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

Long-Range Enhancer Interactions Are Prevalent in Mouse Embryonic Stem Cells and Are Reorganized upon Pluripotent State Transition.
Novo CL, Javierre BM, Cairns J, Segonds-Pichon A, Wingett SW, Freire-Pritchett P, Furlan-Magaril M, Schoenfelder S, Fraser P, Rugg-Gunn PJ

Transcriptional enhancers, including super-enhancers (SEs), form physical interactions with promoters to regulate cell-type-specific gene expression. SEs are characterized by high transcription factor occupancy and large domains of active chromatin, and they are commonly assigned to target promoters using computational predictions. How promoter-SE interactions change upon cell state transitions, and whether transcription factors maintain SE interactions, have not been reported. Here, we used promoter-capture Hi-C to identify promoters that interact with SEs in mouse embryonic stem cells (ESCs). We found that SEs form complex, spatial networks in which individual SEs contact multiple promoters, and a rewiring of promoter-SE interactions occurs between pluripotent states. We also show that long-range promoter-SE interactions are more prevalent in ESCs than in epiblast stem cells (EpiSCs) or Nanog-deficient ESCs. We conclude that SEs form cell-type-specific interaction networks that are partly dependent on core transcription factors, thereby providing insights into the gene regulatory organization of pluripotent cells.

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Cell reports, 22, 2211-1247, 2615-2627, 2018

PMID: 29514091


Open Access

Molecular profiling of aged neural progenitors identifies Dbx2 as a candidate regulator of age-associated neurogenic decline.
Lupo G, Nisi PS, Esteve P, Paul YL, Novo CL, Sidders B, Khan MA, Biagioni S, Liu HK, Bovolenta P, Cacci E, Rugg-Gunn PJ

Adult neurogenesis declines with aging due to the depletion and functional impairment of neural stem/progenitor cells (NSPCs). An improved understanding of the underlying mechanisms that drive age-associated neurogenic deficiency could lead to the development of strategies to alleviate cognitive impairment and facilitate neuroregeneration. An essential step towards this aim is to investigate the molecular changes that occur in NSPC aging on a genomewide scale. In this study, we compare the transcriptional, histone methylation and DNA methylation signatures of NSPCs derived from the subventricular zone (SVZ) of young adult (3 months old) and aged (18 months old) mice. Surprisingly, the transcriptional and epigenomic profiles of SVZ-derived NSPCs are largely unchanged in aged cells. Despite the global similarities, we detect robust age-dependent changes at several hundred genes and regulatory elements, thereby identifying putative regulators of neurogenic decline. Within this list, the homeobox gene Dbx2 is upregulated in vitro and in vivo, and its promoter region has altered histone and DNA methylation levels, in aged NSPCs. Using functional in vitro assays, we show that elevated Dbx2 expression in young adult NSPCs promotes age-related phenotypes, including the reduced proliferation of NSPC cultures and the altered transcript levels of age-associated regulators of NSPC proliferation and differentiation. Depleting Dbx2 in aged NSPCs caused the reverse gene expression changes. Taken together, these results provide new insights into the molecular programmes that are affected during mouse NSPC aging, and uncover a new functional role for Dbx2 in promoting age-related neurogenic decline.

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Aging cell, , 1474-9726, , 2018

PMID: 29504228


Open Access

Gene expression hallmarks of cellular ageing.
Frenk S, Houseley J

Ageing leads to dramatic changes in the physiology of many different tissues resulting in a spectrum of pathology. Nonetheless, many lines of evidence suggest that ageing is driven by highly conserved cell intrinsic processes, and a set of unifying hallmarks of ageing has been defined. Here, we survey reports of age-linked changes in basal gene expression across eukaryotes from yeast to human and identify six gene expression hallmarks of cellular ageing: downregulation of genes encoding mitochondrial proteins; downregulation of the protein synthesis machinery; dysregulation of immune system genes; reduced growth factor signalling; constitutive responses to stress and DNA damage; dysregulation of gene expression and mRNA processing. These encompass widely reported features of ageing such as increased senescence and inflammation, reduced electron transport chain activity and reduced ribosome synthesis, but also reveal a surprising lack of gene expression responses to known age-linked cellular stresses. We discuss how the existence of conserved transcriptomic hallmarks relates to genome-wide epigenetic differences underlying ageing clocks, and how the changing transcriptome results in proteomic alterations where data is available and to variations in cell physiology characteristic of ageing. Identification of gene expression events that occur during ageing across distant organisms should be informative as to conserved underlying mechanisms of ageing, and provide additional biomarkers to assess the effects of diet and other environmental factors on the rate of ageing.

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Biogerontology, , 1573-6768, , 2018

PMID: 29492790


Open Access

Neuronal Cell Death.
Fricker M, Tolkovsky AM, Borutaite V, Coleman M, Brown GC

Neuronal cell death occurs extensively during development and pathology, where it is especially important because of the limited capacity of adult neurons to proliferate or be replaced. The concept of cell death used to be simple as there were just two or three types, so we just had to work out which type was involved in our particular pathology and then block it. However, we now know that there are at least a dozen ways for neurons to die, that blocking a particular mechanism of cell death may not prevent the cell from dying, and that non-neuronal cells also contribute to neuronal death. We review here the mechanisms of neuronal death by intrinsic and extrinsic apoptosis, oncosis, necroptosis, parthanatos, ferroptosis, sarmoptosis, autophagic cell death, autosis, autolysis, paraptosis, pyroptosis, phagoptosis, and mitochondrial permeability transition. We next explore the mechanisms of neuronal death during development, and those induced by axotomy, aberrant cell-cycle reentry, glutamate (excitoxicity and oxytosis), loss of connected neurons, aggregated proteins and the unfolded protein response, oxidants, inflammation, and microglia. We then reassess which forms of cell death occur in stroke and Alzheimer's disease, two of the most important pathologies involving neuronal cell death. We also discuss why it has been so difficult to pinpoint the type of neuronal death involved, if and why the mechanism of neuronal death matters, the molecular overlap and interplay between death subroutines, and the therapeutic implications of these multiple overlapping forms of neuronal death.

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Physiological reviews, 98, 1522-1210, 813-880, 2018

PMID: 29488822


scNMT-seq enables joint profiling of chromatin accessibility DNA methylation and transcription in single cells.
Clark SJ, Argelaguet R, Kapourani CA, Stubbs TM, Lee HJ, Alda-Catalinas C, Krueger F, Sanguinetti G, Kelsey G, Marioni JC, Stegle O, Reik W

Parallel single-cell sequencing protocols represent powerful methods for investigating regulatory relationships, including epigenome-transcriptome interactions. Here, we report a single-cell method for parallel chromatin accessibility, DNA methylation and transcriptome profiling. scNMT-seq (single-cell nucleosome, methylation and transcription sequencing) uses a GpC methyltransferase to label open chromatin followed by bisulfite and RNA sequencing. We validate scNMT-seq by applying it to differentiating mouse embryonic stem cells, finding links between all three molecular layers and revealing dynamic coupling between epigenomic layers during differentiation.

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Nature communications, 9, 2041-1723, 781, 2018

PMID: 29472610


ERK1/2 inhibitors: New weapons to inhibit the RAS-regulated RAF-MEK1/2-ERK1/2 pathway.
Kidger AM, Sipthorp J, Cook SJ

The RAS-regulated RAF-MEK1/2-ERK1/2 signalling pathway is de-regulated in a variety of cancers due to mutations in receptor tyrosine kinases (RTKs), negative regulators of RAS (such as NF1) and core pathway components themselves (RAS, BRAF, CRAF, MEK1 or MEK2). This has driven the development of a variety of pharmaceutical agents to inhibit RAF-MEK1/2-ERK1/2 signalling in cancer and both RAF and MEK inhibitors are now approved and used in the clinic. There is now much interest in targeting at the level of ERK1/2 for a variety of reasons. First, since the pathway is linear from RAF-to-MEK-to-ERK then ERK1/2 are validated as targets per se. Second, innate resistance to RAF or MEK inhibitors involves relief of negative feedback and pathway re-activation with all signalling going through ERK1/2, validating the use of ERK inhibitors with RAF or MEK inhibitors as an up-front combination. Third, long-term acquired resistance to RAF or MEK inhibitors involves a variety of mechanisms (KRAS or BRAF amplification, MEK mutation, etc.) which re-instate ERK activity, validating the use of ERK inhibitors to forestall acquired resistance to RAF or MEK inhibitors. The first potent highly selective ERK1/2 inhibitors have now been developed and are entering clinical trials. They have one of three discrete mechanisms of action - catalytic, "dual mechanism" or covalent - which could have profound consequences for how cells respond and adapt. In this review we describe the validation of ERK1/2 as anti-cancer drug targets, consider the mechanism of action of new ERK1/2 inhibitors and how this may impact on their efficacy, anticipate factors that will determine how tumour cells respond and adapt to ERK1/2 inhibitors and consider ERK1/2 inhibitor drug combinations.

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Pharmacology & therapeutics, , 1879-016X, , 2018

PMID: 29454854


Quick tips for creating effective and impactful biological pathways using the Systems Biology Graphical Notation.
Touré V, Le Novère N, Waltemath D, Wolkenhauer O

PLoS computational biology, 14, 1553-7358, e1005740, 2018

PMID: 29447151


Open Access

Thrombopoietin signaling to chromatin elicits rapid and pervasive epigenome remodeling within poised chromatin architectures.
Comoglio F, Park HJ, Schoenfelder S, Barozzi I, Bode D, Fraser P, Green AR

Thrombopoietin (TPO) is a critical cytokine regulating hematopoietic stem cell maintenance and differentiation into the megakaryocytic lineage. However, the transcriptional and chromatin dynamics elicited by TPO signaling are poorly understood. Here, we study the immediate early transcriptional and cis-regulatory responses to TPO in hematopoietic stem/progenitor cells (HSPCs) and use this paradigm of cytokine signaling to chromatin to dissect the relation between cis- regulatory activity and chromatin architecture. We show that TPO profoundly alters the transcriptome of HSPCs, with key hematopoietic regulators being transcriptionally repressed within 30 minutes of TPO. By examining cis-regulatory dynamics and chromatin architectures, we demonstrate that these changes are accompanied by rapid and extensive epigenome remodeling of cis-regulatory landscapes that is spatially coordinated within topologically associating domains (TADs). Moreover, TPO-responsive enhancers are spatially clustered and engage in preferential homotypic intra- and inter-TAD interactions that are largely refractory to TPO signaling. By further examining the link between cis-regulatory dynamics and chromatin looping, we show that rapid modulation of cis-regulatory activity is largely independent of chromatin looping dynamics. Finally, we show that, although activated and repressed cis-regulatory elements share remarkably similar DNA sequence compositions, transcription factor binding patterns accurately predict rapid cis-regulatory responses to TPO.

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Genome research, , 1549-5469, , 2018

PMID: 29429976


Open Access

Epigenetic control of CD8+ T cell differentiation.
Henning AN, Roychoudhuri R, Restifo NP

Upon stimulation, small numbers of naive CD8+ T cells proliferate and differentiate into a variety of memory and effector cell types. CD8+ T cells can persist for years and kill tumour cells and virally infected cells. The functional and phenotypic changes that occur during CD8+ T cell differentiation are well characterized, but the epigenetic states that underlie these changes are incompletely understood. Here, we review the epigenetic processes that direct CD8+ T cell differentiation and function. We focus on epigenetic modification of DNA and associated histones at genes and their regulatory elements. We also describe structural changes in chromatin organization that affect gene expression. Finally, we examine the translational potential of epigenetic interventions to improve CD8+ T cell function in individuals with chronic infections and cancer.

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Nature reviews. Immunology, , 1474-1741, , 2018

PMID: 29379213


The RNA-binding protein PTBP1 is necessary for B cell selection in germinal centers.
Monzón-Casanova E, Screen M, Díaz-Muñoz MD, Coulson RMR, Bell SE, Lamers G, Solimena M, Smith CWJ, Turner M

Antibody affinity maturation occurs in germinal centers (GCs), where B cells cycle between the light zone (LZ) and the dark zone. In the LZ, GC B cells bearing immunoglobulins with the highest affinity for antigen receive positive selection signals from helper T cells, which promotes their rapid proliferation. Here we found that the RNA-binding protein PTBP1 was needed for the progression of GC B cells through late S phase of the cell cycle and for affinity maturation. PTBP1 was required for proper expression of the c-MYC-dependent gene program induced in GC B cells receiving T cell help and directly regulated the alternative splicing and abundance of transcripts that are increased during positive selection to promote proliferation.

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Nature immunology, , 1529-2916, , 2018

PMID: 29358707


PI3K induces B-cell development and regulates B cell identity.
Abdelrasoul H, Werner M, Setz CS, Okkenhaug K, Jumaa H

Phosphoinositide-3 kinase (PI3K) signaling is important for the survival of numerous cell types and class IA of PI3K is specifically required for the development of B cells but not for T cell development. Here, we show that class IA PI3K-mediated signals induce the expression of the transcription factor Pax5, which plays a central role in B cell commitment and differentiation by activating the expression of central B cell-specific signaling proteins such as SLP-65 and CD19. Defective class IA PI3K function leads to reduction in Pax5 expression and prevents B cell development beyond the stage expressing the precursor B cell receptor (pre-BCR). Investigating the mechanism of PI3K-induced Pax5 expression revealed that it involves a network of transcription factors including FoxO1 and Irf4 that directly binds to the Pax5 gene. Together, our results suggest that PI3K signaling links survival and differentiation of developing B cells with B cell identity and that decreased PI3K activity in pre-B cells results in reduced Pax5 expression and lineage plasticity.

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Scientific reports, 8, 2045-2322, 1327, 2018

PMID: 29358580


Open Access

RNA-binding proteins control gene expression and cell fate in the immune system.
Turner M, Díaz-Muñoz MD

RNA-binding proteins (RBPs) are essential for the development and function of the immune system. They interact dynamically with RNA to control its biogenesis and turnover by transcription-dependent and transcription-independent mechanisms. In this Review, we discuss the molecular mechanisms by which RBPs allow gene expression changes to occur at different speeds and to varying degrees, and which RBPs regulate the diversity of the transcriptome and proteome. These proteins are nodes for integration of transcriptional and signaling networks and are intimately linked to intermediary metabolism. They are essential components of regulatory feedback mechanisms that maintain immune tolerance and limit inflammation. The role of RBPs in malignancy and autoimmunity has led to their emergence as targets for the development of new therapeutic modalities.

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Nature immunology, , 1529-2916, , 2018

PMID: 29348497


MLL2 conveys transcription-independent H3K4 trimethylation in oocytes.
Hanna CW, Taudt A, Huang J, Gahurova L, Kranz A, Andrews S, Dean W, Stewart AF, Colomé-Tatché M, Kelsey G

Histone 3 K4 trimethylation (depositing H3K4me3 marks) is typically associated with active promoters yet paradoxically occurs at untranscribed domains. Research to delineate the mechanisms of targeting H3K4 methyltransferases is ongoing. The oocyte provides an attractive system to investigate these mechanisms, because extensive H3K4me3 acquisition occurs in nondividing cells. We developed low-input chromatin immunoprecipitation to interrogate H3K4me3, H3K27ac and H3K27me3 marks throughout oogenesis. In nongrowing oocytes, H3K4me3 was restricted to active promoters, but as oogenesis progressed, H3K4me3 accumulated in a transcription-independent manner and was targeted to intergenic regions, putative enhancers and silent H3K27me3-marked promoters. Ablation of the H3K4 methyltransferase gene Mll2 resulted in loss of transcription-independent H3K4 trimethylation but had limited effects on transcription-coupled H3K4 trimethylation or gene expression. Deletion of Dnmt3a and Dnmt3b showed that DNA methylation protects regions from acquiring H3K4me3. Our findings reveal two independent mechanisms of targeting H3K4me3 to genomic elements, with MLL2 recruited to unmethylated CpG-rich regions independently of transcription.

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Nature structural & molecular biology, 25, 1545-9985, 73-82, 2018

PMID: 29323282


Microbiota derived short chain fatty acids promote histone crotonylation in the colon through histone deacetylases.
Fellows R, Denizot J, Stellato C, Cuomo A, Jain P, Stoyanova E, Balázsi S, Hajnády Z, Liebert A, Kazakevych J, Blackburn H, Corrêa RO, Fachi JL, Sato FT, Ribeiro WR, Ferreira CM, Perée H, Spagnuolo M, Mattiuz R, Matolcsi C, Guedes J, Clark J, Veldhoen M, Bonaldi T, Vinolo MAR, Varga-Weisz P

The recently discovered histone post-translational modification crotonylation connects cellular metabolism to gene regulation. Its regulation and tissue-specific functions are poorly understood. We characterize histone crotonylation in intestinal epithelia and find that histone H3 crotonylation at lysine 18 is a surprisingly abundant modification in the small intestine crypt and colon, and is linked to gene regulation. We show that this modification is highly dynamic and regulated during the cell cycle. We identify class I histone deacetylases, HDAC1, HDAC2, and HDAC3, as major executors of histone decrotonylation. We show that known HDAC inhibitors, including the gut microbiota-derived butyrate, affect histone decrotonylation. Consistent with this, we find that depletion of the gut microbiota leads to a global change in histone crotonylation in the colon. Our results suggest that histone crotonylation connects chromatin to the gut microbiota, at least in part, via short-chain fatty acids and HDACs.

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Nature communications, 9, 2041-1723, 105, 2018

PMID: 29317660


Open Access

The WD40 domain of ATG16L1 is required for its non-canonical role in lipidation of LC3 at single membranes.
Fletcher K, Ulferts R, Jacquin E, Veith T, Gammoh N, Arasteh JM, Mayer U, Carding SR, Wileman T, Beale R, Florey O

A hallmark of macroautophagy is the covalent lipidation of LC3 and insertion into the double-membrane phagophore, which is driven by the ATG16L1/ATG5-ATG12 complex. In contrast, non-canonical autophagy is a pathway through which LC3 is lipidated and inserted into single membranes, particularly endolysosomal vacuoles during cell engulfment events such as LC3-associated phagocytosis. Factors controlling the targeting of ATG16L1 to phagophores are dispensable for non-canonical autophagy, for which the mechanism of ATG16L1 recruitment is unknown. Here we show that the WD repeat-containing C-terminal domain (WD40 CTD) of ATG16L1 is essential for LC3 recruitment to endolysosomal membranes during non-canonical autophagy, but dispensable for canonical autophagy. Using this strategy to inhibit non-canonical autophagy specifically, we show a reduction of MHC class II antigen presentation in dendritic cells from mice lacking the WD40 CTD Further, we demonstrate activation of non-canonical autophagy dependent on the WD40 CTD during influenza A virus infection. This suggests dependence on WD40 CTD distinguishes between macroautophagy and non-canonical use of autophagy machinery.

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The EMBO journal, , 1460-2075, , 2018

PMID: 29317426


Open Access

De-RSKing ERK - regulation of ERK1/2-RSK dissociation by phosphorylation within a disordered motif.
Kidger AM, Cook SJ

The protein kinases ERK1/2 and RSK associate in unstimulated cells but must separate to target other substrates. In this issue, Gógl et al. show that phosphorylation of RSK by active ERK1/2 culminates in the formation of an intramolecular charge clamp between Lys729 and the phosphate group on Ser732. This promotes the dissociation of ERK1/2 from RSK allowing them to engage with other targets.

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The FEBS journal, 285, 1742-4658, 42-45, 2018

PMID: 29314599


Calcium phosphate particles stimulate interleukin-1β release from human vascular smooth muscle cells: A role for spleen tyrosine kinase and exosome release.
Dautova Y, Kapustin AN, Pappert K, Epple M, Okkenhaug H, Cook SJ, Shanahan CM, Bootman MD, Proudfoot D

Calcium phosphate (CaP) particle deposits are found in several inflammatory diseases including atherosclerosis and osteoarthritis. CaP, and other forms of crystals and particles, can promote inflammasome formation in macrophages leading to caspase-1 activation and secretion of mature interleukin-1β (IL-1β). Given the close association of small CaP particles with vascular smooth muscle cells (VSMCs) in atherosclerotic fibrous caps, we aimed to determine if CaP particles affected pro-inflammatory signalling in human VSMCs.

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Journal of molecular and cellular cardiology, , 1095-8584, , 2017

PMID: 29274344


In-depth PtdIns(3,4,5)P3 signalosome analysis identifies DAPP1 as a negative regulator of GPVI-driven platelet function.
Durrant TN, Hutchinson JL, Heesom KJ, Anderson KE, Stephens LR, Hawkins PT, Marshall AJ, Moore SF, Hers I

The class I phosphoinositide 3-kinase (PI3K) isoforms play important roles in platelet priming, activation, and stable thrombus formation. Class I PI3Ks predominantly regulate cell function through their catalytic product, the signaling phospholipid phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3], which coordinates the localization and/or activity of a diverse range of binding proteins. Notably, the complete repertoire of these class I PI3K effectors in platelets remains unknown, limiting mechanistic understanding of class I PI3K-mediated control of platelet function. We measured robust agonist-driven PtdIns (3,4,5)P3 generation in human platelets by lipidomic mass spectrometry (MS), and then used affinity-capture coupled to high-resolution proteomic MS to identify the targets of PtdIns (3,4,5)P3 in these cells. We reveal for the first time a diverse platelet PtdIns(3,4,5)P3 interactome, including kinases, signaling adaptors, and regulators of small GTPases, many of which are previously uncharacterized in this cell type. Of these, we show dual adaptor for phosphotyrosine and 3-phosphoinositides (DAPP1) to be regulated by Src-family kinases and PI3K, while platelets from DAPP1-deficient mice display enhanced thrombus formation on collagen in vitro. This was associated with enhanced platelet α/δ granule secretion and αIIbβ3 integrin activation downstream of the collagen receptor glycoprotein VI. Thus, we present the first comprehensive analysis of the PtdIns(3,4,5)P3 signalosome of human platelets and identify DAPP1 as a novel negative regulator of platelet function. This work provides important new insights into how class I PI3Ks shape platelet function.

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Blood advances, 1, 2473-9529, 918-932, 2017

PMID: 29242851


Open Access

Clearance of senescent decidual cells by uterine natural killer cells in cycling human endometrium.
Brighton PJ, Maruyama Y, Fishwick K, Vrljicak P, Tewary S, Fujihara R, Muter J, Lucas ES, Yamada T, Woods L, Lucciola R, Hou Lee Y, Takeda S, Ott S, Hemberger M, Quenby S, Brosens JJ

In cycling human endometrium, menstruation is followed by rapid estrogen-dependent growth. Upon ovulation, progesterone and rising cellular cAMP levels activate the transcription factor Forkhead box O1 (FOXO1) in endometrial stromal cells (EnSCs), leading to cell cycle exit and differentiation into decidual cells that control embryo implantation. Here we show that FOXO1 also causes acute senescence of a subpopulation of decidualizing EnSCs in an IL-8 dependent manner. Selective depletion or enrichment of this subpopulation revealed that decidual senescence drives the transient inflammatory response associated with endometrial receptivity. Further, senescent cells prevent differentiation of endometrial mesenchymal stem cells in decidualizing cultures. As the cycle progresses, IL-15 activated uterine natural killer (uNK) cells selectively target and clear senescent decidual cells through granule exocytosis. Our findings reveal that acute decidual senescence governs endometrial rejuvenation and remodeling at embryo implantation, and suggest a critical role for uNK cells in maintaining homeostasis in cycling endometrium.

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eLife, 6, 2050-084X, , 2017

PMID: 29227245


Open Access

Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins.
Wutz G, Várnai C, Nagasaka K, Cisneros DA, Stocsits RR, Tang W, Schoenfelder S, Jessberger G, Muhar M, Hossain MJ, Walther N, Koch B, Kueblbeck M, Ellenberg J, Zuber J, Fraser P, Peters JM

Mammalian genomes are spatially organized into compartments, topologically associating domains (TADs), and loops to facilitate gene regulation and other chromosomal functions. How compartments, TADs, and loops are generated is unknown. It has been proposed that cohesin forms TADs and loops by extruding chromatin loops until it encounters CTCF, but direct evidence for this hypothesis is missing. Here, we show that cohesin suppresses compartments but is required for TADs and loops, that CTCF defines their boundaries, and that the cohesin unloading factor WAPL and its PDS5 binding partners control the length of loops. In the absence of WAPL and PDS5 proteins, cohesin forms extended loops, presumably by passing CTCF sites, accumulates in axial chromosomal positions (vermicelli), and condenses chromosomes. Unexpectedly, PDS5 proteins are also required for boundary function. These results show that cohesin has an essential genome-wide function in mediating long-range chromatin interactions and support the hypothesis that cohesin creates these by loop extrusion, until it is delayed by CTCF in a manner dependent on PDS5 proteins, or until it is released from DNA by WAPL.

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The EMBO journal, , 1460-2075, , 2017

PMID: 29217591


Science Forum: The Human Cell Atlas.
Regev A, Teichmann SA, Lander ES, Amit I, Benoist C, Birney E, Bodenmiller B, Campbell PJ, Carninci P, Clatworthy M, Clevers H, Deplancke B, Dunham I, Eberwine J, Eils R, Enard W, Farmer A, Fugger L, Göttgens B, Hacohen N, Haniffa M, Hemberg M, Kim SK, Klenerman P, Kriegstein A, Lein E, Linnarsson S, Lundberg E, Lundeberg J, Majumder P, Marioni JC, Merad M, Mhlanga M, Nawijn M, Netea M, Nolan G, Pe'er D, Phillipakis A, Ponting CP, Quake SR, Reik W, Rozenblatt-Rosen O, Sanes JR, Satija R, Schumacher TN, Shalek AK, Shapiro E, Sharma P, Shin JW, Stegle O, Stratton MR, Stubbington MJT, Theis FJ, Uhlen M, van Oudenaarden A, Wagner A, Watt FM, Weissman JS, Wold BJ, Xavier RJ, Yosef N,

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community.

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eLife, 6, 2050-084X, , 2017

PMID: 29206104


Open Access

Local Chromatin Features Including PU.1 and IKAROS Binding and H3K4 Methylation Shape the Repertoire of Immunoglobulin Kappa Genes Chosen for V(D)J Recombination.
Matheson LS, Bolland DJ, Chovanec P, Krueger F, Andrews S, Koohy H, Corcoran AE

V(D)J recombination is essential for the generation of diverse antigen receptor (AgR) repertoires. In B cells, immunoglobulin kappa (Igκ) light chain recombination follows immunoglobulin heavy chain (Igh) recombination. We recently developed the DNA-based VDJ-seq assay for the unbiased quantitation of Igh VH and DH repertoires. Integration of VDJ-seq data with genome-wide datasets revealed that two chromatin states at the recombination signal sequence (RSS) of VH genes are highly predictive of recombination in mouse pro-B cells. It is unknown whether local chromatin states contribute to Vκ gene choice during Igκ recombination. Here we adapt VDJ-seq to profile the Igκ VκJκ repertoire and present a comprehensive readout in mouse pre-B cells, revealing highly variable Vκ gene usage. Integration with genome-wide datasets for histone modifications, DNase hypersensitivity, transcription factor binding and germline transcription identified PU.1 binding at the RSS, which was unimportant for Igh, as highly predictive of whether a Vκ gene will recombine or not, suggesting that it plays a binary, all-or-nothing role, priming genes for recombination. Thereafter, the frequency with which these genes recombine was shaped both by the presence and level of enrichment of several other chromatin features, including H3K4 methylation and IKAROS binding. Moreover, in contrast to the Igh locus, the chromatin landscape of the promoter, as well as of the RSS, contributes to Vκ gene recombination. Thus, multiple facets of local chromatin features explain much of the variation in Vκ gene usage. Together, these findings reveal shared and divergent roles for epigenetic features and transcription factors in AgR V(D)J recombination and provide avenues for further investigation of chromatin signatures that may underpin V(D)J-mediated chromosomal translocations.

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Frontiers in immunology, 8, 1664-3224, 1550, 2017

PMID: 29204143


Open Access

Regulatory T Cell Migration Is Dependent on Glucokinase-Mediated Glycolysis.
Kishore M, Cheung KCP, Fu H, Bonacina F, Wang G, Coe D, Ward EJ, Colamatteo A, Jangani M, Baragetti A, Matarese G, Smith DM, Haas R, Mauro C, Wraith DC, Okkenhaug K, Catapano AL, De Rosa V, Norata GD, Marelli-Berg FM

Migration of activated regulatory T (Treg) cells to inflamed tissue is crucial for their immune-modulatory function. While metabolic reprogramming during Treg cell differentiation has been extensively studied, the bioenergetics of Treg cell trafficking remains undefined. We have investigated the metabolic demands of migrating Treg cells in vitro and in vivo. We show that glycolysis was instrumental for their migration and was initiated by pro-migratory stimuli via a PI3K-mTORC2-mediated pathway culminating in induction of the enzyme glucokinase (GCK). Subsequently, GCK promoted cytoskeletal rearrangements by associating with actin. Treg cells lacking this pathway were functionally suppressive but failed to migrate to skin allografts and inhibit rejection. Similarly, human carriers of a loss-of-function GCK regulatory protein gene-leading to increased GCK activity-had reduced numbers of circulating Treg cells. These cells displayed enhanced migratory activity but similar suppressive function, while conventional T cells were unaffected. Thus, GCK-dependent glycolysis regulates Treg cell migration.

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Immunity, 47, 1097-4180, 875-889.e10, 2017

PMID: 29166588


Open Access

Coupling shRNA screens with single-cell RNA-seq identifies a dual role for mTOR in reprogramming-induced senescence.
Aarts M, Georgilis A, Beniazza M, Beolchi P, Banito A, Carroll T, Kulisic M, Kaemena DF, Dharmalingam G, Martin N, Reik W, Zuber J, Kaji K, Chandra T, Gil J

Expression of the transcription factors OCT4, SOX2, KLF4, and cMYC (OSKM) reprograms somatic cells into induced pluripotent stem cells (iPSCs). Reprogramming is a slow and inefficient process, suggesting the presence of safeguarding mechanisms that counteract cell fate conversion. One such mechanism is senescence. To identify modulators of reprogramming-induced senescence, we performed a genome-wide shRNA screen in primary human fibroblasts expressing OSKM. In the screen, we identified novel mediators of OSKM-induced senescence and validated previously implicated genes such as CDKN1A We developed an innovative approach that integrates single-cell RNA sequencing (scRNA-seq) with the shRNA screen to investigate the mechanism of action of the identified candidates. Our data unveiled regulation of senescence as a novel way by which mechanistic target of rapamycin (mTOR) influences reprogramming. On one hand, mTOR inhibition blunts the induction of cyclin-dependent kinase (CDK) inhibitors (CDKIs), including p16(INK4a), p21(CIP1), and p15(INK4b), preventing OSKM-induced senescence. On the other hand, inhibition of mTOR blunts the senescence-associated secretory phenotype (SASP), which itself favors reprogramming. These contrasting actions contribute to explain the complex effect that mTOR has on reprogramming. Overall, our study highlights the advantage of combining functional screens with scRNA-seq to accelerate the discovery of pathways controlling complex phenotypes.

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Genes & development, , 1549-5477, , 2017

PMID: 29138277


Open Access

BioModels: expanding horizons to include more modelling approaches and formats.
Glont M, Nguyen TVN, Graesslin M, Hälke R, Ali R, Schramm J, Wimalaratne SM, Kothamachu VB, Rodriguez N, Swat MJ, Eils J, Eils R, Laibe C, Malik-Sheriff RS, Chelliah V, Le Novère N, Hermjakob H

BioModels serves as a central repository of mathematical models representing biological processes. It offers a platform to make mathematical models easily shareable across the systems modelling community, thereby supporting model reuse. To facilitate hosting a broader range of model formats derived from diverse modelling approaches and tools, a new infrastructure for BioModels has been developed that is available at http://www.ebi.ac.uk/biomodels. This new system allows submitting and sharing of a wide range of models with improved support for formats other than SBML. It also offers a version-control backed environment in which authors and curators can work collaboratively to curate models. This article summarises the features available in the current system and discusses the potential benefit they offer to the users over the previous system. In summary, the new portal broadens the scope of models accepted in BioModels and supports collaborative model curation which is crucial for model reproducibility and sharing.

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Nucleic acids research, , 1362-4962, , 2017

PMID: 29106614