The establishment and maintenance of central tolerance depends to a large extent on the ability of medullary thymic epithelial cells to express a variety of tissue-restricted antigens, the so-called promiscuous gene [removed]pGE). Autoimmune regulator (Aire) is to date the best characterised transcriptional regulator known to at least partially coordinate pGE. There is accruing evidence that the expression of Aire-dependent and -independent genes is modulated by higher order chromatin configuration, epigenetic modifications and post-transcriptional control. Given the involvement of microRNAs (miRNAs) as potent post-transcriptional modulators of gene expression, we investigated their role in the regulation of pGE in purified mouse and human thymic epithelial cells (TECs). Microarray profiling of TEC subpopulations revealed evolutionarily conserved cell type and differentiation-specific miRNA signatures with a subset of miRNAs being significantly upregulated during terminal medullary thymic epithelial cell differentiation. The differential regulation of this subset of miRNAs was correlated with Aire expression and some of these miRNAs were misexpressed in the Aire knockout thymus. In turn, the specific absence of miRNAs in TECs resulted in a progressive reduction of Aire expression and pGE, affecting both Aire-dependent and -independent genes. In contrast, the absence of miR-29a only affected the Aire-dependent gene pool. These findings reveal a mutual interdependence of miRNA and Aire.

The MIRIAM Registry (http://www.ebi.ac.uk/miriam/) records information about collections of data in the life sciences, as well as where it can be obtained. This information is used, in combination with the resolving infrastructure of Identifiers.org (http://identifiers.org/), to generate globally unique identifiers, in the form of Uniform Resource Identifier. These identifiers are now widely used to provide perennial cross-references and annotations. The growing demand for these identifiers results in a significant increase in curational efforts to maintain the underlying registry. This requires the design and implementation of an economically viable and sustainable solution able to cope with such expansion. We briefly describe the Registry, the current curation duties entailed, and our plans to extend and distribute this workload through collaborative and community efforts.

Mammalian cells can express up to four different class I phosphatidylinositide 3-kinase (PI3K) isoforms, each of which is engaged by tyrosine kinases or G protein-coupled receptors (GPCRs) to generate the second messenger signaling molecule PtdIns(3,4,5)P₃ (PIP₃). The p110α and p110β isoforms are relatively widely expressed, whereas p110γ and p110δ are more highly expressed in cells of the immune system than in other cell types. Nevertheless, each of the four class I PI3Ks have been shown to participate in the orchestration of the signaling events that lead to immune cell development and control of gene expression, skewing toward individual cell lineage subsets and proliferation.

MicroRNAs (miRNAs) are a newly recognized class of regulatory genes which repress the expression of protein-coding genes. Numerous studies have uncovered a complex role for miRNAs regulating many aspects of a variety of cellular processes including cell growth, differentiation, and lineage commitment. In the immune system, miR-155 is unique in its ability to shape the transcriptome of activated myeloid and lymphoid cells controlling diverse biological functions ranging from inflammation to immunological memory. Not surprisingly, a tight control of miR-155 expression is required to avoid malignant transformation, as evidenced by miR-155 overexpression in many cancers of B-cell origin. In this review, we discuss the potential of miR-155 as a molecular target for therapeutic intervention and discuss the function of miR-155 in the context of protective immunity. We first look back into the emergence of miR-155 in evolution, which is coincidental with the emergence of the ancestors of the antigen receptors. We then summarize what we have learned about the role of miR-155 in the regulation of lymphoid subsets at the cellular and molecular level in the context of recent progress in this field.

MicroRNAs are short, 19-24 nucleotide long, RNA molecules capable of regulating the longevity and, to a lesser extent, translation of messenger RNA (mRNA) species. The function of the microRNA network, and indeed, even that of individual microRNA species, can have profoundly different roles in even a single cell type as the microRNA/mRNA composition evolves. As the role of microRNA within T cells has come under increasing scrutiny, several distinct checkpoints have been demonstrated to have a particular reliance on microRNA regulation. MicroRNAs are arguably most important in T cells during the earliest and last stages in T-cell biology. The first stages of early thymic differentiation have a crucial reliance on the microRNA network, while later stages and peripheral homeostasis are largely, although not completely, microRNA-independent. The most profound effects on T cells are in the activation of effector and regulatory functions of conventional and regulatory T cells, where microRNA deficiency results in a near-complete loss of function. In this review, we focus on integrating the research on individual microRNA into a more global understanding of the function of the microRNA regulatory network in T cells.

There is a need for a standardized, practical annotation for structures of lipid species derived from mass spectrometric approaches; i.e., for high-throughput data obtained from instruments operating in either high- or low-resolution modes. This proposal is based on common, officially accepted terms and builds upon the LIPID MAPS terminology. It aims to add defined levels of information below the LIPID MAPS nomenclature, as detailed chemical structures, including stereochemistry, are usually not automatically provided by mass spectrometric analysis. To this end, rules for lipid species annotation were developed that reflect the structural information derived from the analysis. For example, commonly used head group-specific analysis of glycerophospholipids (GP) by low-resolution instruments is neither capable of differentiating the fatty acids linked to the glycerol backbone nor able to define their bond type (ester, alkyl-, or alk-1-enyl-ether). This and other missing structural information is covered by the proposed shorthand notation presented here. Beyond GPs, we provide shorthand notation for fatty acids/acyls (FA), glycerolipids (GL), sphingolipids (SP), and sterols (ST). In summary, this defined shorthand nomenclature provides a standard methodology for reporting lipid species from mass spectrometric analysis and for constructing databases.

Pluripotent cells, when fused with somatic cells, have the dominant ability to reprogram the somatic genome. Work by Piccolo et al. (2013) shows that the Tet1 and Tet2 hydroxylases are important for DNA methylation reprogramming of pluripotency genes and parental imprints.

The development of functional T cells requires receptor-mediated transition through multiple checkpoints in the thymus. Double negative 3 (DN3) thymocytes are selected for the presence of a rearranged TCR beta chain in a process termed β-selection which requires signalling via the pre-TCR, Notch1 and CXCL12. Signal integration by these receptors converges on core pathways including the Phosphatidylinositol-3-kinase (PI3K) pathway. Glycogen Synthase Kinase 3 (GSK3) is generally thought to be negatively regulated by the PI3K pathway but its role in β-selection has not been characterised. Here we show that developmental progression of DN3 thymocytes is promoted following inhibition of GSK3 by the synthetic compound CHIR99021. CHIR99021 allows differentiation in the absence of pre-TCR-, Notch1- or CXCL12-mediated signalling. It antagonizes IL-7-mediated inhibition of DP thymocyte differentiation and increases IL-7-promoted cell recovery. These data indicate a potentially important role for inactivation of GSK3 during β-selection. They might help to establish an in vitro stromal cell-free culture system of thymocyte development and offer a new platform for screening regulators of proliferation, differentiation and apoptosis.

Cooperativity of ligand binding to allosteric receptors can be quantified using the Hill coefficient (nH) to measure the sigmoidal character of the binding curve. However, for measurements of the transition between conformational states, nH values can be misleading due to ambiguity of the reference state. For cooperative ligand binding, the reference state is a hyperbolic curve for a monomer with a single binding site characterized by nH=1. Therefore, binding curves with nH>1 provide a direct measure of cooperativity. For the dependence of the conformational state on ligand concentration, curves with nH>1 are observed, but in virtually all cases, the equivalent allosteric monomer has a value of nH<1. The ratio of the two nH values defines the effective cooperativity and always corresponds to nH=N (the number of protomers in the oligomer) for concerted transitions as specified by the Monod-Wyman-Changeux model. Dose-response curves for homopentameric α7 nicotinic receptors illustrate this relationship for both wild-type and mutant forms. For functional allosteric monomers such as G-protein-coupled receptors, normalization stretches the dose-response curve along the y-axis, thereby masking the "allosteric range" and increasing the apparent cooperativity to a limit for monomers of nH =1. The concepts of equivalent monomer and allosteric range were originally proposed in 1965 by Crick and Wyman in a manuscript circulated among the proponents of allostery, but only now published for the first time in this special issue.

International efforts to test gene function in the mouse by the systematic knockout of each gene are creating many lines in which embryonic development is compromised. These homozygous lethal mutants represent a potential treasure trove for the biomedical community. Developmental biologists could exploit them in their studies of tissue differentiation and organogenesis; for clinical researchers they offer a powerful resource for investigating the origins of developmental diseases that affect newborns. Here, we outline a new programme of research in the UK aiming to kick-start research with embryonic lethal mouse lines. The 'Deciphering the Mechanisms of Developmental Disorders' (DMDD) programme has the ambitious goal of identifying all embryonic lethal knockout lines made in the UK over the next 5 years, and will use a combination of comprehensive imaging and transcriptomics to identify abnormalities in embryo structure and development. All data will be made freely available, enabling individual researchers to identify lines relevant to their research. The DMDD programme will coordinate its work with similar international efforts through the umbrella of the International Mouse Phenotyping Consortium [see accompanying Special Article (Adams et al., 2013)] and, together, these programmes will provide a novel database for embryonic development, linking gene identity with molecular profiles and morphology phenotypes.

In response to a 100-word footnote in the 1965 article by Monod, Wyman, and Changeux, a detailed manuscript signed by Francis Crick and Jeffries Wyman with 6000 words and 30 equations entitled "A Footnote on Allostery" circulated in 1965 among a limited group of scientists interested in allosteric interactions. This interesting and provocative document is published in this special issue for the first time. An intriguing equation in their text relates the difference between n (the number of ligand binding sites) and n' (the Hill coefficient) to the ratio of the saturation functions Y¯, for oligomers with n-1 and n binding sites. A compact derivation of this equation was not provided by Crick and Wyman, but one is presented here based on a definition of Y¯ involving the binding polynomial and its first derivative.

Rotaviruses (RVs) cause acute gastroenteritis in infants and young children, and are globally distributed. Within the infected host cell, RVs establish replication complexes in viroplasms ('viral factories') to which lipid droplet organelles are recruited. To further understand this recently discovered phenomenon, the lipidomes of RV-infected and uninfected MA104 cells were investigated. Cell lysates were subjected to equilibrium ultracentrifugation through iodixanol gradients. Fourteen different classes of lipids were differentiated by mass spectrometry. The concentrations of virtually all lipids were elevated in RV-infected cells. Fractions of low density (1.11-1.15 g ml⁻¹), in which peaks of the RV dsRNA genome and lipid droplet- and viroplasm-associated proteins were observed, contained increased amounts of lipids typically found concentrated in the cellular organelle lipid droplets, confirming the close interaction of lipid droplets with viroplasms. A decrease in the ratio of the amounts of surface to internal components of lipid droplets upon RV infection suggested that the lipid droplet-viroplasm complexes became enlarged.

DNA methylation is a carrier of important regulatory information that undergoes global reprogramming in the mammalian germ line, including pre-implantation embryos and primordial germ cells (PGCs). A flurry of recent studies have employed technical advances to generate global profiles of methylation and hydroxymethylation in these cells, unravelling the dynamics of methylation erasure at single locus resolution. Active demethylation in the zygote, involving extensive oxidation, is followed by passive loss over early cell divisions. Certain gamete-contributed methylation marks appear to have evolved non-canonical mechanisms for targeted maintenance of methylation in the face of these processes. These protected sequences include the imprinting control regions (ICRs) required for parental imprinting but also a surprising number of other regions. Such targeted maintenance mechanisms may also operate at certain sequences during early PGC migration when global passive demethylation occurs. In later gonadal PGCs, imprints must be reset and this may be achieved through the targeting of active mechanisms including oxidation. Thus, emerging evidence paints a complex picture whereby active and passive demethylation pathways operate synergistically and in parallel to ensure robust erasure in the early embryo and PGCs.

Protein kinase clients are recruited to the Hsp90 molecular chaperone system via Cdc37, which simultaneously binds Hsp90 and kinases and regulates the Hsp90 chaperone cycle. Pharmacological inhibition of Hsp90 in vivo results in degradation of kinase clients, with a therapeutic effect in dependent tumors. We show here that Cdc37 directly antagonizes ATP binding to client kinases, suggesting a role for the Hsp90-Cdc37 complex in controlling kinase activity. Unexpectedly, we find that Cdc37 binding to protein kinases is itself antagonized by ATP-competitive kinase inhibitors, including vemurafenib and lapatinib. In cancer cells, these inhibitors deprive oncogenic kinases such as B-Raf and ErbB2 of access to the Hsp90-Cdc37 complex, leading to their degradation. Our results suggest that at least part of the efficacy of ATP-competitive inhibitors of Hsp90-dependent kinases in tumor cells may be due to targeted chaperone deprivation.

Association studies have implicated common variants in the 12q14.1 region containing CYP27B1 in multiple sclerosis (MS). Rare CYP27B1 mutations cause autosomal recessive vitamin D-dependent rickets type 1, and it has recently been reported that heterozygous CYP27B1 mutations are associated with increased MS susceptibility and lower active vitamin D levels. By sequencing CYP27B1 in 134 multiplex families and genotyping the most common variant R389H in 2,608 MS patients and 1,987 controls from Italy and Belgium (a total of 4,729 individuals), we were unable to replicate these observations. These results provide evidence against a major role for CYP27B1 mutations in MS.

We disrupted the gene encoding lysophosphatidylinositol-acyltransferase-1 (LPIAT1) in the mouse with the aim of understanding its role in determining cellular phosphoinositide content. LPIAT1(-/-) mice were born at lower than Mendelian ratios and exhibited a severe developmental brain defect. We compared the phospholipid content of livers and brains from LPIAT1(-/-) and LPIAT1(+/+) littermates by LC-ESI/MS. In accord with previous studies, the most abundant molecular species of each phosphoinositide class (PtdIns, PtdInsP, PtdInsP2 and PtdInsP3) possessed a C38∶4 complement of fatty-acyl esters (C18∶0 and C20∶4 are usually assigned to the sn-1 and sn-2 positions, respectively). LPIAT1(-/-) liver and brain contained relatively less of the C38∶4 species of PtdIns, PtdInsP and PtdInsP2 (dropping from 95-97% to 75-85% of the total species measured for each lipid class) and relatively more of the less abundant species (PtdInsP3 less abundant species were below our quantification levels). The increases in the less abundant PtdIns and PtdInsP2 species did not compensate for the loss in C38∶4 species, resulting in a 26-44% reduction in total PtdIns and PtdInsP2 levels in both brain and liver. LPIAT1(-/-) brain and liver also contained increased levels of C18∶0 lyso-PtdIns (300% and 525% respectively) indicating a defect in the reacylation of this molecule. LPIAT1(-/-) brain additionally contained significantly reduced C38∶4 PC and PE levels (by 47% and 55% respectively), possibly contributing to the phenotype in this organ. The levels of all other molecular species of PC, PE, PS and PA measured in the brain and liver were very similar between LPIAT1(-/-) and LPIAT1(+/+) samples. These results suggest LPIAT1 activity plays a non-redundant role in maintaining physiological levels of PtdIns within an active deacylation/reacylation cycle in mouse tissues. They also suggest that this pathway must act in concert with other, as yet unidentified, mechanisms to achieve the enrichment observed in C38∶4 molecular species of phosphoinositides.

The establishment of the epigenetic mark H4K20me1 (monomethylation of H4K20) by PR-Set7 during G2/M directly impacts S-phase progression and genome stability. However, the mechanisms involved in the regulation of this event are not well understood. Here we show that SirT2 regulates H4K20me1 deposition through the deacetylation of H4K16Ac (acetylation of H4K16) and determines the levels of H4K20me2/3 throughout the cell cycle. SirT2 binds and deacetylates PR-Set7 at K90, modulating its chromatin localization. Consistently, SirT2 depletion significantly reduces PR-Set7 chromatin levels, alters the size and number of PR-Set7 foci, and decreases the overall mitotic deposition of H4K20me1. Upon stress, the interaction between SirT2 and PR-Set7 increases along with the H4K20me1 levels, suggesting a novel mitotic checkpoint mechanism. SirT2 loss in mice induces significant defects associated with defective H4K20me1-3 levels. Accordingly, SirT2-deficient animals exhibit genomic instability and chromosomal aberrations and are prone to tumorigenesis. Our studies suggest that the dynamic cross-talk between the environment and the genome during mitosis determines the fate of the subsequent cell cycle.

Multiple models of human metabolism have been reconstructed, but each represents only a subset of our knowledge. Here we describe Recon 2, a community-driven, consensus 'metabolic reconstruction', which is the most comprehensive representation of human metabolism that is applicable to computational modeling. Compared with its predecessors, the reconstruction has improved topological and functional features, including ∼2× more reactions and ∼1.7× more unique metabolites. Using Recon 2 we predicted changes in metabolite biomarkers for 49 inborn errors of metabolism with 77% accuracy when compared to experimental data. Mapping metabolomic data and drug information onto Recon 2 demonstrates its potential for integrating and analyzing diverse data types. Using protein expression data, we automatically generated a compendium of 65 cell type-specific models, providing a basis for manual curation or investigation of cell-specific metabolic properties. Recon 2 will facilitate many future biomedical studies and is freely available at http://humanmetabolism.org/.

The enormous antigen receptor loci in lymphocytes are a paradigm of dynamic nuclear organisation, which is integral to their need to move extensively in 3D space to achieve distal gene synapse for V(D)J recombination and allelic exclusion. The loci undergo extensive 3D looping to bring distal genes together, controlled by several tissue-specific and ubiquitous factors, but how these factors achieve looping, synapsis and V(D)J recombination has been a mystery. Now several studies provide evidence that non-coding transcription, often proposed to play a role, is indeed an important driver, and furthermore has a specific nuclear destination for recombination. Both local transcription-independent looping and longer range factor-mediated transcription-dependent looping play separate roles in altering AgR architecture to enable V(D)J recombination.

Viviparity has many evolutionary advantages but brings with it the problem of the semi-allogeneic foetus having to coexist with the mother for the duration of pregnancy. In species with haemochorial placentation this problem is particularly evident as foetal trophoblast cells are extensively intermingled with maternal tissue and are directly exposed to maternal blood. Fascinating adaptations on both the foetal and maternal side have allowed for this interaction to be re-directed away from an immune rejection response not only towards immunotolerance, but in fact towards actively supporting reproductive success. Recent data have shown that some of these remarkable adaptations are conserved between mice and humans. Thus, a subset of trophoblast cells that is directly exposed to the maternal uterine environment shares the feature of expressing an unusual antigen repertoire on their surface. Paternal antigens can be recognized by maternal immune cells, in particular uterine natural killer cells that express cognate receptors, to regulate the extensive remodelling events that take place at the implantation site. Detailed genetic dissection experiments in the mouse have further demonstrated the direct impact of antigenic dissimilarity on foetal growth. With the availability of inbred strains, in vitro culture systems of trophoblast stem cells, and in-depth genetic, genomic and epigenomic data the mouse will be a valuable model system to study the intricate immune crosstalk at the foeto-maternal boundary. These insights will pave the way towards unravelling the mutual and synergistic interactions between trophoblast and its surrounding maternal environment, and in doing so help understand pregnancy pathologies.

DNA methylation in the oocyte has a particular significance: it may contribute to gene regulation in the oocyte and marks specific genes for activity in the embryo, as in the case of imprinted genes. Despite the fundamental importance of DNA methylation established in the oocyte, knowledge of the mechanisms by which it is conferred and how much is stably maintained in the embryo has remained very limited. Next generation sequencing approaches have dramatically altered our views on DNA methylation in oocytes. They have revealed that most methylation occurs in gene bodies in the oocyte. This observation ties in with genetic evidence showing that transcription is essential for methylation of imprinted genes, and is consistent with a model in which DNA methyltransferases are recruited by the histone modification patterns laid down by transcription events. These findings lead to a new perspective that transcription events dictate the placing and timing of methylation in specific genes and suggest a mechanism by which methylation could be coordinated by the events and factors regulating oocyte growth. With these new insights into the de novo methylation mechanism and new methods that allow high resolution profiling of DNA methylation in oocytes, we should be in a position to investigate whether and how DNA methylation errors could arise in association with assisted reproduction technologies or in response to exposure to environmental toxins.

Molecular control of the pluripotent state is thought to reside in a core circuitry of master transcription factors including the homeodomain-containing protein NANOG, which has an essential role in establishing ground state pluripotency during somatic cell reprogramming. Whereas the genomic occupancy of NANOG has been extensively investigated, comparatively little is known about NANOG-associated proteins and their contribution to the NANOG-mediated reprogramming process. Using enhanced purification techniques and a stringent computational algorithm, we identify 27 high-confidence protein interaction partners of NANOG in mouse embryonic stem cells. These consist of 19 previously unknown partners of NANOG that have not been reported before, including the ten-eleven translocation (TET) family methylcytosine hydroxylase TET1. We confirm physical association of NANOG with TET1, and demonstrate that TET1, in synergy with NANOG, enhances the efficiency of reprogramming. We also find physical association and reprogramming synergy of TET2 with NANOG, and demonstrate that knockdown of TET2 abolishes the reprogramming synergy of NANOG with a catalytically deficient mutant of TET1. These results indicate that the physical interaction between NANOG and TET1/TET2 proteins facilitates reprogramming in a manner that is dependent on the catalytic activity of TET1/TET2. TET1 and NANOG co-occupy genomic loci of genes associated with both maintenance of pluripotency and lineage commitment in embryonic stem cells, and TET1 binding is reduced upon NANOG depletion. Co-expression of NANOG and TET1 increases 5-hydroxymethylcytosine levels at the top-ranked common target loci Esrrb and Oct4 (also called Pou5f1), resulting in priming of their expression before reprogramming to naive pluripotency. We propose that TET1 is recruited by NANOG to enhance the expression of a subset of key reprogramming target genes. These results provide an insight into the reprogramming mechanism of NANOG and uncover a new role for 5-methylcytosine hydroxylases in the establishment of naive pluripotency.

A blood test may be an effective means of improving the appropriateness of referrals for symptomatic patients referred to specialist colorectal clinics. We evaluated the accuracy of a serum matrix metalloproteinase (MMP9) test in indicating colorectal cancer or its precursor conditions in a symptomatic population.