Life Sciences Research for Lifelong Health

Publications

The Babraham Institute Publications database contains details of all publications resulting from our research groups and scientific services.

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Title / Authors / Details Open Access Download

The RNA-Binding Proteins Zfp36l1 and Zfp36l2 Enforce the Thymic β-Selection Checkpoint by Limiting DNA Damage Response Signaling and Cell Cycle Progression.
Vogel KU, Bell LS, Galloway A, Ahlfors H, Turner M

The RNA-binding proteins Zfp36l1 and Zfp36l2 act redundantly to enforce the β-selection checkpoint during thymopoiesis, yet their molecular targets remain largely unknown. In this study, we identify these targets on a genome-wide scale in primary mouse thymocytes and show that Zfp36l1/l2 regulate DNA damage response and cell cycle transcripts to ensure proper β-selection. Double-negative 3 thymocytes lacking Zfp36l1/l2 share a gene expression profile with postselected double-negative 3b cells despite the absence of intracellular TCRβ and reduced IL-7 signaling. Our findings show that in addition to controlling the timing of proliferation at β-selection, posttranscriptional control by Zfp36l1/l2 limits DNA damage responses, which are known to promote thymocyte differentiation. Zfp36l1/l2 therefore act as posttranscriptional safeguards against chromosomal instability and replication stress by integrating pre-TCR and IL-7 signaling with DNA damage and cell cycle control.

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Journal of immunology (Baltimore, Md. : 1950), , 1550-6606, , 2016

PMID: 27566829


Oxygen Sensing by T Cells Establishes an Immunologically Tolerant Metastatic Niche.
Clever D, Roychoudhuri R, Constantinides MG, Askenase MH, Sukumar M, Klebanoff CA, Eil RL, Hickman HD, Yu Z, Pan JH, Palmer DC, Phan AT, Goulding J, Gattinoni L, Goldrath AW, Belkaid Y, Restifo NP

Cancer cells must evade immune responses at distant sites to establish metastases. The lung is a frequent site for metastasis. We hypothesized that lung-specific immunoregulatory mechanisms create an immunologically permissive environment for tumor colonization. We found that T-cell-intrinsic expression of the oxygen-sensing prolyl-hydroxylase (PHD) proteins is required to maintain local tolerance against innocuous antigens in the lung but powerfully licenses colonization by circulating tumor cells. PHD proteins limit pulmonary type helper (Th)-1 responses, promote CD4(+)-regulatory T (Treg) cell induction, and restrain CD8(+) T cell effector function. Tumor colonization is accompanied by PHD-protein-dependent induction of pulmonary Treg cells and suppression of IFN-γ-dependent tumor clearance. T-cell-intrinsic deletion or pharmacological inhibition of PHD proteins limits tumor colonization of the lung and improves the efficacy of adoptive cell transfer immunotherapy. Collectively, PHD proteins function in T cells to coordinate distinct immunoregulatory programs within the lung that are permissive to cancer metastasis. PAPERCLIP.

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Cell, 166, 1097-4172, 1117-1131.e14, 2016

PMID: 27565342


Exosomes bind autotaxin and act as a physiological delivery mechanism to stimulate LPA receptor signalling in cells.
Jethwa SA, Leah EJ, Zhang Q, Bright NA, Oxley D, Bootman MD, Rudge SA, Wakelam MJ

Autotaxin (ATX) the lysophospholipase responsible for generating the lipid receptor agonist lysophosphatidic acid (LPA) is a secreted enzyme. Here we show that once secreted it can bind to the surface of cell secreted exosomes. Exosome-bound ATX is catalytically active and carries generated LPA. Once bound to a cell, through specific integrin interaction, ATX releases the LPA to activate cell surface G-protein coupled LPA receptors; inhibition of signaling by the receptor antagonist Ki1642 suggests these are either LPAR1 or LPAR3. The binding stimulates downstream signaling including AKT and MAPK phosphorylation, the release of intracellular stored calcium and cell migration. We propose that exosomal binding of LPA-loaded ATX provides a means of efficiently delivering the lipid agonist to cell surface receptors to promote signalling. We further propose that this is a means whereby autotaxin-LPA signaling operates physiologically.

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Journal of cell science, , 1477-9137, , 2016

PMID: 27557622


Clinical spectrum and features of activated phosphoinositide 3-kinase δ syndrome: A large patient cohort study.
Coulter TI, Chandra A, Bacon CM, Babar J, Curtis J, Screaton N, Goodlad JR, Farmer G, Steele CL, Leahy TR, Doffinger R, Baxendale H, Bernatoniene J, Edgar JD, Longhurst HJ, Ehl S, Speckmann C, Grimbacher B, Sediva A, Milota T, Faust SN, Williams AP, Hayman G, Kucuk ZY, Hague R, French P, Brooker R, Forsyth P, Herriot R, Cancrini C, Palma P, Ariganello P, Conlon N, Feighery C, Gavin PJ, Jones A, Imai K, Ibrahim MA, Markelj G, Abinun M, Rieux-Laucat F, Latour S, Pellier I, Fischer A, Touzot F, Casanova JL, Durandy A, Burns SO, Savic S, Kumararatne DS, Moshous D, Kracker S, Vanhaesebroeck B, Okkenhaug K, Picard C, Nejentsev S, Condliffe AM, Cant AJ

Activated phosphoinositide 3-kinase δ syndrome (APDS) is a recently described combined immunodeficiency resulting from gain-of-function mutations in PIK3CD, the gene encoding the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ).

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The Journal of allergy and clinical immunology, , 1097-6825, , 2016

PMID: 27555459


Open Access

Identification of novel regulators of developmental hematopoiesis using endoglin regulatory elements as molecular probes.
Nasrallah R, Fast EM, Solaimani P, Knezevic K, Eliades A, Patel R, Thambyrajah R, Unnikrishnan A, Thoms J, Beck D, Vink CS, Smith A, Wong J, Shepherd M, Kent D, Roychoudhuri R, Paul F, Klippert J, Hammes A, Willnow T, Göttgens B, Dzierzak E, Zon LI, Lacaud G, Kouskoff V, Pimanda JE

Enhancers are the primary determinants of cell identity and specific promoter/enhancer combinations of Endoglin (ENG) have been shown to target blood and endothelium in the embryo. Here, we generated a series of embryonic stem cell lines, each targeted with reporter constructs driven by specific promoter/enhancer combinations of ENG, to evaluate their discriminative potential and value as molecular probes of the corresponding transcriptome. The Eng promoter (P) in combination with the -8/+7/+9kb enhancers, targeted cells in FLK1 mesoderm that were enriched for blast colony forming potential, whereas the P/-8kb enhancer targeted TIE2+/c-KIT+/CD41- endothelial cells that were enriched for hematopoietic potential. These fractions were isolated using reporter expression and their transcriptomes profiled by RNA-seq. There was high concordance between our signatures and those from embryos with defects at corresponding stages of hematopoiesis. Of the six genes that were up-regulated in both hemogenic mesoderm and hemogenic endothelial fractions targeted by the reporters, LRP2, a multiligand receptor, was the only gene that had not previously been associated with hematopoiesis. We show that LRP2 is indeed involved in definitive hematopoiesis and by doing so validate the use of reporter gene coupled enhancers as probes to gain insights into transcriptional changes that facilitate cell fate transitions.

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Blood, , 1528-0020, , 2016

PMID: 27554085


Coincident signals from GPCRs and receptor tyrosine kinases are uniquely transduced by PI3Kβ in myeloid cells.
Houslay DM, Anderson KE, Chessa T, Kulkarni S, Fritsch R, Downward J, Backer JM, Stephens LR, Hawkins PT

Class I phosphoinositide 3-kinases (PI3Ks) catalyze production of the lipid messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3), which plays a central role in a complex signaling network regulating cell growth, survival, and movement. This network is overactivated in cancer and inflammation, and there is interest in determining the PI3K catalytic subunit (p110α, p110β, p110γ, or p110δ) that should be targeted in different therapeutic contexts. Previous studies have defined unique regulatory inputs for p110β, including direct interaction with Gβγ subunits, Rac, and Rab5. We generated mice with knock-in mutations of p110β that selectively blocked the interaction with Gβγ and investigated its contribution to the PI3K isoform dependency of receptor tyrosine kinase (RTK) and G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) responses in primary macrophages and neutrophils. We discovered a unique role for p110β in supporting synergistic PIP3 formation in response to the coactivation of macrophages by macrophage colony-stimulating factor (M-CSF) and the complement protein C5a. In contrast, we found partially redundant roles for p110α, p110β, and p110δ downstream of M-CSF alone and a nonredundant role for p110γ downstream of C5a alone. This role for p110β completely depended on direct interaction with Gβγ, suggesting that p110β transduces GPCR signals in the context of coincident activation by an RTK. The p110β-Gβγ interaction was also required for neutrophils to generate reactive oxygen species in response to the Fcγ receptor-dependent recognition of immune complexes and for their β2 integrin-mediated adhesion to fibrinogen or poly-RGD+, directly implicating heterotrimeric G proteins in these two responses.

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Science signaling, 9, 1937-9145, ra82, 2016

PMID: 27531651


Open Access

Autophagy initiation by ULK complex assembly on ER tubulovesicular regions marked by ATG9 vesicles.
Karanasios E, Walker SA, Okkenhaug H, Manifava M, Hummel E, Zimmermann H, Ahmed Q, Domart MC, Collinson L, Ktistakis NT

Autophagosome formation requires sequential translocation of autophagy-specific proteins to membranes enriched in PI3P and connected to the ER. Preceding this, the earliest autophagy-specific structure forming de novo is a small punctum of the ULK1 complex. The provenance of this structure and its mode of formation are unknown. We show that the ULK1 structure emerges from regions, where ATG9 vesicles align with the ER and its formation requires ER exit and coatomer function. Super-resolution microscopy reveals that the ULK1 compartment consists of regularly assembled punctate elements that cluster in progressively larger spherical structures and associates uniquely with the early autophagy machinery. Correlative electron microscopy after live imaging shows tubulovesicular membranes present at the locus of this structure. We propose that the nucleation of autophagosomes occurs in regions, where the ULK1 complex coalesces with ER and the ATG9 compartment.

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Nature communications, 7, 2041-1723, 12420, 2016

PMID: 27510922


Open Access

MYC activation and BCL2L11 silencing by a tumour virus through the large-scale reconfiguration of enhancer-promoter hubs.
Wood CD, Veenstra H, Khasnis S, Gunnell A, Webb HM, Shannon-Lowe C, Andrews S, Osborne CS, West MJ

Lymphomagenesis in the presence of deregulated MYC requires suppression of MYC-driven apoptosis, often through downregulation of the pro-apoptotic BCL2L11 gene (Bim). Transcription factors (EBNAs) encoded by the lymphoma-associated Epstein-Barr virus (EBV) activate MYC and silence BCL2L11. We show that the EBNA2 transactivator activates multiple MYC enhancers and reconfigures the MYC locus to increase upstream and decrease downstream enhancer-promoter interactions. EBNA2 recruits the BRG1 ATPase of the SWI/SNF remodeller to MYC enhancers and BRG1 is required for enhancer-promoter interactions in EBV-infected cells. At BCL2L11, we identify a haematopoietic enhancer hub that is inactivated by the EBV repressors EBNA3A and EBNA3C through recruitment of the H3K27 methyltransferase EZH2. Reversal of enhancer inactivation using an EZH2 inhibitor upregulates BCL2L11 and induces apoptosis. EBV therefore drives lymphomagenesis by hijacking long-range enhancer hubs and specific cellular co-factors. EBV-driven MYC enhancer activation may contribute to the genesis and localisation of MYC-Immunoglobulin translocation breakpoints in Burkitt's lymphoma.

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eLife, 5, 2050-084X, , 2016

PMID: 27490482


Open Access

KIF1A mediates axonal transport of BACE1 and identification of independently moving cargoes in living SCG neurons.
Hung CO, Coleman MP

Neurons rely heavily on axonal transport to deliver materials from the sites of synthesis to the axon terminals over distances that can be many centimetres long. KIF1A is the neuron-specific kinesin with the fastest reported anterograde motor activity. Previous studies have shown that KIF1A transports a subset of synaptic proteins, neurofilaments and dense-core vesicles. Using two-colour live imaging, we showed that BACE1-mCherry moves together with KIF1A-GFP in both the anterograde and retrograde directions in SCG neurons. We confirmed that KIF1A is functionally required for BACE1 transport by using KIF1A siRNA and a KIF1A mutant construct (KIF1A-T312M) to impair its motor activity. We further identified several cargoes that have little or no co-migration with KIF1A-GFP and also move independently from BACE1-mCherry. Together, these findings support a primary role for KIF1A in the anterograde transport of BACE1 and suggest that axonally transported cargoes are sorted into different classes of carrier vesicles in the cell body and are transported by cargo-specific motor proteins through the axon.

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Traffic (Copenhagen, Denmark), , 1600-0854, , 2016

PMID: 27484852


C13orf31 (FAMIN) is a central regulator of immunometabolic function.
Cader MZ, Boroviak K, Zhang Q, Assadi G, Kempster SL, Sewell GW, Saveljeva S, Ashcroft JW, Clare S, Mukhopadhyay S, Brown KP, Tschurtschenthaler M, Raine T, Doe B, Chilvers ER, Griffin JL, Kaneider NC, Floto RA, D'Amato M, Bradley A, Wakelam MJ, Dougan G, Kaser A

Single-nucleotide variations in C13orf31 (LACC1) that encode p.C284R and p.I254V in a protein of unknown function (called 'FAMIN' here) are associated with increased risk for systemic juvenile idiopathic arthritis, leprosy and Crohn's disease. Here we set out to identify the biological mechanism affected by these coding variations. FAMIN formed a complex with fatty acid synthase (FASN) on peroxisomes and promoted flux through de novo lipogenesis to concomitantly drive high levels of fatty-acid oxidation (FAO) and glycolysis and, consequently, ATP regeneration. FAMIN-dependent FAO controlled inflammasome activation, mitochondrial and NADPH-oxidase-dependent production of reactive oxygen species (ROS), and the bactericidal activity of macrophages. As p.I254V and p.C284R resulted in diminished function and loss of function, respectively, FAMIN determined resilience to endotoxin shock. Thus, we have identified a central regulator of the metabolic function and bioenergetic state of macrophages that is under evolutionary selection and determines the risk of inflammatory and infectious disease.

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Nature immunology, , 1529-2916, , 2016

PMID: 27478939


3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy.
Russell MR, Lerner TR, Burden JJ, Nkwe DO, Pelchen-Matthews A, Domart MC, Durgan J, Weston A, Jones ML, Peddie CJ, Carzaniga R, Florey O, Marsh M, Gutierrez MG, Collinson LM

The processes of life take place in multiple dimensions, but imaging these processes in even three dimensions is challenging. Here we describe a workflow for 3D correlative light and electron microscopy (CLEM) of cell monolayers using fluorescence microscopy to identify and follow biological events, combined with serial blockface scanning electron microscopy to analyse the underlying ultrastructure. The workflow encompasses all steps from cell culture to sample processing, imaging strategy and 3D image processing/analysis. We demonstrate successful application of the workflow to three studies, each aiming to better understand complex and dynamic biological processes, including bacterial and viral infections of cultured cells and formation of entotic cell-in-cell structures commonly observed in tumours. Our workflow revealed new insight into the replicative niche of Mycobacterium tuberculosis in primary human lymphatic endothelial cells, HIV-1 in human monocyte-derived macrophages, and the composition of the entotic vacuole. The broad application of this 3D CLEM technique will make it a useful addition to the correlative imaging toolbox for biomedical research.

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Journal of cell science, , 1477-9137, , 2016

PMID: 27445312


Open Access

SNPsplit: Allele-specific splitting of alignments between genomes with known SNP genotypes.
Krueger F, Andrews SR

Sequencing reads overlapping polymorphic sites in diploid mammalian genomes may be assigned to one allele or the other. This holds the potential to detect gene expression, chromatin modifications, DNA methylation or nuclear interactions in an allele-specific fashion. SNPsplit is an allele-specific alignment sorter designed to read files in SAM/BAM format and determine the allelic origin of reads or read-pairs that cover known single nucleotide polymorphic (SNP) positions. For this to work libraries must have been aligned to a genome in which all known SNP positions were masked with the ambiguity base 'N' and aligned using a suitable mapping program such as Bowtie2, TopHat, STAR, HISAT2, HiCUP or Bismark. SNPsplit also provides an automated solution to generate N-masked reference genomes for hybrid mouse strains based on the variant call information provided by the Mouse Genomes Project. The unique ability of SNPsplit to work with various different kinds of sequencing data including RNA-Seq, ChIP-Seq, Bisulfite-Seq or Hi-C opens new avenues for the integrative exploration of allele-specific data.

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F1000Research, 5, , 1479, 2016

PMID: 27429743


Open Access

Integrating epigenomic data and 3D genomic structure with a new measure of chromatin assortativity.
Pancaldi V, Carrillo-de-Santa-Pau E, Javierre BM, Juan D, Fraser P, Spivakov M, Valencia A, Rico D

Network analysis is a powerful way of modeling chromatin interactions. Assortativity is a network property used in social sciences to identify factors affecting how people establish social ties. We propose a new approach, using chromatin assortativity, to integrate the epigenomic landscape of a specific cell type with its chromatin interaction network and thus investigate which proteins or chromatin marks mediate genomic contacts.

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Genome biology, 17, 1474-760X, 152, 0

PMID: 27391817


Open Access

In vivo genome-wide profiling reveals a tissue-specific role for 5-formylcytosine.
Iurlaro M, McInroy GR, Burgess HE, Dean W, Raiber EA, Bachman M, Beraldi D, Balasubramanian S, Reik W

Genome-wide methylation of cytosine can be modulated in the presence of TET and thymine DNA glycosylase (TDG) enzymes. TET is able to oxidise 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). TDG can excise the oxidative products 5fC and 5caC, initiating base excision repair. These modified bases are stable and detectable in the genome, suggesting that they could have epigenetic functions in their own right. However, functional investigation of the genome-wide distribution of 5fC has been restricted to cell culture-based systems, while its in vivo profile remains unknown.

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Genome biology, 17, 1474-760X, 141, 2016

PMID: 27356509


Open Access

CHiCAGO: robust detection of DNA looping interactions in Capture Hi-C data.
Cairns J, Freire-Pritchett P, Wingett SW, Várnai C, Dimond A, Plagnol V, Zerbino D, Schoenfelder S, Javierre BM, Osborne C, Fraser P, Spivakov M

Capture Hi-C (CHi-C) is a method for profiling chromosomal interactions involving targeted regions of interest, such as gene promoters, globally and at high resolution. Signal detection in CHi-C data involves a number of statistical challenges that are not observed when using other Hi-C-like techniques. We present a background model and algorithms for normalisation and multiple testing that are specifically adapted to CHi-C experiments. We implement these procedures in CHiCAGO ( http://regulatorygenomicsgroup.org/chicago ), an open-source package for robust interaction detection in CHi-C. We validate CHiCAGO by showing that promoter-interacting regions detected with this method are enriched for regulatory features and disease-associated SNPs.

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Genome biology, 17, 1474-760X, 127, 2016

PMID: 27306882


Open Access

Synaptophysin depletion and intraneuronal Aβ in organotypic hippocampal slice cultures from huAPP transgenic mice.
Harwell CS, Coleman MP

To date, there are no effective disease-modifying treatments for Alzheimer's disease (AD). In order to develop new therapeutics for stages where they are most likely to be effective, it is important to identify the first pathological alterations in the disease cascade. Changes in Aβ concentration have long been reported as one of the first steps, but understanding the source, and earliest consequences, of pathology requires a model system that represents all major CNS cell types, is amenable to repeated observation and sampling, and can be readily manipulated. In this regard, long term organotypic hippocampal slice cultures (OHSCs) from neonatal amyloid mice offer an excellent compromise between in vivo and primary culture studies, largely retaining the cellular composition and neuronal architecture of the in vivo hippocampus, but with the in vitro advantages of accessibility to live imaging, sampling and intervention.

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Molecular neurodegeneration, 11, 1750-1326, 44, 2016

PMID: 27287430


Systems Biology Approaches for Understanding Genome Architecture.
Sewitz S, Lipkow K

The linear and three-dimensional arrangement and composition of chromatin in eukaryotic genomes underlies the mechanisms directing gene regulation. Understanding this organization requires the integration of many data types and experimental results. Here we describe the approach of integrating genome-wide protein-DNA binding data to determine chromatin states. To investigate spatial aspects of genome organization, we present a detailed description of how to run stochastic simulations of protein movements within a simulated nucleus in 3D. This systems level approach enables the development of novel questions aimed at understanding the basic mechanisms that regulate genome dynamics.

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Methods in molecular biology (Clifton, N.J.), 1431, 1940-6029, 109-26, 2016

PMID: 27283305


Two Mutually Exclusive Local Chromatin States Drive Efficient V(D)J Recombination.
Bolland DJ, Koohy H, Wood AL, Matheson LS, Krueger F, Stubbington MJ, Baizan-Edge A, Chovanec P, Stubbs BA, Tabbada K, Andrews SR, Spivakov M, Corcoran AE

Variable (V), diversity (D), and joining (J) (V(D)J) recombination is the first determinant of antigen receptor diversity. Understanding how recombination is regulated requires a comprehensive, unbiased readout of V gene usage. We have developed VDJ sequencing (VDJ-seq), a DNA-based next-generation-sequencing technique that quantitatively profiles recombination products. We reveal a 200-fold range of recombination efficiency among recombining V genes in the primary mouse Igh repertoire. We used machine learning to integrate these data with local chromatin profiles to identify combinatorial patterns of epigenetic features that associate with active VH gene recombination. These features localize downstream of VH genes and are excised by recombination, revealing a class of cis-regulatory element that governs recombination, distinct from expression. We detect two mutually exclusive chromatin signatures at these elements, characterized by CTCF/RAD21 and PAX5/IRF4, which segregate with the evolutionary history of associated VH genes. Thus, local chromatin signatures downstream of VH genes provide an essential layer of regulation that determines recombination efficiency.

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Cell reports, 15, 2211-1247, 2475-87, 2016

PMID: 27264181


Open Access

Impairment of DNA Methylation Maintenance Is the Main Cause of Global Demethylation in Naive Embryonic Stem Cells.
von Meyenn F, Iurlaro M, Habibi E, Liu NQ, Salehzadeh-Yazdi A, Santos F, Petrini E, Milagre I, Yu M, Xie Z, Kroeze LI, Nesterova TB, Jansen JH, Xie H, He C, Reik W, Stunnenberg HG

Global demethylation is part of a conserved program of epigenetic reprogramming to naive pluripotency. The transition from primed hypermethylated embryonic stem cells (ESCs) to naive hypomethylated ones (serum-to-2i) is a valuable model system for epigenetic reprogramming. We present a mathematical model, which accurately predicts global DNA demethylation kinetics. Experimentally, we show that the main drivers of global demethylation are neither active mechanisms (Aicda, Tdg, and Tet1-3) nor the reduction of de novo methylation. UHRF1 protein, the essential targeting factor for DNMT1, is reduced upon transition to 2i, and so is recruitment of the maintenance methylation machinery to replication foci. Concurrently, there is global loss of H3K9me2, which is needed for chromatin binding of UHRF1. These mechanisms synergistically enforce global DNA hypomethylation in a replication-coupled fashion. Our observations establish the molecular mechanism for global demethylation in naive ESCs, which has key parallels with those operating in primordial germ cells and early embryos.

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Molecular cell, , 1097-4164, , 2016

PMID: 27237052


Open Access

The RNA-binding protein TTP is a global post-transcriptional regulator of feedback control in inflammation.
Tiedje C, Diaz-Muñoz MD, Trulley P, Ahlfors H, Laaß K, Blackshear PJ, Turner M, Gaestel M

RNA-binding proteins (RBPs) facilitate post-transcriptional control of eukaryotic gene expression at multiple levels. The RBP tristetraprolin (TTP/Zfp36) is a signal-induced phosphorylated anti-inflammatory protein guiding unstable mRNAs of pro-inflammatory proteins for degradation and preventing translation. Using iCLIP, we have identified numerous mRNA targets bound by wild-type TTP and by a non-MK2-phosphorylatable TTP mutant (TTP-AA) in 1 h LPS-stimulated macrophages and correlated their interaction with TTP to changes at the level of mRNA abundance and translation in a transcriptome-wide manner. The close similarity of the transcriptomes of TTP-deficient and TTP-expressing macrophages upon short LPS stimulation suggested an effective inactivation of TTP by MK2, whereas retained RNA-binding capacity of TTP-AA to 3'UTRs caused profound changes in the transcriptome and translatome, altered NF-κB-activation and induced cell death. Increased TTP binding to the 3'UTR of feedback inhibitor mRNAs, such as Ier3, Dusp1 or Tnfaip3, in the absence of MK2-dependent TTP neutralization resulted in a strong reduction of their protein synthesis contributing to the deregulation of the NF-κB-signaling pathway. Taken together, our study uncovers a role of TTP as a suppressor of feedback inhibitors of inflammation and highlights the importance of fine-tuned TTP activity-regulation by MK2 in order to control the pro-inflammatory response.

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Nucleic acids research, , 1362-4962, , 2016

PMID: 27220464


Open Access

Tbet or Continued RORγt Expression Is Not Required for Th17-Associated Immunopathology.
Brucklacher-Waldert V, Ferreira C, Innocentin S, Kamdar S, Withers DR, Kullberg MC, Veldhoen M

The discovery of Th17 cell plasticity, in which CD4(+) IL-17-producing Th17 cells give rise to IL-17/IFN-γ double-producing cells and Th1-like IFNγ(+) ex-Th17 lymphocytes, has raised questions regarding which of these cell types contribute to immunopathology during inflammatory diseases. In this study, we show using Helicobacter hepaticus-induced intestinal inflammation that IL-17A(Cre)- or Rag1(Cre)-mediated deletion of Tbx21 has no effect on the generation of IL-17/IFN-γ double-producing cells, but leads to a marked absence of Th1-like IFNγ(+) ex-Th17 cells. Despite the lack of Th1-like ex-Th17 cells, the degree of H. hepaticus-triggered intestinal inflammation in mice in which Tbx21 was excised in IL-17-producing or Rag1-expressing cells is indistinguishable from that observed in control mice. In stark contrast, using experimental autoimmune encephalomyelitis, we show that IL-17A(Cre)-mediated deletion of Tbx21 prevents the conversion of Th17 cells to IL-17A/IFN-γ double-producing cells as well as Th1-like IFN-γ(+) ex-Th17 cells. However, IL-17A(Cre)-mediated deletion of Tbx21 has only limited effects on disease course in this model and is not compensated by Ag-specific Th1 cells. IL-17A(Cre)-mediated deletion of Rorc reveals that RORγt is essential for the maintenance of the Th17 cell lineage, but not immunopathology during experimental autoimmune encephalomyelitis. These results show that neither the single Th17 subset, nor its progeny, is solely responsible for immunopathology or autoimmunity.

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Journal of immunology (Baltimore, Md. : 1950), , 1550-6606, , 2016

PMID: 27183623


Open Access

BACH2 regulates CD8(+) T cell differentiation by controlling access of AP-1 factors to enhancers.
Roychoudhuri R, Clever D, Li P, Wakabayashi Y, Quinn KM, Klebanoff CA, Ji Y, Sukumar M, Eil RL, Yu Z, Spolski R, Palmer DC, Pan JH, Patel SJ, Macallan DC, Fabozzi G, Shih HY, Kanno Y, Muto A, Zhu J, Gattinoni L, O'Shea JJ, Okkenhaug K, Igarashi K, Leonard WJ, Restifo NP

T cell antigen receptor (TCR) signaling drives distinct responses depending on the differentiation state and context of CD8(+) T cells. We hypothesized that access of signal-dependent transcription factors (TFs) to enhancers is dynamically regulated to shape transcriptional responses to TCR signaling. We found that the TF BACH2 restrains terminal differentiation to enable generation of long-lived memory cells and protective immunity after viral infection. BACH2 was recruited to enhancers, where it limited expression of TCR-driven genes by attenuating the availability of activator protein-1 (AP-1) sites to Jun family signal-dependent TFs. In naive cells, this prevented TCR-driven induction of genes associated with terminal differentiation. Upon effector differentiation, reduced expression of BACH2 and its phosphorylation enabled unrestrained induction of TCR-driven effector programs.

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Nature immunology, , 1529-2916, , 2016

PMID: 27158840


Application of virtual screening to the discovery of novel nicotinamide phosphoribosyltransferase (NAMPT) inhibitors with potential for the treatment of cancer and axonopathies.
Clark DE, Waszkowycz B, Wong M, Lockey PM, Adalbert R, Gilley J, Clark J, Coleman MP

NAMPT may represent a novel target for drug discovery in various therapeutic areas, including oncology and inflammation. Additionally, recent work has suggested that targeting NAMPT has potential in treating axon degeneration. In this work, publicly available X-ray co-crystal structures of NAMPT and the structures of two known NAMPT inhibitors were used as the basis for a structure- and ligand-based virtual screening campaign. From this, two novel series of NAMPT inhibitors were identified, one of which showed a statistically significant protective effect when tested in a cellular model of axon degeneration.

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Bioorganic & medicinal chemistry letters, , 1464-3405, , 2016

PMID: 27158141


Open Access

CHiCP: a web-based tool for the integrative and interactive visualization of promoter capture Hi-C datasets.
Schofield EC, Carver T, Achuthan P, Freire-Pritchett P, Spivakov M, Todd JA, Burren OS

Promoter capture Hi-C (PCHi-C) allows the genome-wide interrogation of physical interactions between distal DNA regulatory elements and gene promoters in multiple tissue contexts. Visual integration of the resultant chromosome interaction maps with other sources of genomic annotations can provide insight into underlying regulatory mechanisms. We have developed Capture HiC Plotter (CHiCP), a web-based tool that allows interactive exploration of PCHi-C interaction maps and integration with both public and user-defined genomic datasets.

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Bioinformatics (Oxford, England), 32, 1367-4811, 2511-3, 2016

PMID: 27153610


Open Access

The pluripotency factor Nanog regulates pericentromeric heterochromatin organization in mouse embryonic stem cells.
Novo CL, Tang C, Ahmed K, Djuric U, Fussner E, Mullin NP, Morgan NP, Hayre J, Sienerth AR, Elderkin S, Nishinakamura R, Chambers I, Ellis J, Bazett-Jones DP, Rugg-Gunn PJ

An open and decondensed chromatin organization is a defining property of pluripotency. Several epigenetic regulators have been implicated in maintaining an open chromatin organization, but how these processes are connected to the pluripotency network is unknown. Here, we identified a new role for the transcription factor NANOG as a key regulator connecting the pluripotency network with constitutive heterochromatin organization in mouse embryonic stem cells. Deletion of Nanog leads to chromatin compaction and the remodeling of heterochromatin domains. Forced expression of NANOG in epiblast stem cells is sufficient to decompact chromatin. NANOG associates with satellite repeats within heterochromatin domains, contributing to an architecture characterized by highly dispersed chromatin fibers, low levels of H3K9me3, and high major satellite transcription, and the strong transactivation domain of NANOG is required for this organization. The heterochromatin-associated protein SALL1 is a direct cofactor for NANOG, and loss of Sall1 recapitulates the Nanog-null phenotype, but the loss of Sall1 can be circumvented through direct recruitment of the NANOG transactivation domain to major satellites. These results establish a direct connection between the pluripotency network and chromatin organization and emphasize that maintaining an open heterochromatin architecture is a highly regulated process in embryonic stem cells.

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Genes & development, , 1549-5477, , 2016

PMID: 27125671


Open Access