The discovery of cytosine hydroxymethylation (5hmC) as a mechanism that potentially controls DNA methylation changes typical of neoplasia prompted us to investigate its behaviour in colon cancer. 5hmC is globally reduced in proliferating cells such as colon tumours and the gut crypt progenitors, from which tumours can arise.

The function of mast cells in allergic and organ-specific autoimmune responses is highly controversial. In the current study, we aimed to dissect the role of mast cells in systemic autoimmunity in the B6(lpr/lpr) mouse, a spontaneous model of systemic lupus erythematosus. B6(lpr/lpr) mice were interbred with C57Bl/6-Kit(W-sh/W-sh) (Wsh) mice, resulting in mast cell deficiency. The offspring from this cross (Lpr/Wsh mice) developed symptoms of lupus of the same severity as B6(lpr/lpr) mice. Loss of mast cells on the Lpr background did not alter autoantibody production, proteinuria, the composition of T and B cell populations or autoimmune pathology. Reduced c-Kit expression did drive expanded splenomegaly and impeded interleukin-4 production by CD4(+) cells, suggesting minor functions for mast cells. In general, we conclude that mast cell deficiency and c-Kit deficiency do not play a role in the pathogenesis of lupus in B6(lpr/lpr) mice.

SARM1 function and nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) loss both promote axon degeneration, but their relative relationship in the process is unknown. Here, we show that NMNAT2 loss and resultant changes to NMNAT metabolites occur in injured SARM1-deficient axons despite their delayed degeneration and that axon degeneration specifically induced by NMNAT2 depletion requires SARM1. Strikingly, SARM1 deficiency also corrects axon outgrowth in mice lacking NMNAT2, independently of NMNAT metabolites, preventing perinatal lethality. Furthermore, NAMPT inhibition partially restores outgrowth of NMNAT2-deficient axons, suggesting that the NMNAT substrate, NMN, contributes to this phenotype. NMNAT2-depletion-dependent degeneration of established axons and restricted extension of developing axons are thus both SARM1 dependent, and SARM1 acts either downstream of NMNAT2 loss and NMN accumulation in a linear pathway or in a parallel branch of a convergent pathway. Understanding the pathway will help establish relationships with other modulators of axon survival and facilitate the development of effective therapies for axonopathies.

The TNF family cytokine TL1A (Tnfsf15) costimulates T cells and type 2 innate lymphocytes (ILC2) through its receptor DR3 (Tnfrsf25). DR3-deficient mice have reduced T cell accumulation at the site of inflammation and reduced ILC2-dependent immune responses in a number of models of autoimmune and allergic diseases. In allergic lung disease models, immunopathology and local Th2 and ILC2 accumulation is reduced in DR3-deficient mice despite normal systemic priming of Th2 responses and generation of T cells secreting IL-13 and IL-4, prompting the question of whether TL1A promotes the development of other T cell subsets that secrete cytokines to drive allergic disease. In this study, we find that TL1A potently promotes generation of murine T cells producing IL-9 (Th9) by signaling through DR3 in a cell-intrinsic manner. TL1A enhances Th9 differentiation through an IL-2 and STAT5-dependent mechanism, unlike the TNF-family member OX40, which promotes Th9 through IL-4 and STAT6. Th9 differentiated in the presence of TL1A are more pathogenic, and endogenous TL1A signaling through DR3 on T cells is required for maximal pathology and IL-9 production in allergic lung inflammation. Taken together, these data identify TL1A-DR3 interactions as a novel pathway that promotes Th9 differentiation and pathogenicity. TL1A may be a potential therapeutic target in diseases dependent on IL-9.

The TNF family cytokine TL1A (Tnfsf15) costimulates T cells and type 2 innate lymphocytes (ILC2) through its receptor DR3 (Tnfrsf25). DR3-deficient mice have reduced T cell accumulation at the site of inflammation and reduced ILC2-dependent immune responses in a number of models of autoimmune and allergic diseases. In allergic lung disease models, immunopathology and local Th2 and ILC2 accumulation is reduced in DR3-deficient mice despite normal systemic priming of Th2 responses and generation of T cells secreting IL-13 and IL-4, prompting the question of whether TL1A promotes the development of other T cell subsets that secrete cytokines to drive allergic disease. In this study, we find that TL1A potently promotes generation of murine T cells producing IL-9 (Th9) by signaling through DR3 in a cell-intrinsic manner. TL1A enhances Th9 differentiation through an IL-2 and STAT5-dependent mechanism, unlike the TNF-family member OX40, which promotes Th9 through IL-4 and STAT6. Th9 differentiated in the presence of TL1A are more pathogenic, and endogenous TL1A signaling through DR3 on T cells is required for maximal pathology and IL-9 production in allergic lung inflammation. Taken together, these data identify TL1A-DR3 interactions as a novel pathway that promotes Th9 differentiation and pathogenicity. TL1A may be a potential therapeutic target in diseases dependent on IL-9.

The Hedgehog pathway is critical for animal development and has been implicated in multiple human malignancies. Despite great interest in targeting the pathway pharmacologically, many of the principles underlying the signal transduction cascade remain poorly understood. Hedgehog ligands are recognized by a unique receptor system that features the transporter-like protein Patched and the G protein-coupled receptor (GPCR)-like Smoothened (SMO). The biochemical interaction between these transmembrane proteins is the subject of intensive efforts. Recent structural and functional studies have provided great insight into the small-molecule regulation of SMO through identification of two distinct ligand-binding sites. In this Perspective, we review these recent findings and relate them to potential mechanisms for the endogenous regulation of SMO.

To identify the underlying genetic defect in a 16-year-old girl with severe early-onset and refractory systemic lupus erythematosus (SLE), IgA deficiency, and mild lower limb spasticity without neuroradiologic manifestations.

The third annual meeting of the EpiGeneSys network brought together epigenetics and systems biologists to report on collaborative projects that apply quantitative approaches to understanding complex epigenetic processes. The figure shown represents one meeting highlight, which was the unexpected emergence of genotype versus epigenotype in control of cell state.

Macrophages provide a bridge linking innate and adaptive immunity. An increased frequency of macrophages and other myeloid cells paired with excessive cytokine production is commonly seen in the aging immune system, known as 'inflamm-aging'. It is presently unclear how healthy macrophages are maintained throughout life and what connects inflammation with myeloid dysfunction during aging. Autophagy, an intracellular degradation mechanism, has known links with aging and lifespan extension. Here, we show for the first time that autophagy regulates the acquisition of major aging features in macrophages. In the absence of the essential autophagy gene Atg7, macrophage populations are increased and key functions such as phagocytosis and nitrite burst are reduced, while the inflammatory cytokine response is significantly increased - a phenotype also observed in aged macrophages. Furthermore, reduced autophagy decreases surface antigen expression and skews macrophage metabolism toward glycolysis. We show that macrophages from aged mice exhibit significantly reduced autophagic flux compared to young mice. These data demonstrate that autophagy plays a critical role in the maintenance of macrophage homeostasis and function, regulating inflammation and metabolism and thereby preventing immunosenescence. Thus, autophagy modulation may prevent excess inflammation and preserve macrophage function during aging, improving immune responses and reducing the morbidity and mortality associated with inflamm-aging. © 2015 S. Karger AG, Basel.

The Computational Modeling in Biology Network (COMBINE) is a consortium of groups involved in the development of open community standards and formats used in computational modeling in biology. COMBINE's aim is to act as a coordinator, facilitator, and resource for different standardization efforts whose domains of use cover related areas of the computational biology space. In this perspective article, we summarize COMBINE, its general organization, and the community standards and other efforts involved in it. Our goals are to help guide readers toward standards that may be suitable for their research activities, as well as to direct interested readers to relevant communities where they can best expect to receive assistance in how to develop interoperable computational models.

Smoothened (SMO) inhibitors are under clinical investigation for the treatment of several cancers. Vismodegib is approved for the treatment of locally advanced and metastatic basal cell carcinoma (BCC). Most BCC patients experience significant clinical benefit on vismodegib, but some develop resistance. Genomic analysis of tumor biopsies revealed that vismodegib resistance is associated with Hedgehog (Hh) pathway reactivation, predominantly through mutation of the drug target SMO and to a lesser extent through concurrent copy number changes in SUFU and GLI2. SMO mutations either directly impaired drug binding or activated SMO to varying levels. Furthermore, we found evidence for intra-tumor heterogeneity, suggesting that a combination of therapies targeting components at multiple levels of the Hh pathway is required to overcome resistance.

The mammalian genome harbors up to one million regulatory elements often located at great distances from their target genes. Long-range elements control genes through physical contact with promoters and can be recognized by the presence of specific histone modifications and transcription factor binding. Linking regulatory elements to specific promoters genome-wide is currently impeded by the limited resolution of high-throughput chromatin interaction assays. Here we apply a sequence capture approach to enrich Hi-C libraries for >22,000 annotated mouse promoters to identify statistically significant, long-range interactions at restriction fragment resolution, assigning long-range interacting elements to their target genes genome-wide in embryonic stem cells and fetal liver cells. The distal sites contacting active genes are enriched in active histone modifications and transcription factor occupancy, whereas inactive genes contact distal sites with repressive histone marks, demonstrating the regulatory potential of the distal elements identified. Furthermore, we find that coregulated genes cluster nonrandomly in spatial interaction networks correlated with their biological function and expression level. Interestingly, we find the strongest gene clustering in ES cells between transcription factor genes that control key developmental processes in embryogenesis. The results provide the first genome-wide catalog linking gene promoters to their long-range interacting elements and highlight the complex spatial regulatory circuitry controlling mammalian gene expression.

Contrasting phenotypes arise from similar genomes through a combination of losses, gains, co-option and modifications of inherited genomic material. Understanding the molecular basis of this phenotypic diversity is a fundamental challenge in modern evolutionary biology. Comparisons of the genes and their expression patterns underlying traits in closely related species offer an unrivaled opportunity to evaluate the extent to which genomic material is reorganized to produce novel traits. Advances in molecular methods now allow us to dissect the molecular machinery underlying phenotypic diversity in almost any organism, from single-celled entities to the most complex vertebrates. Here we discuss how comparisons of social parasites and their free-living hosts may provide unique insights into the molecular basis of phenotypic evolution. Social parasites evolve from a eusocial ancestor and are specialized to exploit the socially acquired resources of their closely-related eusocial host. Molecular comparisons of such species pairs can reveal how genomic material is re-organized in the loss of ancestral traits (i.e., of free-living traits in the parasites) and the gain of new ones (i.e., specialist traits required for a parasitic lifestyle). We define hypotheses on the molecular basis of phenotypes in the evolution of social parasitism and discuss their wider application in our understanding of the molecular basis of phenotypic diversity within the theoretical framework of phenotypic plasticity and shifting reaction norms. Currently there are no data available to test these hypotheses, and so we also provide some proof of concept data using the paper wasp social parasite/host system (Polistes sulcifer-Polistes dominula). This conceptual framework and first empirical data provide a spring-board for directing future genomic analyses on exploiting social parasites as a route to understanding the evolution of phenotypic specialization.

Regulatory T (Treg) cells suppress effector T (Teff) cells and prevent immune-mediated rejection of cancer. Much less appreciated are mechanisms by which Teff cells antagonize Treg cells. Herein, we consider how complex reciprocal interactions between Teff and Treg cells shape their population dynamics within tumors. Under states of tolerance, including during tumor escape, suppressed Teff cells support Treg cell populations through antigen-dependent provision of interleukin (IL)-2. During immune activation, Teff cells can lose this supportive capacity and directly antagonize Treg cell populations to neutralize their immunosuppressive function. While this latter state is rarely achieved spontaneously within tumors, we propose that therapeutic induction of immune activation has the potential to stably disrupt immunosuppressive population states resulting in durable cancer regression.

Myelin is essential for efficient signal transduction in the nervous system comprising of multiple proteins. The intricacies of the regulation of the formation of myelin, and its components, are not fully understood. Here, we describe the characterization of a novel myelin basic protein (Mbp) mutant mouse, mbp(jive), which spontaneously occurred in our mouse colony. These mice displayed the onset of a shaking gait before 3 weeks of age and seizure onset before 2 months of age. Due to a progressive increase of seizure intensity, mbp(jive) mice experienced premature lethality at around 3 months of age. Mbp mRNA transcript or protein was undetectable and, accordingly, genetic analysis demonstrated a homozygous loss of exons 3 to 6 of Mbp. Peripheral nerve conductance was mostly unimpaired. Additionally, we observed grave structural changes in white matter predominant structures were detected by T1, T2 and diffusion weighted magnetic resonance imaging. We additionally observed that Mbp-deficiency results in an upregulation of Qkl, Mag and Cnp, suggestive of a regulatory feedback mechanism whereby compensatory increases in Qkl have downstream effects on Mag and Cnp. Further research will clarify the role and specifications of this myelin feedback loop, as well as determine its potential role in therapeutic strategies for demyelinating disorders.

Post-transcriptional regulation of mRNA by the RNA-binding protein HuR (encoded by Elavl1) is required in B cells for the germinal center reaction and for the production of class-switched antibodies in response to thymus-independent antigens. Transcriptome-wide examination of RNA isoforms and their abundance and translation in HuR-deficient B cells, together with direct measurements of HuR-RNA interactions, revealed that HuR-dependent splicing of mRNA affected hundreds of transcripts, including that encoding dihydrolipoamide S-succinyltransferase (Dlst), a subunit of the 2-oxoglutarate dehydrogenase (α-KGDH) complex. In the absence of HuR, defective mitochondrial metabolism resulted in large amounts of reactive oxygen species and B cell death. Our study shows how post-transcriptional processes control the balance of energy metabolism required for the proliferation and differentiation of B cells.

Multiple regulatory elements distant from their targets on the linear genome can influence the expression of a single gene through chromatin looping. Chromosome conformation capture implemented in Hi-C allows for genome-wide agnostic characterization of chromatin contacts. However, detection of functional enhancer-promoter interactions is precluded by its effective resolution that is determined by both restriction fragmentation and sensitivity of the experiment. Here we develop a capture Hi-C (cHi-C) approach to allow an agnostic characterization of these physical interactions on a genome-wide scale. Single-nucleotide polymorphisms associated with complex diseases often reside within regulatory elements and exert effects through long-range regulation of gene expression. Applying this cHi-C approach to 14 colorectal cancer risk loci allows us to identify key long-range chromatin interactions in cis and trans involving these loci.

Immunologists studying the relationship between nutrition and immunological function face many challenges. We discuss here some of the historical skepticism with which nutritional research has often been faced and the complexities that need to be overcome in order to provide meaningful mechanistic insights.

Enhancers regulate spatiotemporal gene expression and impart cell-specific transcriptional outputs that drive cell identity. Super-enhancers (SEs), also known as stretch-enhancers, are a subset of enhancers especially important for genes associated with cell identity and genetic risk of disease. CD4(+) T cells are critical for host defence and autoimmunity. Here we analysed maps of mouse T-cell SEs as a non-biased means of identifying key regulatory nodes involved in cell specification. We found that cytokines and cytokine receptors were the dominant class of genes exhibiting SE architecture in T cells. Nonetheless, the locus encoding Bach2, a key negative regulator of effector differentiation, emerged as the most prominent T-cell SE, revealing a network in which SE-associated genes critical for T-cell biology are repressed by BACH2. Disease-associated single-nucleotide polymorphisms for immune-mediated disorders, including rheumatoid arthritis, were highly enriched for T-cell SEs versus typical enhancers or SEs in other cell lineages. Intriguingly, treatment of T cells with the Janus kinase (JAK) inhibitor tofacitinib disproportionately altered the expression of rheumatoid arthritis risk genes with SE structures. Together, these results indicate that genes with SE architecture in T cells encompass a variety of cytokines and cytokine receptors but are controlled by a 'guardian' transcription factor, itself endowed with an SE. Thus, enumeration of SEs allows the unbiased determination of key regulatory nodes in T cells, which are preferentially modulated by pharmacological intervention.

Post-transcriptional mRNA regulation by RNA binding proteins (RBPs) associated with AU-rich elements (AREs) present in the 3' untranslated region (3'UTR) of specific mRNAs modulates transcript stability and translation in eukaryotic cells. Here we have functionally characterised the importance of the AREs present within the Bcl2 3'UTR in order to maintain Bcl2 expression. Gene targeting deletion of 300 nucleotides of the Bcl2 3'UTR rich in AREs diminishes Bcl2 mRNA stability and protein levels in primary B cells, decreasing cell lifespan. Generation of chimeric mice indicates that Bcl2-ARE∆/∆ B cells have an intrinsic competitive disadvantage compared to wild type cells. Biochemical assays and predictions using a bioinformatics approach show that several RBPs bind to the Bcl2 AREs, including AUF1 and HuR proteins. Altogether, association of RBPs to Bcl2 AREs contributes to Bcl2 protein expression by stabilizing Bcl2 mRNA and promotes B cell maintenance.

Drosophila phototransduction is mediated via a G-protein-coupled PLC cascade. Recent evidence, including the demonstration that light evokes rapid contractions of the photoreceptors, suggested that the light-sensitive channels (TRP and TRPL) may be mechanically gated, together with protons released by PLC-mediated PIP2 hydrolysis. If mechanical gating is involved we predicted that the response to light should be influenced by altering the physical properties of the membrane. To achieve this, we used diet to manipulate the degree of saturation of membrane phospholipids. In flies reared on a yeast diet, lacking polyunsaturated fatty acids (PUFAs), mass spectrometry showed that the proportion of polyunsaturated phospholipids was sevenfold reduced (from 38 to ∼5%) but rescued by adding a single species of PUFA (linolenic or linoleic acid) to the diet. Photoreceptors from yeast-reared flies showed a 2- to 3-fold increase in latency and time to peak of the light response, without affecting quantum bump waveform. In the absence of Ca(2+) influx or in trp mutants expressing only TRPL channels, sensitivity to light was reduced up to ∼10-fold by the yeast diet, and essentially abolished in hypomorphic G-protein mutants (Gαq). PLC activity appeared little affected by the yeast diet; however, light-induced contractions measured by atomic force microscopy or the activation of ectopic mechanosensitive gramicidin channels were also slowed ∼2-fold. The results are consistent with mechanosensitive gating and provide a striking example of how dietary fatty acids can profoundly influence sensory performance in a classical G-protein-coupled signaling cascade.