The t(12;21) translocation that generates the ETV6-RUNX1 (TEL-AML1) fusion gene, is the most common chromosomal rearrangement in childhood cancer and is exclusively associated with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The translocation arises in utero and is necessary but insufficient for the development of leukemia. Single-nucleotide polymorphism array analysis of ETV6-RUNX1 patient samples has identified multiple additional genetic alterations; however, the role of these lesions in leukemogenesis remains undetermined. Moreover, murine models of ETV6-RUNX1 ALL that faithfully recapitulate the human disease are lacking. To identify novel genes that cooperate with ETV6-RUNX1 in leukemogenesis, we generated a mouse model that uses the endogenous Etv6 locus to coexpress the Etv6-RUNX1 fusion and Sleeping Beauty transposase. An insertional mutagenesis screen was performed by intercrossing these mice with those carrying a Sleeping Beauty transposon array. In contrast to previous models, a substantial proportion (20%) of the offspring developed BCP-ALL. Isolation of the transposon insertion sites identified genes known to be associated with BCP-ALL, including Ebf1 and Epor, in addition to other novel candidates. This is the first mouse model of ETV6-RUNX1 to develop BCP-ALL and provides important insight into the cooperating genetic alterations in ETV6-RUNX1 leukemia.

The Bcl-2 protein, best known for its ability to inhibit apoptosis, interacts with the inositol 1,4,5-trisphosphate receptor (IP(3)R) Ca(2+) channel to regulate IP(3)-mediated Ca(2+) release from the endoplasmic reticulum. This review summarizes the current state of knowledge regarding the interaction of Bcl-2, and also its homologue Bcl-xl, with the IP(3)R and how these interactions regulate Ca(2+) signaling. The dual role of these interactions in promoting prosurvival Ca(2+) signals, while at the same time inhibiting proapoptotic Ca(2+) signals, is discussed. Moreover, this review will elucidate the recently recognized importance of the Bcl-2-IP(3)R interaction in human disease.

NAD(+) synthesizing enzyme NMNAT1 constitutes most of the sequence of neuroprotective protein Wld(S), which delays axon degeneration by 10-fold. NMNAT1 activity is necessary but not sufficient for Wld(S) neuroprotection in mice and 70 amino acids at the N-terminus of Wld(S), derived from polyubiquitination factor Ube4b, enhance axon protection by NMNAT1. NMNAT1 activity can confer neuroprotection when redistributed outside the nucleus or when highly overexpressed in vitro and partially in Drosophila. However, the role of endogenous NMNAT1 in normal axon maintenance and in Wallerian degeneration has not been elucidated yet. To address this question we disrupted the Nmnat1 locus by gene targeting. Homozygous Nmnat1 knockout mice do not survive to birth, indicating that extranuclear NMNAT isoforms cannot compensate for its loss. Heterozygous Nmnat1 knockout mice develop normally and do not show spontaneous neurodegeneration or axon pathology. Wallerian degeneration after sciatic nerve lesion is neither accelerated nor delayed in these mice, consistent with the proposal that other endogenous NMNAT isoforms play a principal role in Wallerian degeneration.

In mammals, silencing of one of the two X chromosomes in female cells compensates for the different number of X chromosomes between the sexes. The noncoding Xist RNA initiates X chromosome inactivation. Xist spreads from its transcription site over the X chromosome territory and triggers the formation of a repressive chromatin domain. To understand localization of Xist over one X chromosome we aimed to develop a system for investigating Xist in living cells. Here we report successful visualization of transgenically expressed MS2-tagged Xist in mouse embryonic stem cells. Imaging of Xist during an entire cell cycle shows that Xist spreads from a single point to a steady state when the chromosome is covered with a constant amount of Xist. Photobleaching experiments of the established Xist cluster indicate that chromosome-bound Xist is dynamic and turns over on the fully Xist covered chromosome. It appears that in interphase the loss of bound Xist and newly produced Xist are in equilibrium. We also show that the turnover of bound Xist requires transcription, and Xist binding becomes stable when transcription is inhibited. Our data reveal a strategy for visualizing Xist and indicate that spreading over the chromosome might involve dynamic binding and displacement.

The signalling lipid phosphatidic acid (PA) is generated by the hydrolysis of phosphatidylcholine (PC), which is catalysed by phospholipase D (PLD) enzymes. Neutrophils, important cells of the innate immune system, maintain the body's defence against infection. Previous studies have implicated PLD-generated PA in neutrophil function; these have relied heavily on the use of primary alcohols to act as inhibitors of PA production. The recent development of isoform-selective small molecule inhibitors and the generation of a knockout mouse model provide us with accurate tools to study the role of PLDs in neutrophil responses. We show that PLD1 is a regulator of phorbol-ester-, chemoattractant, adhesion-dependent and Fcγ-receptor-stimulated production of reactive oxygen species (ROS) in neutrophils. Significantly we found that this role of PLD is isoform specific: the absence of PLD2 does not negatively affect these processes. Contrary to expectation, other functions required for an efficient immune response operate effectively in Pld2-deficient neutrophils or when both isoforms are inhibited pharmacologically. We conclude that although PLD1 does have important regulatory roles in neutrophils, the field has been confused by the use of primary alcohols; now that gold standard Pld-knockout mouse models are available, previous work might need to be reassessed.

DNA methylation is one of the most extensively studied, and one of the most stable, of all epigenetic modifications. Two drugs that target DNA methyltransferase enzymes are licensed for clinical use in oncology but relatively little attention has focused on the enzymatic pathways by which DNA methylation can be reversed. Recent breakthroughs have identified at least two classes of enzymes that can achieve functional reversal. This review discusses the significance of DNA demethylation in a range of human diseases, the candidate proteins that mediate the demethylation and the opportunities and challenges in targeting these candidates to develop new therapeutics.

Defective V(D)J rearrangement of immunoglobulin heavy or light chain (IgH or IgL) or class switch recombination (CSR) can initiate chromosomal translocations. The DNA-damage kinase ATM is required for the suppression of chromosomal translocations but ATM regulation is incompletely understood. Here, we show that mice lacking the ATM cofactor ATMIN in B cells (ATMIN(ΔB/ΔB)) have impaired ATM signaling and develop B cell lymphomas. Notably, ATMIN(ΔB/ΔB) cells exhibited defective peripheral V(D)J rearrangement and CSR, resulting in translocations involving the Igh and Igl loci, indicating that ATMIN is required for efficient repair of DNA breaks generated during somatic recombination. Thus, our results identify a role for ATMIN in regulating the maintenance of genomic stability and tumor suppression in B cells.

CD4(+) T helper (T(H)) cells play a critical role in orchestrating a pleiotropy of immune activities against a large variety of pathogens. It is generally thought that this is achieved through the acquisition of highly specialized functions after activation followed by the differentiation into various functional subsets. The differentiation process of naive precursor T(H) cells into defined effector subsets is controlled by cells of the innate immune system and their complex array of effector molecules such as secreted cytokines and membrane bound costimulatory molecules. These provide a unique quantitative or qualitative signal initiating T(H) development, which is subsequently reinforced via T cell-mediated feedback signals and selective survival and proliferative cues, ultimately resulting in the predominance of a particular T cell subset. In recent years, the number of defined T(H)cell subsets has expanded and the once rigid division of labor among them has been blurred with reports of plasticity among the subsets. In this chapter, we summarize and speculate on the current knowledge of the differentiation requirements of T(H) cell lineages, with particular focus on the T(H)17 subset.

Epigenetic marks such as posttranslational histone modifications specify the functional states of underlying DNA sequences, though how they are maintained after their disruption during DNA replication remains a critical question. We identify the mammalian SWI/SNF-like protein SMARCAD1 as a key factor required for the re-establishment of repressive chromatin. The ATPase activity of SMARCAD1 is necessary for global deacetylation of histones H3/H4. In this way, SMARCAD1 promotes methylation of H3K9, the establishment of heterochromatin, and faithful chromosome segregation. SMARCAD1 associates with transcriptional repressors including KAP1, histone deacetylases HDAC1/2 and the histone methyltransferase G9a/GLP and modulates the interaction of HDAC1 and KAP1 with heterochromatin. SMARCAD1 directly interacts with PCNA, a central component of the replication machinery, and is recruited to sites of DNA replication. Our findings suggest that chromatin remodeling by SMARCAD1 ensures that silenced loci, such as pericentric heterochromatin, are correctly perpetuated.

The Rap family of small GTPases regulate the adhesion of cells to extracellular matrices. Several Rap-binding proteins have been shown to function as effectors that mediate Rap-induced adhesion. However, little is known regarding the relationships between these effectors, or about other proteins that are downstream of or act in parallel to the effectors. To establish whether an array of effectors was required for Rap-induced cell adhesion and spreading, and to find new components involved in Rap-signal transduction, we performed a small-scale siRNA screen in A549 lung epithelial cells. Of the Rap effectors tested, only Radil blocked Rap-induced spreading. Additionally, we identified a novel role for Ezrin downstream of Rap1. Ezrin was necessary for Rap-induced cell spreading, but not Rap-induced cell adhesion or basal adhesion processes. Furthermore, Ezrin depletion inhibited Rap-induced cell spreading in several cell lines, including primary human umbilical vein endothelial cells. Interestingly, Radixin and Moesin, two proteins with high homology to Ezrin, are not required for Rap-induced cell spreading and cannot compensate for loss of Ezrin to rescue Rap-induced cell spreading. Here, we present a novel function for Ezrin in Rap1-induced cell spreading and evidence of a non-redundant role of an ERM family member.

Heavy chain diseases (HCDs) are B-cell proliferative disorders characterized by the production of monoclonal, incomplete, immunoglobulin (Ig) heavy chains (HCs) without associated light chains (LCs). These abnormal HCs are produced as a consequence of HC gene alterations in the neoplastic B cells. HC gene alterations will also impact on surface HC, which is part of the B-cell receptor (BCR), a crucial player in lymphocyte activation by antigen. The selective advantage conferred to mutant cells by abnormal BCR without an antigen-binding domain may be explained by activation of ligand-independent signaling, in analogy to what has been shown for mutated oncogenic growth factor receptors. Here we review data obtained from mouse models showing abnormal, constitutive activity of HCD-BCR, and we discuss the possible mechanism involved, namely, aberrant spontaneous self-aggregation. This self-aggregation might occur as a consequence of escape from the chaperone immunoglobulin binding protein (BiP) and from the anti-aggregation effect of LC association. The concept of misfolding-induced signaling elaborated here may extend to other pathologies termed conformational diseases.

To investigate the role of Aire in thymic selection, we examined the cellular requirements for generation of ovalbumin (OVA)-specific CD4 and CD8 T cells in mice expressing OVA under the control of the rat insulin promoter. Aire deficiency reduced the number of mature single-positive OVA-specific CD4(+) or CD8(+) T cells in the thymus, independent of OVA expression. Importantly, it also contributed in 2 ways to OVA-dependent negative selection depending on the T-cell type. Aire-dependent negative selection of OVA-specific CD8 T cells correlated with Aire-regulated expression of OVA. By contrast, for OVA-specific CD4 T cells, Aire affected tolerance induction by a mechanism that operated independent of the level of OVA expression, controlling access of antigen presenting cells to medullary thymic epithelial cell (mTEC)-expressed OVA. This study supports the view that one mechanism by which Aire controls thymic negative selection is by regulating the indirect presentation of mTEC-derived antigens by thymic dendritic cells. It also indicates that mTECs can mediate tolerance by direct presentation of Aire-regulated antigens to both CD4 and CD8 T cells.

DNA methylation is involved in a number of important processes such as maintaining genome stability, silencing of retrotransposons, co-ordinating mono-alleleic expression of parentally imprinted genes and ensuring transcriptional repression of genes on the inactive X chromosome. Further, correct DNA methylation patterns are necessary for normal development and lineage commitment. DNA methylation provides a stable and heritable epigenetic mark. However, it can be removed, either actively or passively, during periods of reprogramming in primordial germ cells and preimplantation embryos. By combining immunofluorescence data with recent insights from genome-wide studies utilizing techniques such as Bisulphite-seq and MeDIP-ChIP, a clearer picture of the dynamic patterns of DNA methylation throughout gametogenesis, preimplantation development and early lineage commitment is beginning to emerge. The continuing use of these next-generation technologies to elucidate genome-wide methylation patterns in a variety of cellular contexts will further understanding of how this epigenetic mark contributes to lineage commitment, differentiation and pluripotency and, ultimately, to human health and disease.

The mysterious secrets of long noncoding RNAs, often referred to as the Dark Matter of the genome, are gradually coming to light. Several recent papers dig deep to reveal surprisingly complex and diverse functions of these enigmatic molecules.

A combination of bisulfite treatment of DNA and high-throughput sequencing (BS-Seq) can capture a snapshot of a cell's epigenomic state by revealing its genome-wide cytosine methylation at single base resolution. Bismark is a flexible tool for the time-efficient analysis of BS-Seq data which performs both read mapping and methylation calling in a single convenient step. Its output discriminates between cytosines in CpG, CHG and CHH context and enables bench scientists to visualize and interpret their methylation data soon after the sequencing run is completed. Availability and implementation: Bismark is released under the GNU GPLv3+ licence. The source code is freely available from www.bioinformatics.bbsrc.ac.uk/projects/bismark/.

Tauopathies are characterized by hyperphosphorylation of the microtubule-associated protein tau and its accumulation into fibrillar aggregates. Toxic effects of aggregated tau and/or dysfunction of soluble tau could both contribute to neural defects in these neurodegenerative diseases. We have generated a novel knockin mouse model of an inherited tauopathy, frontotemporal dementia with parkinsonism linked to tau mutations on chromosome 17 (FTDP-17T). We incorporated a single mutation, homologous to the common FTDP-17T P301L mutation, directly into the endogenous mouse gene, mimicking the human disease situation. These mice express P301L-equivalent mutant tau at normal physiological levels from the knockin allele. Importantly, in contrast to existing transgenic mouse models that overexpress human P301L mutant tau, no overt tau pathology developed during the normal lifespan of the knockin mice. In fact, overall phosphorylation of tau was reduced, perhaps due to reduced microtubule binding. However, homozygous knockin mice did display intriguing age-dependent changes in axonal transport of mitochondria, and increased spontaneous locomotor activity in old age. These could represent early consequences of the tau dysfunction that eventually precipitates pathogenesis in humans.

Neutrophils form a vital part of the innate immune response, but at the same time their inappropriate activation contributes to autoimmune diseases. Many molecular components are involved in fine-tuning neutrophil function. We report here the first characterization of the role of ARAP3, a PI3K and Rap-regulated GTPase-activating protein for RhoA and Arf6 in murine neutrophils. We show that neutrophils lacking ARAP3 are preactivated in vitro and in vivo, exhibiting increased β2 integrin affinity and avidity. ARAP3-deficient neutrophils are hyperresponsive in several adhesion-dependent situations in vitro, including the formation of reactive oxygen species, adhesion, spreading, and granule release. ARAP3-deficient cells adhere more firmly under flow conditions in vitro and to the vessel wall in vivo. Finally, loss of ARAP3 interferes with integrin-dependent neutrophil chemotaxis. The results of the present study suggest an important function of ARAP3 downstream of Rap. By modulating β2 integrin activity, ARAP3 guards neutrophils in their quiescent state unless activated.

Mice lacking either CD19 or p110δ have reduced numbers of marginal zone and B1 B cells but normal numbers of naïve B2 cells which occupy the follicles of the lymphoid organs. We show here that mice lacking both CD19 and p110δ have normal B cell development in the bone marrow but have a significant reduction in the number of naïve B2 cells in the bone marrow, spleen and lymph nodes. These p110δ/CD19 double mutant B cells show a survival defect and reduced responsiveness to the pro-survival cytokine BAFF despite normal NFκB2/p100 processing and elevated expression of Bcl-2. Although the combined loss of p110δ and CD19 did not increase switching to Ig-lambda in immature B cells, mature B lymphocytes from the lymph nodes of p110δ/CD19 double mutant mice express highly elevated levels of mRNA encoding RAG-1 and RAG-2, which confirms the existing synergy between CD19 and p110δ-mediated signaling.

A mutation in the rat GIMAP5 gene predisposes for autoimmunity, most famously in the BB rat model of autoimmune type 1 diabetes mellitus. This mutation is associated with severe peripheral T lymphopenia, as is mutation of the same gene in mice, but the mechanism by which GIMAP5 normally protects T cells from death is unknown. GIMAP5 is a putative small GTPase, a class of proteins which often fulfil their functions in the vicinity of cellular membranes. The objective of this study was to determine the normal intracellular location of GIMAP5 in lymphoid cells. Combining studies in rat, mouse and human systems, novel monoclonal antibodies (mAbs) were used to examine the localization of GIMAP5 and the closely-related protein, GIMAP1, in lymphoid cells by means of confocal microscopy and sub-cellular fractionation combined with immunoblotting. Additionally, human Jurkat T cells that inducibly express epitope-tagged GIMAP5 were established and used in electron microscopy (EM). Endogenous GIMAP5 was found to be located in a membraneous compartment/s which was also detected by established markers of lysosomes. GIMAP1, by contrast, was found to be located in the Golgi apparatus. EM studies of the inducible Jurkat T cells also found GIMAP5 in lysosomes and, in addition, in multivesicular bodies. This study establishes that the endogenous location of GIMAP5 is in lysosomes and related compartments and provides a clearer context for hypotheses about its mechanism of action.

Neutrophils are activated by immunoglobulin G (IgG)-containing immune complexes through receptors that recognize the Fc portion of IgG (FcγRs). Here, we used genetic and pharmacological approaches to define a selective role for the β isoform of phosphoinositide 3-kinase (PI3Kβ) in FcγR-dependent activation of mouse neutrophils by immune complexes of IgG and antigen immobilized on a plate surface. At low concentrations of immune complexes, loss of PI3Kβ alone substantially inhibited the production of reactive oxygen species (ROS) by neutrophils, whereas at higher doses, similar suppression of ROS production was achieved only by targeting both PI3Kβ and PI3Kδ, suggesting that this pathway displays stimulus strength-dependent redundancy. Activation of PI3Kβ by immune complexes involved cooperation between FcγRs and BLT1, the receptor for the endogenous proinflammatory lipid leukotriene B₄. Coincident activation by a tyrosine kinase-coupled receptor (FcγR) and a heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (BLT1) may provide a rationale for the preferential activation of the β isoform of PI3K. PI3Kβ-deficient mice were highly protected in an FcγR-dependent model of autoantibody-induced skin blistering and were partially protected in an FcγR-dependent model of inflammatory arthritis, whereas combined deficiency of PI3Kβ and PI3Kδ resulted in near-complete protection in the latter case. These results define PI3Kβ as a potential therapeutic target in inflammatory disease.

PECAM-1 (CD31) is a cellular adhesion and signaling receptor that is highly expressed at endothelial cell-cell junctions in confluent vascular beds. Previous studies have implicated PECAM-1 in the maintenance of vascular barrier integrity; however, the mechanisms behind PECAM-1-mediated barrier protection are still poorly understood. The goal of the present study, therefore, was to examine the pertinent biological properties of PECAM-1 (i.e. adhesion and/or signaling) that allow it to support barrier integrity. We found that, compared with PECAM-1-deficient endothelial cells, PECAM-1-expressing endothelial cell monolayers exhibit increased steady-state barrier function, as well as more rapid restoration of barrier integrity following thrombin-induced perturbation of the endothelial cell monolayer. The majority of PECAM-1-mediated barrier protection was found to be due to the ability of PECAM-1 to interact homophilically and become localized to cell-cell junctions, because a homophilic binding-crippled mutant form of PECAM-1 was unable to support efficient barrier function when re-expressed in cells. By contrast, cells expressing PECAM-1 variants lacking residues known to be involved in PECAM-1-mediated signal transduction exhibited normal to near-normal barrier integrity. Taken together, these studies suggest that PECAM-1-PECAM-1 homophilic interactions are more important than its signaling function for maintaining the integrity of endothelial cell junctions.

In eukaryotes transcription is complicated by the DNA being packed in nucleosomes and by supercoils induced by opening of the DNA double helix during elongation. Here we discuss our recent genome-wide work regarding topoisomerases and their role in chromatin remodeling during the transcription cycle and we report a novel function for topoisomerases in transcription termination.