylation and does not support transmigration. Together, these data support a model whereby engagement of leukocyte PECAM induces its transient tyrosine phosphorylation and induction of downstream signals that drive transmigration. These signals are then downregulated following PECAM translocation to DRMs.

Reporter genes are widely used in biology and only a limited number are available. We present a new reporter gene for the localization of mammalian cells and transgenic tissues based on detection of the bglA (SYNbglA) gene of Caldocellum saccharolyticum that encodes a thermophilic beta-glucosidase.

Lymphocyte development and function are regulated by tyrosine kinase and G-protein coupled receptors. Each of these classes of receptors activates phosphoinositide 3-kinase (PI3K). In this chapter, we summarize current understanding of how PI3K contributes to key aspects of the adaptive immune system.

In Drosophila melanogaster, gustatory receptor genes (Grs) encode putative G-protein-coupled receptors (GPCRs) that are expressed in gustatory receptor neurons (GRNs). One of the Gr genes, Gr5a, encodes a receptor for trehalose that is expressed in a subset of GRNs. Although a role for the G protein, Gsα, has been shown in Gr5a-expressing taste neurons, there is the residual responses to trehalose in Gsα mutants which could suggest additional transduction mechanisms. Expression and genetic analysis of the heterotrimeric G-protein subunit, Gq, shown here suggest involvement of this Gα subunit in trehalose perception in Drosophila. A green fluorescent protein reporter of Gq expression is detected in gustatory neurons in the labellum, tarsal segments, and wing margins. Animals heterozygous for dgq mutations and RNA interference-mediated knockdown of dgq showed reduced responses to trehalose in the proboscis extension reflex assay and feeding behavior assay. These defects were rescued by targeted expression of the wild-type dgqα transgene in the GRNs. These data together with observations from other mutants in phospholipid signaling provide insights into the mechanisms of taste transduction in Drosophila.

T cells develop in the thymus and are critical for adaptive immunity. Natural killer (NK) lymphocytes constitute an essential component of the innate immune system in tumor surveillance, reproduction, and defense against microbes and viruses. Here, we show that the transcription factor Bcl11b was expressed in all T cell compartments and was indispensable for T lineage development. When Bcl11b was deleted, T cells from all developmental stages acquired NK cell properties and concomitantly lost or decreased T cell-associated gene expression. These induced T-to-natural killer (ITNK) cells, which were morphologically and genetically similar to conventional NK cells, killed tumor cells in vitro, and effectively prevented tumor metastasis in vivo. Therefore, ITNKs may represent a new cell source for cell-based therapies.

In vitro antibody generation technologies have now been available for two decades. Research reagents prepared via phage display are becoming available and several recent studies have demonstrated that these technologies are now sufficiently advanced to facilitate generation of a comprehensive renewable resource of antibodies for any protein encoded by the approximately 22,500 human protein-coding genes. Antibody selection in vitro offers properties not available in animal-based antibody generation methods. By adjusting the biochemical milieu during selection, it is possible to control the antigen conformation recognized, the antibody affinity or unwanted cross-reactivity. For larger-scale antibody generation projects, the handling, transport and storage logistics and bacterial production offer cost benefits. Because the DNA sequence encoding the antibody is available, modifications, such as site-specific in vivo biotinylation and multimerization, are only a cloning step away. This opinion article summarizes opportunities for the generation of antibodies for proteome research using in vitro technologies.

This is the concluding article in a series of 3 studies that investigate the anatomical determinants of thalamocortical (TC) input to excitatory neurons in a cortical column of rat primary somatosensory cortex (S1). We used viral synaptophysin-enhanced green fluorescent protein expression in thalamic neurons and reconstructions of biocytin-labeled cortical neurons in TC slices to quantify the number and distribution of boutons from the ventral posterior medial (VPM) and posteromedial (POm) nuclei potentially innervating dendritic arbors of excitatory neurons located in layers (L)2-6 of a cortical column in rat somatosensory cortex. We found that 1) all types of excitatory neurons potentially receive substantial TC input (90-580 boutons per neuron); 2) pyramidal neurons in L3-L6 receive dual TC input from both VPM and POm that is potentially of equal magnitude for thick-tufted L5 pyramidal neurons (ca. 300 boutons each from VPM and POm); 3) L3, L4, and L5 pyramidal neurons have multiple (2-4) subcellular TC innervation domains that match the dendritic compartments of pyramidal cells; and 4) a subtype of thick-tufted L5 pyramidal neurons has an additional VPM innervation domain in L4. The multiple subcellular TC innervation domains of L5 pyramidal neurons may partly explain their specific action potential patterns observed in vivo. We conclude that the substantial potential TC innervation of all excitatory neuron types in a cortical column constitutes an anatomical basis for the initial near-simultaneous representation of a sensory stimulus in different neuron types.

Th cell functional subsets have unique transcriptional programs that form the molecular basis for T cell differentiation and functions. T follicular helper (TFH) cells have emerged as the main providers of T cell help to B cells during the germinal center (GC) reaction, where B cells undergo selection events through competition for Ag and for access to GC T cell-mediated prosurvival and differentiation signals. Because T cell help is one limiting factor for GC B cells, the molecular mechanisms controlling TFH cell abundance and functionality are central to the GC reaction and generation of long-term humoral immunity. Two signaling pathways are absolutely critical for TFH cells: phosphoinositide-3 kinase pathway and the signaling lymphocyte activation molecule-associated protein. In this review, the molecular mechanisms constituting the signaling network in TFH cells will be explored.

MicroRNAs 125a and 125b are predicted to be able to bind to the B lymphocyte-induced maturation protein-1 (BLIMP-1) and IFN regulatory protein-4 (IRF-4) transcription factors, which are essential for plasma cell differentiation. A computational survey of the human and mouse genomes revealed that miR-125a and miR-125b are members of a multigene family located in paralogous clusters. The miR-125a cluster on chromosome 19 in humans includes miR-99b and let-7e, whereas the miR-125b cluster on chromosome 21 includes miR-99a and miR-let-7c. Our analysis of the expression profiles for these six miRs during B lineage differentiation indicated that mature miR-125a, miR-125b, miR-99b and let-7e transcripts are preferentially expressed by the actively dividing centroblasts in germinal centers (GC). However, miR-99b and let-7e are not predicted to bind BLIMP-1 or IRF-4 transcripts, and binding to the untranslated region of BLIMP-1 and IRF-4 messenger RNAs could be confirmed only for miR-125b. When the effect of miR-125b over-expression on terminal B cell differentiation was evaluated in an LPS-responsive B cell line, the induction of BLIMP-1 expression and IgM secretion was inhibited in this model system. Furthermore, miR-125b over-expression inhibited the differentiation of primary B cells and compromised the survival of cultured myeloma cells. These findings suggest that miR-125b promotes B lymphocyte diversification in GC by inhibiting premature utilization of essential transcription factors for plasma cell differentiation.

Schizophrenia is a multifactorial complex disease with a large impact on society. Many hypotheses have been proposed over the years to explain its causes, and genomics and functional genomic approaches may shed light on the reason behind these controversies and discrepancies. We give an overview of several approaches that have been used to identify the genetic causes and molecular phenotypes of the disease. We focus on a recent microarray analysis by Torkamani and colleagues on the evolution of regulatory networks in normal and schizophrenic brains. Combining the conclusion of that study with the prevalent hypotheses of schizophrenia, we suggest that the schizophrenic brain might resemble a juvenile brain.

An open chromatin architecture devoid of compact chromatin is thought to be associated with pluripotency in embryonic stem cells. Establishing this distinct epigenetic state may also be required for somatic cell reprogramming. However, there has been little direct examination of global structural domains of chromatin during the founding and loss of pluripotency that occurs in preimplantation mouse development. Here, we used electron spectroscopic imaging to examine large-scale chromatin structural changes during the transition from one-cell to early postimplantation stage embryos. In one-cell embryos chromatin was extensively dispersed with no noticeable accumulation at the nuclear envelope. Major changes were observed from one-cell to two-cell stage embryos, where chromatin became confined to discrete blocks of compaction and with an increased concentration at the nuclear envelope. In eight-cell embryos and pluripotent epiblast cells, chromatin was primarily distributed as an extended meshwork of uncompacted fibres and was indistinguishable from chromatin organization in embryonic stem cells. In contrast, lineage-committed trophectoderm and primitive endoderm cells, and the stem cell lines derived from these tissues, displayed higher levels of chromatin compaction, suggesting an association between developmental potential and chromatin organisation. We examined this association in vivo and found that deletion of Oct4, a factor required for pluripotency, caused the formation of large blocks of compact chromatin in putative epiblast cells. Together, these studies show that an open chromatin architecture is established in the embryonic lineages during development and is sufficient to distinguish pluripotent cells from tissue-restricted progenitor cells.

A unique property of the mammalian embryo is that stem cells can be derived from its early tissue lineages. These lineages will give rise to the fetus as well as essential extraembryonic tissues. Understanding how chromatin regulation participates in establishment of these lineages in the embryo and their derived stem cells provides insight that will critically inform our understanding of embryogenesis and stem cell biology. Here, we compare the genomewide location of active and repressive histone modifications in embryonic stem cells, trophoblast stem cells, and extraembryonic endoderm stem cells from the mouse. Our results show that the active modification H3K4me3 has a similar role in the three stem cell types, but the repressive modification H3K27me3 varies in abundance and genomewide distribution. Thus, alternative mechanisms mediate transcriptional repression in stem cells from the embryo. In addition, using carrier chromatin immunoprecipitation we show that bivalent histone domains seen in embryonic stem cells exist in pluripotent cells of the early embryo. However, the epigenetic status of extraembryonic progenitor cells in the embryo did not entirely reflect the extraembryonic stem cell lines. These studies indicate that histone modification mechanisms may differ between early embryo lineages and emphasize the importance of examining in vivo and in vitro progenitor cells.

Epidemiological studies have shown an association between statin use and a decreased risk of dementia. However, the mechanism by which this beneficial effect is brought about is unclear. In the context of Alzheimer's disease, at least three possibilities have been studied; reduction in amyloid beta peptide (Abeta) production, the promotion of alpha-secretase cleavage and positive effects on neurite outgrowth. By investigating the effects of mevalonate pathway blockade on neurite outgrowth using real-time imaging, we found that rather than promote the production of neurite extensions, inhibition rapidly induced cell rounding. Crucially, neurite-like structures were generated through the persistence of cell-cell and cell-substrate adhesions and not through a mechanism of positive outgrowth. This effect can be strikingly enhanced by the over-expression of human amyloid precursor protein and is isoprenoid rather than cholesterol dependent.

Polycomb Repressor Complexes (PRCs) are important regulators of embryogenesis. In embryonic stem (ES) cells many genes that regulate subsequent stages in development are enriched at their promoters for PRC1, PRC2 and Ser 5-phosphorylated RNA Polymerase II (RNAP), and contain domains of 'bivalent' chromatin (enriched for H3K4me3; histone H3 di- or trimethylated at Lys 4 and H3K27me3; histone H3 trimethylated at Lys 27). Loss of individual PRC components in ES cells can lead to gene de-repression and to unscheduled differentiation. Here we show that Jarid2 is a novel subunit of PRC2 that is required for the co-recruitment of PRC1 and RNAP to genes that regulate development in ES cells. Jarid2-deficient ES cells showed reduced H3K4me2/me3 and H3K27me3 marking and PRC1/PRC2 recruitment, and did not efficiently establish Ser 5-phosporylated RNAP at target genes. ES cells lacking Jarid2, in contrast to previously characterized PRC1 and PRC2 mutants, did not inappropriately express PRC2 target genes. Instead, they show a severely compromised capacity for successful differentiation towards neural or mesodermal fates and failed to correctly initiate lineage-specific gene expression in vitro. Collectively, these data indicate that transcriptional priming of bivalent genes in pluripotent ES cells is Jarid2-dependent, and suggests that priming is critical for subsequent multi-lineage differentiation.

Adaptive behavior in dynamic environments critically depends on the ability to learn rapidly and flexibly from the outcomes of prior choices. In social environments, facial expressions of emotion often serve as performance feedback and thereby guide declarative learning. Abundant evidence implicates beta-noradrenergic signaling in the modulatory influence of emotion on declarative learning. It is currently unclear whether a similar mechanism also mediates a guidance of declarative learning by social-emotional feedback administered in the form of facial expressions. We therefore conducted a double-blind randomized placebo-controlled trial to test the effects of a 40-mg single oral dose of the nonspecific beta-noradrenergic antagonist propranolol in a behavioral task that required gradual declarative learning of item-category associations from either social-emotional (happy vs. angry faces) or nonsocial (green vs. red color signals) trial-by-trial feedback. As predicted on the basis of our previous experiments, learning from social-emotional feedback was more effective than learning from nonsocial feedback in placebo-treated subjects. This advantage of social-emotional over nonsocial feedback was abolished by propranolol treatment. Propranolol had no effect on learning during the nonsocial feedback condition. Our findings suggest that a facilitation of declarative learning by social-emotional feedback critically involves signaling via beta-noradrenergic receptors.

Autoantibodies to gastric cellular antigens and glycoproteins including mucins and Lewis X and Y antigens have been implicated in the induction of autoimmune gastritis. Monoclonal antibody D10 (D10 MAb) recognizes a highly conserved mucin expressed in the foregut of mammals and other vertebrates. The objective of this study was to biochemically characterize the autoantigen identified by D10 MAb and examine its autoimmunogenicity in the mouse. Characterization of the mucin autoantigen was undertaken following purification, by amino acid and carbohydrate analyses, deglycosylation, SDS-PAGE, and immunoblotting using D10 MAb. Autoimmune reactivity and specificity of D10 MAb were validated by immunohistochemistry and ELISA using mouse tissue. Induction of autoimmune gastritis was investigated following immunization of mice with D10 MAb-reactive heterologous mucin. D10 MAb was shown to be a murine anti-mucin autoantibody with a unique pattern of immunohistochemical staining of Brunner's glands of the duodenum and the cardiac glands, mucous neck cells, and pyloric glands of the stomach from inbred Balb/c mice in patterns identical to that previously reported in human tissue. Amino acid and carbohydrate analysis of purified D10 mucin reflected a compositional profile of a typical mucin molecule. Confirmation that D10 MAb recognizes a mucin was also provided by demonstration that the carbohydrate epitope resides on a high molecular weight (>1x10(6)Da), high-density (>1.40 g/mL) molecule comprised of greater than 60% carbohydrate. Mice immunized with D10 MAb-reactive, purified, heterologous mucin produced autoantibodies of identical specificity to the original D10 MAb. These data demonstrate the autoimmunogenic properties of a novel foregut mucin and raise the potential of anti-mucin autoantibodies in the induction of autoimmune gastritis.

Size-dependent protein segregation at the cell-cell contact interface has been suggested to be critical for regulation of lymphocyte function. We investigated the role of ligand dimensions in regulation of mouse NK-cell activation and inhibition. Elongated forms of H60a, a mouse NKG2D ligand, were generated and expressed stably in the RMA cell line. RMA cells expressing the normal size H60a were lysed efficiently by both freshly isolated and IL-2 stimulated C57BL/6 mouse-derived NK cells; however the level of lysis decreased as the H60a ligand size increased. Importantly, H60a elongation did not affect NKG2D binding, as determined by soluble NKG2D tetramer staining, and by examining NK-cell target cell conjugate formation. CHO cells are efficient at activating NK cells from C57BL/6 mice, and expression of a single chain form of H-2K(b), a ligand for the mouse inhibitory receptor Ly49C, strongly inhibited such activation of Ly49C/I positive NK cells. Elongation of H-2K(b) resulted in decreased inhibition of both lysis and IFN-gamma production by NK cells. These results establish that small ligand dimensions are important for both NK-cell activation and inhibition, and suggest that there are shared features between the mechanisms of receptor triggering on different types of lymphocytes.