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1.  Interplay of dFOXO and Two ETS-Family Transcription Factors Determines Lifespan in Drosophila melanogaster 
PLoS Genetics  2014;10(9):e1004619.
Forkhead box O (FoxO) transcription factors (TFs) are key drivers of complex transcriptional programmes that determine animal lifespan. FoxOs regulate a number of other TFs, but how these TFs in turn might mediate the anti-ageing programmes orchestrated by FoxOs in vivo is unclear. Here, we identify an E-twenty six (ETS)-family transcriptional repressor, Anterior open (Aop), as regulated by the single Drosophila melanogaster FoxO (dFOXO) in the adult gut. AOP, the functional orthologue of the human Etv6/Tel protein, binds numerous genomic sites also occupied by dFOXO and counteracts the activity of an ETS activator, Pointed (Pnt), to prevent the lifespan-shortening effects of co-activation of dFOXO and PNT. This detrimental synergistic effect of dFOXO and PNT appears to stem from a mis-regulation of lipid metabolism. At the same time, AOP activity in another fly organ, the fat body, has further beneficial roles, regulating genes in common with dfoxo, such as the secreted, non-sensory, odorant binding protein (Obp99b), and robustly extending lifespan. Our study reveals a complex interplay between evolutionarily conserved ETS factors and dFOXO, the functional significance of which may extend well beyond animal lifespan.
Author Summary
Despite the apparent complexity of ageing, animal lifespan can be extended. Activity of Forkhead Box O (FoxO) transcription factors can prolong survival of organisms ranging from the budding yeast to the fruit fly, and FoxO gene variants are linked to human longevity. FoxOs extend lifespan by driving complex, widespread changes in gene expression. Their primary targets include a second tier of transcriptional regulators, but it remains unclear how these secondary regulators are involved in the anti-ageing programmes orchestrated by FoxOs in vivo. To elucidate the role of this second tier, we identify a transcription factor called Anterior open (Aop) as directly regulated by the single Drosophila melanogaster FoxO protein (dFOXO) in the adult fly gut. Under certain circumstances, such as co-activation of the Pointed (PNT) transcription factor, dFOXO can be detrimental to lifespan. The role of Aop is to protect from this negative synergistic effect. Additionally, activation of AOP in the fly adipose tissue can robustly extend lifespan. Our study reveals a complex interplay between two evolutionarily conserved transcriptional regulators and dFOXO in lifespan. This significance of this interplay may extend to other physiological processes where these transcription factors play important roles.
PMCID: PMC4169242  PMID: 25232726
2.  Design, purification and characterization of a soluble variant of the integral membrane protein MotB for structural studies 
The bacterial flagellar motor is an intricate nanomachine powered by a transmembrane electrochemical gradient. Rotation is driven by the cumulative action of several peptidoglycan-anchored stator complexes on the rotor. In proton-motive force-driven motors, the stator complex is composed of a motility protein B (MotB) dimer surrounded by four copies of MotA, where both MotA and MotB are integral membrane proteins. The lack of full-length MotA and MotB structures hinders understanding of the mechanism of torque generation. Given the low levels of expression and low stability of detergent-solubilized MotB, a soluble chimaeric variant was engineered, where the two transmembrane helices of the MotB dimer were replaced by a leucine zipper. The biochemical and biophysical analysis of the resultant protein showed that it was properly folded, stable, behaved as a monodisperse dimer at low pH, had molecular dimensions close to those expected for native MotB and yielded reproducible crystals. The chimaeric protein is, therefore, a good candidate for structural studies. This ‘solubilization by design’ approach may be generally applicable to the production of soluble forms of other dimeric, trimeric and tetrameric single-span membrane proteins for functional and structural studies.
PMCID: PMC3565690  PMID: 23193105
flagellar motor; membrane proteins; motility protein B; protein engineering
3.  Recognition of H2-M3 by Ly49A regulates natural killer cell licensing and activation 
Nature immunology  2012;13(12):1171-1177.
Natural killer cell development and function is regulated by the interaction of inhibitory Ly49 receptors with distinct peptide-laden Major Histocompatibility Complex allotypes (pMHC-I), although whether Ly49 could bind to other MHC-I like molecules was unclear. We show the prototypic inhibitory receptor Ly49A binds the highly conserved non-classical class I molecule, pH2-M3 with similar affinity to that with H-2Dd. Ly49A specific recognition of H2-M3 regulates NK cell licensing and mediates missing-self recognition of H2-M3 deficient-bone marrow. Host-peptide H2-M3 was required for optimal NK cell activity against experimental metastases and carcinogenesis. Accordingly, non-classical MHC-I molecules can act as cognate ligands for Ly49 molecules and provide insight into the various mechanisms that lead to NK cell tolerance.
PMCID: PMC3913127  PMID: 23142773
4.  The RNA-binding protein hnRNPLL induces a T cell alternative splicing program delineated by differential intron retention in polyadenylated RNA 
Genome Biology  2014;15(1):R26.
Retention of a subset of introns in spliced polyadenylated mRNA is emerging as a frequent, unexplained finding from RNA deep sequencing in mammalian cells.
Here we analyze intron retention in T lymphocytes by deep sequencing polyadenylated RNA. We show a developmentally regulated RNA-binding protein, hnRNPLL, induces retention of specific introns by sequencing RNA from T cells with an inactivating Hnrpll mutation and from B lymphocytes that physiologically downregulate Hnrpll during their differentiation. In Ptprc mRNA encoding the tyrosine phosphatase CD45, hnRNPLL induces selective retention of introns flanking exons 4 to 6; these correspond to the cassette exons containing hnRNPLL binding sites that are skipped in cells with normal, but not mutant or low, hnRNPLL. We identify similar patterns of hnRNPLL-induced differential intron retention flanking alternative exons in 14 other genes, representing novel elements of the hnRNPLL-induced splicing program in T cells. Retroviral expression of a normally spliced cDNA for one of these targets, Senp2, partially corrects the survival defect of Hnrpll-mutant T cells. We find that integrating a number of computational methods to detect genes with differentially retained introns provides a strategy to enrich for alternatively spliced exons in mammalian RNA-seq data, when complemented by RNA-seq analysis of purified cells with experimentally perturbed RNA-binding proteins.
Our findings demonstrate that intron retention in mRNA is induced by specific RNA-binding proteins and suggest a biological significance for this process in marking exons that are poised for alternative splicing.
PMCID: PMC4053824  PMID: 24476532
5.  Rasgrp1 mutation increases naïve T-cell CD44 expression and drives mTOR-dependent accumulation of Helios+ T cells and autoantibodies 
eLife  2013;2:e01020.
Missense variants are a major source of human genetic variation. Here we analyze a new mouse missense variant, Rasgrp1Anaef, with an ENU-mutated EF hand in the Rasgrp1 Ras guanine nucleotide exchange factor. Rasgrp1Anaef mice exhibit anti-nuclear autoantibodies and gradually accumulate a CD44hi Helios+ PD-1+ CD4+ T cell population that is dependent on B cells. Despite reduced Rasgrp1-Ras-ERK activation in vitro, thymocyte selection in Rasgrp1Anaef is mostly normal in vivo, although CD44 is overexpressed on naïve thymocytes and T cells in a T-cell-autonomous manner. We identify CD44 expression as a sensitive reporter of tonic mTOR-S6 kinase signaling through a novel mouse strain, chino, with a reduction-of-function mutation in Mtor. Elevated tonic mTOR-S6 signaling occurs in Rasgrp1Anaef naïve CD4+ T cells. CD44 expression, CD4+ T cell subset ratios and serum autoantibodies all returned to normal in Rasgrp1AnaefMtorchino double-mutant mice, demonstrating that increased mTOR activity is essential for the Rasgrp1Anaef T cell dysregulation.
eLife digest
Our DNA contains more than three billion nucleotides. Each of these nucleotides can be an A, C, G or T, and groups of three neighboring nucleotides within our DNA are used to represent the 20 amino acids that are used to make proteins. This means that changing just one nucleotide can cause one amino acid to be replaced by a different amino acid in the protein encoded by that stretch of DNA: AAA and AAG code for the amino acid lysine, for example, but AAC and AAT code for asparagine. Known as missense gene variants, these changes can also increase or decrease the expression of the gene.
Every person has thousands of missense gene variants, including about 12,000 inherited from their parents. Sometimes these variants have no consequence, but they can be harmful if replacing the correct amino acid with a different amino acid prevents the protein from performing an important task. In particular, missense gene variants in genes that encode immune system proteins are likely to play a role in diseases of the immune system. For example, variants near a gene called Rasgrp1 have been linked to two autoimmune diseases – type 1 diabetes and Graves’ disease—in which the immune system mistakenly attacks the body’s own cells and tissues.
Now Daley et al. have shed new light on the mechanism by which a missense gene variant in Rasgrp1 can cause autoimmune diseases. Mice with this mutation show signs of autoimmune disease, but their T cells—white blood cells that have a central role in the immune system – develop normally despite this mutation. Instead, Daley et al. found that a specific type of T cell, called T helper cells, accumulated in large numbers in the mutant mice and stimulated cells of a third type—immune cells called B cells—to produce autoantibodies. The production of autoantibodies is a common feature of autoimmune diseases.
Daley et al. traced the origins of the T helper cells to excessive activity on a signaling pathway that involves a protein called mTOR, and went on to show that treatment with the drug rapamycin counteracted the accumulation of the T helper cells and reduced the level of autoimmune activity. In addition to exemplifying how changing just one amino acid change can have a profound effect, the work of Daley et al. is an attractive model for exploring how missense gene variants in people can contribute to autoimmune diseases.
PMCID: PMC3858598  PMID: 24336796
autoimmunity; signaling; RasGRP1; mTOR; T lymphoctes; ENU mutant; Mouse
7.  B cell survival, surface BCR and BAFFR expression, CD74 metabolism, and CD8− dendritic cells require the intramembrane endopeptidase SPPL2A 
Mice lacking activity of the intramembrane protease SPPL2A exhibit humoral immunodeficiency and lack mature B cell subsets.
Druggable proteins required for B lymphocyte survival and immune responses are an emerging source of new treatments for autoimmunity and lymphoid malignancy. In this study, we show that mice with an inactivating mutation in the intramembrane protease signal peptide peptidase–like 2A (SPPL2A) unexpectedly exhibit profound humoral immunodeficiency and lack mature B cell subsets, mirroring deficiency of the cytokine B cell–activating factor (BAFF). Accumulation of Sppl2a-deficient B cells was rescued by overexpression of the BAFF-induced survival protein B cell lymphoma 2 (BCL2) but not BAFF and was distinguished by low surface BAFF receptor and IgM and IgD B cell receptors. CD8-negative dendritic cells were also greatly decreased. SPPL2A deficiency blocked the proteolytic processing of CD74 MHC II invariant chain in both cell types, causing dramatic build-up of the p8 product of Cathepsin S and interfering with earlier steps in CD74 endosomal retention and processing. The findings illuminate an important role for the final step in the CD74–MHC II pathway and a new target for protease inhibitor treatment of B cell diseases.
PMCID: PMC3549710  PMID: 23267016
8.  Cancer immunoediting by the innate immune system in the absence of adaptive immunity 
The Journal of Experimental Medicine  2012;209(10):1869-1882.
In the absence of adaptive immunity, NK cells polarize M1 macrophages to facilitate cancer immunoediting.
Cancer immunoediting is the process whereby immune cells protect against cancer formation by sculpting the immunogenicity of developing tumors. Although the full process depends on innate and adaptive immunity, it remains unclear whether innate immunity alone is capable of immunoediting. To determine whether the innate immune system can edit tumor cells in the absence of adaptive immunity, we compared the incidence and immunogenicity of 3′methylcholanthrene-induced sarcomas in syngeneic wild-type, RAG2−/−, and RAG2−/−x γc−/− mice. We found that innate immune cells could manifest cancer immunoediting activity in the absence of adaptive immunity. This activity required natural killer (NK) cells and interferon γ (IFN-γ), which mediated the induction of M1 macrophages. M1 macrophages could be elicited by administration of CD40 agonists, thereby restoring editing activity in RAG2−/−x γc−/− mice. Our results suggest that in the absence of adaptive immunity, NK cell production of IFN-γ induces M1 macrophages, which act as important effectors during cancer immunoediting.
PMCID: PMC3457735  PMID: 22927549
9.  Non-classical MHC Class I molecules regulating natural killer cell function 
Oncoimmunology  2013;2(3):e23336.
Natural killer (NK) cells possess effector and immunoregulatory functions that are controlled by a myriad of receptor-ligand pairs, including human killer inhibitory receptor (KIR) and mouse Ly49-MHC class I interactions. We have recently shown that the NK cell inhibitory molecule Ly49A binds the non-classical MHC molecule H2-M3, thus regulating host innate immune responses to tumor initiation and metastasis.
PMCID: PMC3661161  PMID: 23802076
education; inhibitory; metastasis; tumor immunosurveillance
10.  Origins and functional impact of copy number variation in the human genome 
Nature  2009;464(7289):704-712.
Structural variations of DNA greater than 1 kilobase in size account for most bases that vary among human genomes, but are still relatively under-ascertained. Here we use tiling oligonucleotide microarrays, comprising 42 million probes, to generate a comprehensive map of 11,700 copy number variations (CNVs) greater than 443 base pairs, of which most (8,599) have been validated independently. For 4,978 of these CNVs, we generated reference genotypes from 450 individuals of European, African or East Asian ancestry. The predominant mutational mechanisms differ among CNV size classes. Retrotransposition has duplicated and inserted some coding and non-coding DNA segments randomly around the genome. Furthermore, by correlation with known trait-associated single nucleotide polymorphisms (SNPs), we identified 30 loci with CNVs that are candidates for influencing disease susceptibility. Despite this, having assessed the completeness of our map and the patterns of linkage disequilibrium between CNVs and SNPs, we conclude that, for complex traits, the heritability void left by genome-wide association studies will not be accounted for by common CNVs.
PMCID: PMC3330748  PMID: 19812545
11.  ATP11c is critical for phosphatidylserine internalization and B lymphocyte differentiation 
Nature Immunology  2011;12(5):441-449.
Subcompartments of the plasma membrane are believed to be critical for lymphocyte responses but few genetic tools exist to test their function. Here we describe a new X-linked B cell deficiency syndrome in mice caused by mutations in Atp11c, a member of the P4 ATPase family thought to serve as flippases concentrating aminophospholipids in the cytoplasmic leaflet of cell membranes. Defective ATP11c decreased the rate of phosphatidylserine translocation in pro-B cells, greatly reduced pre-B and B cell numbers independent of Bcl2-inhibited apoptosis or immunoglobulin gene rearrangement and abolished pre-B cell expansion in response to an Il7 transgene. The only other abnormalities noted were anemia, hyperbilirubinemia and hepatocellular carcinoma. These results identify an intimate connection between phospholipid transport and B lymphocyte function.
PMCID: PMC3272780  PMID: 21423173
12.  Genome-wide dFOXO targets and topology of the transcriptomic response to stress and insulin signalling 
Over 700 direct transcriptional targets of the dFOXO transcription factor are identified in the adult fruit fly. dFOXO-bound genes are conserved between worm and fly, but dFOXO is not the sole mediator of the transcriptional response to changes in insulin signalling in the fly.
More than 700 direct transcriptional targets of dFOXO were determined in the adult Drosophila female, in the wild-type or an insulin-signalling mutant.dFOXO has an important role in the wild-type fly and is important for transcription of numerous signalling components including TOR and Sos.While dFOXO is an important effector of the insulin signalling pathway, it is required for only a portion of the transcriptional changes that occur in response to alterations in the pathway in the fly, and in many cases indirectly.There is strong evolutionary conservation of dFOXO-bound genes between the worm and the fly, specifically enriched in regulatory genes.
Forkhead Box-O (FoxO) transcription factors are crucial players in numerous cellular and organismal processes including metabolism, stress protection, cellular differentiation, cell-cycle arrest, apoptosis and lifespan. FoxOs are regulated by a number of signalling pathways, including negative regulation by insulin/insulin-like growth factor signalling (IIS) (Partridge and Bruning, 2008; Salih and Brunet, 2008). The fruit fly Drosophila melanogaster has a single FoxO orthologue—dFOXO. dFOXO is capable of extending fly lifespan, as well as being required for lifespan extension in response to downregulation of IIS (Giannakou et al, 2004; Hwangbo et al, 2004; Slack et al, 2011). To further our understanding of dFOXO biology, we uncover over 700 direct dFOXO targets in the adult female fly, both in the wild-type fly and in a mutant with reduced IIS activity.
dFOXO is directly required for transcription of several components of IIS and interacting pathways, such as the gene encoding the target of rapamycin (TOR) kinase, in the wild-type fly. Indeed, the removal of dFOXO results in reduced signal through these pathways. The genomic locations occupied by dFOXO in adults are different from those observed by others in larvae or cultured cells (Puig et al, 2003; Teleman et al, 2008), indicating that dFOXO binding is influenced by developmental stage and/or cell type. These locations remain unchanged upon activation of dFOXO by stresses or reduced IIS in the adult, but the binding of dFOXO to the same sites is increased. Genetically induced reduction in IIS results in activation/repression of a greater number of direct dFOXO targets than observed in the wild-type fly.
To determine the relationship between dFOXO and IIS in the adult fly, we identify the part of the IIS transcriptional response that is controlled by dFOXO, both directly and indirectly. We observe that aspects of the transcriptional response to changes in IIS can take place in the absence of dFOXO, indicating that other transcriptional regulators must be involved. This is different to the situation in the worm Caenorhabditis elegans where the worm FoxO, encoded by the daf-16 gene, is required for all the effects of a reduction in IIS (Kenyon et al, 1993; Gems et al, 1998; Murphy et al, 2003). On the other hand, the existence of dFOXO-independent effects is in accordance with genetic experiments in the fly where lifespan extension and xenobiotic resistance caused by a reduction in IIS are dependent on dFOXO, while lowered fecundity and body size, delayed development and resistance to paraquat are not (Slack et al, 2011). Promoter analyses revealed GATA and other forkhead factors as candidate mediators of the dFOXO-independent effects in the fly.
Despite the different topology of the transcriptomic response to IIS changes in the two organisms, there is genome-wide evolutionary conservation of dFOXO targets between the fly and the worm (Figure 9), enriched for a second tier of regulators including the dHR96/daf-12 nuclear hormone receptor.
FoxO transcription factors, inhibited by insulin/insulin-like growth factor signalling (IIS), are crucial players in numerous organismal processes including lifespan. Using genomic tools, we uncover over 700 direct dFOXO targets in adult female Drosophila. dFOXO is directly required for transcription of several IIS components and interacting pathways, such as TOR, in the wild-type fly. The genomic locations occupied by dFOXO in adults are different from those observed in larvae or cultured cells. These locations remain unchanged upon activation by stresses or reduced IIS, but the binding is increased and additional targets activated upon genetic reduction in IIS. We identify the part of the IIS transcriptional response directly controlled by dFOXO and the indirect effects and show that parts of the transcriptional response to IIS reduction do not require dfoxo. Promoter analyses revealed GATA and other forkhead factors as candidate mediators of the indirect and dfoxo-independent effects. We demonstrate genome-wide evolutionary conservation of dFOXO targets between the fly and the worm Caenorhabditis elegans, enriched for a second tier of regulators including the dHR96/daf-12 nuclear hormone receptor.
PMCID: PMC3159968  PMID: 21694719
dFOXO; Drosophila; insulin/insulin-like growth factor signalling; transcription
13.  Crystallographic and Molecular Dynamics Analysis of Loop Motions Unmasking the Peptidoglycan-Binding Site in Stator Protein MotB of Flagellar Motor 
PLoS ONE  2011;6(4):e18981.
The C-terminal domain of MotB (MotB-C) shows high sequence similarity to outer membrane protein A and related peptidoglycan (PG)-binding proteins. It is believed to anchor the power-generating MotA/MotB stator unit of the bacterial flagellar motor to the peptidoglycan layer of the cell wall. We previously reported the first crystal structure of this domain and made a puzzling observation that all conserved residues that are thought to be essential for PG recognition are buried and inaccessible in the crystal structure. In this study, we tested a hypothesis that peptidoglycan binding is preceded by, or accompanied by, some structural reorganization that exposes the key conserved residues.
Methodology/Principal Findings
We determined the structure of a new crystalline form (Form B) of Helicobacter pylori MotB-C. Comparisons with the existing Form A revealed conformational variations in the petal-like loops around the carbohydrate binding site near one end of the β-sheet. These variations are thought to reflect natural flexibility at this site required for insertion into the peptidoglycan mesh. In order to understand the nature of this flexibility we have performed molecular dynamics simulations of the MotB-C dimer. The results are consistent with the crystallographic data and provide evidence that the three loops move in a concerted fashion, exposing conserved MotB residues that have previously been implicated in binding of the peptide moiety of peptidoglycan.
Our structural analysis provides a new insight into the mechanism by which MotB inserts into the peptidoglycan mesh, thus anchoring the power-generating complex to the cell wall.
PMCID: PMC3080404  PMID: 21533052
14.  Innate immunity defines the capacity of antiviral T cells to limit persistent infection 
The Journal of Experimental Medicine  2010;207(6):1333-1343.
Effective immunity requires the coordinated activation of innate and adaptive immune responses. Natural killer (NK) cells are central innate immune effectors, but can also affect the generation of acquired immune responses to viruses and malignancies. How NK cells influence the efficacy of adaptive immunity, however, is poorly understood. Here, we show that NK cells negatively regulate the duration and effectiveness of virus-specific CD4+ and CD8+ T cell responses by limiting exposure of T cells to infected antigen-presenting cells. This impacts the quality of T cell responses and the ability to limit viral persistence. Our studies provide unexpected insights into novel interplays between innate and adaptive immune effectors, and define the critical requirements for efficient control of viral persistence.
PMCID: PMC2882831  PMID: 20513749
15.  Molecular Evolution and Functional Characterization of Drosophila Insulin-Like Peptides 
PLoS Genetics  2010;6(2):e1000857.
Multicellular animals match costly activities, such as growth and reproduction, to the environment through nutrient-sensing pathways. The insulin/IGF signaling (IIS) pathway plays key roles in growth, metabolism, stress resistance, reproduction, and longevity in diverse organisms including mammals. Invertebrate genomes often contain multiple genes encoding insulin-like ligands, including seven Drosophila insulin-like peptides (DILPs). We investigated the evolution, diversification, redundancy, and functions of the DILPs, combining evolutionary analysis, based on the completed genome sequences of 12 Drosophila species, and functional analysis, based on newly-generated knock-out mutations for all 7 dilp genes in D. melanogaster. Diversification of the 7 DILPs preceded diversification of Drosophila species, with stable gene diversification and family membership, suggesting stabilising selection for gene function. Gene knock-outs demonstrated both synergy and compensation of expression between different DILPs, notably with DILP3 required for normal expression of DILPs 2 and 5 in brain neurosecretory cells and expression of DILP6 in the fat body compensating for loss of brain DILPs. Loss of DILP2 increased lifespan and loss of DILP6 reduced growth, while loss of DILP7 did not affect fertility, contrary to its proposed role as a Drosophila relaxin. Importantly, loss of DILPs produced in the brain greatly extended lifespan but only in the presence of the endosymbiontic bacterium Wolbachia, demonstrating a specific interaction between IIS and Wolbachia in lifespan regulation. Furthermore, loss of brain DILPs blocked the responses of lifespan and fecundity to dietary restriction (DR) and the DR response of these mutants suggests that IIS extends lifespan through mechanisms that both overlap with those of DR and through additional mechanisms that are independent of those at work in DR. Evolutionary conservation has thus been accompanied by synergy, redundancy, and functional differentiation between DILPs, and these features may themselves be of evolutionary advantage.
Author Summary
The insulin/IGF signalling (IIS) pathway plays key roles in growth, metabolism, reproduction, and longevity in animals as diverse as flies and mammals. Most multicellular animals contain multiple IIS ligands, including 7 in the fruit fly Drosophila melanogaster (DILP1-7), implying that the diverse functions of IIS could in part be mediated by the functional diversification of the ligands. Although Drosophila is a prime model organism to study IIS, knowledge about the function of individual DILPs is still very limited due to the lack of gene-specific mutants. Therefore, we generated mutants for all 7 dilp genes and systematically analyzed their phenotypes. We show that loss of DILP2 extends lifespan and describe a novel role for DILP6 in growth control. Furthermore, we show that DILPs are evolutionary conserved and can act redundantly, supporting the hypothesis that functional redundancy itself can be of evolutionary advantage. We also describe a specific interaction between IIS and the endosymbiontic bacterium Wolbachia in lifespan regulation. This finding has implications for all longevity studies using IIS mutants in flies and offers the opportunity to study IIS as a mechanism involved in host/symbiont interactions. Finally, we show that DILPs mediate the response of lifespan and fecundity to dietary restriction.
PMCID: PMC2829060  PMID: 20195512
16.  DNAM-1 promotes activation of cytotoxic lymphocytes by nonprofessional antigen-presenting cells and tumors 
The Journal of Experimental Medicine  2008;205(13):2965-2973.
Natural killer (NK) cells and CD8 T cells require adhesion molecules for migration, activation, expansion, differentiation, and effector functions. DNAX accessory molecule 1 (DNAM-1), an adhesion molecule belonging to the immunoglobulin superfamily, promotes many of these functions in vitro. However, because NK cells and CD8 T cells express multiple adhesion molecules, it is unclear whether DNAM-1 has a unique function or is effectively redundant in vivo. To address this question, we generated mice lacking DNAM-1 and evaluated DNAM-1–deficient CD8 T cell and NK cell function in vitro and in vivo. Our results demonstrate that CD8 T cells require DNAM-1 for co-stimulation when recognizing antigen presented by nonprofessional antigen-presenting cells; in contrast, DNAM-1 is dispensable when dendritic cells present the antigen. Similarly, NK cells require DNAM-1 for the elimination of tumor cells that are comparatively resistant to NK cell–mediated cytotoxicity caused by the paucity of other NK cell–activating ligands. We conclude that DNAM-1 serves to extend the range of target cells that can activate CD8 T cell and NK cells and, hence, may be essential for immunosurveillance against tumors and/or viruses that evade recognition by other activating or accessory molecules.
PMCID: PMC2605240  PMID: 19029380
17.  Global variation in copy number in the human genome 
Nature  2006;444(7118):444-454.
Copy number variation (CNV) of DNA sequences is functionally significant but has yet to be fully ascertained. We have constructed a first-generation CNV map of the human genome through the study of 270 individuals from four populations with ancestry in Europe, Africa or Asia (the HapMap collection). DNA from these individuals was screened for CNV using two complementary technologies: single nucleotide polymorphism (SNP) genotyping arrays, and clone-based comparative genomic hybridization. 1,447 copy number variable regions covering 360 megabases (12% of the genome) were identified in these populations; these CNV regions contained hundreds of genes, disease loci, functional elements and segmental duplications. Strikingly, these CNVs encompassed more nucleotide content per genome than SNPs, underscoring the importance of CNV in genetic diversity and evolution. The data obtained delineate linkage disequilibrium patterns for many CNVs, and reveal dramatic variation in copy number among populations. We also demonstrate the utility of this resource for genetic disease studies.
PMCID: PMC2669898  PMID: 17122850
18.  The DNA sequence of the human X chromosome 
Ross, Mark T. | Grafham, Darren V. | Coffey, Alison J. | Scherer, Steven | McLay, Kirsten | Muzny, Donna | Platzer, Matthias | Howell, Gareth R. | Burrows, Christine | Bird, Christine P. | Frankish, Adam | Lovell, Frances L. | Howe, Kevin L. | Ashurst, Jennifer L. | Fulton, Robert S. | Sudbrak, Ralf | Wen, Gaiping | Jones, Matthew C. | Hurles, Matthew E. | Andrews, T. Daniel | Scott, Carol E. | Searle, Stephen | Ramser, Juliane | Whittaker, Adam | Deadman, Rebecca | Carter, Nigel P. | Hunt, Sarah E. | Chen, Rui | Cree, Andrew | Gunaratne, Preethi | Havlak, Paul | Hodgson, Anne | Metzker, Michael L. | Richards, Stephen | Scott, Graham | Steffen, David | Sodergren, Erica | Wheeler, David A. | Worley, Kim C. | Ainscough, Rachael | Ambrose, Kerrie D. | Ansari-Lari, M. Ali | Aradhya, Swaroop | Ashwell, Robert I. S. | Babbage, Anne K. | Bagguley, Claire L. | Ballabio, Andrea | Banerjee, Ruby | Barker, Gary E. | Barlow, Karen F. | Barrett, Ian P. | Bates, Karen N. | Beare, David M. | Beasley, Helen | Beasley, Oliver | Beck, Alfred | Bethel, Graeme | Blechschmidt, Karin | Brady, Nicola | Bray-Allen, Sarah | Bridgeman, Anne M. | Brown, Andrew J. | Brown, Mary J. | Bonnin, David | Bruford, Elspeth A. | Buhay, Christian | Burch, Paula | Burford, Deborah | Burgess, Joanne | Burrill, Wayne | Burton, John | Bye, Jackie M. | Carder, Carol | Carrel, Laura | Chako, Joseph | Chapman, Joanne C. | Chavez, Dean | Chen, Ellson | Chen, Guan | Chen, Yuan | Chen, Zhijian | Chinault, Craig | Ciccodicola, Alfredo | Clark, Sue Y. | Clarke, Graham | Clee, Chris M. | Clegg, Sheila | Clerc-Blankenburg, Kerstin | Clifford, Karen | Cobley, Vicky | Cole, Charlotte G. | Conquer, Jen S. | Corby, Nicole | Connor, Richard E. | David, Robert | Davies, Joy | Davis, Clay | Davis, John | Delgado, Oliver | DeShazo, Denise | Dhami, Pawandeep | Ding, Yan | Dinh, Huyen | Dodsworth, Steve | Draper, Heather | Dugan-Rocha, Shannon | Dunham, Andrew | Dunn, Matthew | Durbin, K. James | Dutta, Ireena | Eades, Tamsin | Ellwood, Matthew | Emery-Cohen, Alexandra | Errington, Helen | Evans, Kathryn L. | Faulkner, Louisa | Francis, Fiona | Frankland, John | Fraser, Audrey E. | Galgoczy, Petra | Gilbert, James | Gill, Rachel | Glöckner, Gernot | Gregory, Simon G. | Gribble, Susan | Griffiths, Coline | Grocock, Russell | Gu, Yanghong | Gwilliam, Rhian | Hamilton, Cerissa | Hart, Elizabeth A. | Hawes, Alicia | Heath, Paul D. | Heitmann, Katja | Hennig, Steffen | Hernandez, Judith | Hinzmann, Bernd | Ho, Sarah | Hoffs, Michael | Howden, Phillip J. | Huckle, Elizabeth J. | Hume, Jennifer | Hunt, Paul J. | Hunt, Adrienne R. | Isherwood, Judith | Jacob, Leni | Johnson, David | Jones, Sally | de Jong, Pieter J. | Joseph, Shirin S. | Keenan, Stephen | Kelly, Susan | Kershaw, Joanne K. | Khan, Ziad | Kioschis, Petra | Klages, Sven | Knights, Andrew J. | Kosiura, Anna | Kovar-Smith, Christie | Laird, Gavin K. | Langford, Cordelia | Lawlor, Stephanie | Leversha, Margaret | Lewis, Lora | Liu, Wen | Lloyd, Christine | Lloyd, David M. | Loulseged, Hermela | Loveland, Jane E. | Lovell, Jamieson D. | Lozado, Ryan | Lu, Jing | Lyne, Rachael | Ma, Jie | Maheshwari, Manjula | Matthews, Lucy H. | McDowall, Jennifer | McLaren, Stuart | McMurray, Amanda | Meidl, Patrick | Meitinger, Thomas | Milne, Sarah | Miner, George | Mistry, Shailesh L. | Morgan, Margaret | Morris, Sidney | Müller, Ines | Mullikin, James C. | Nguyen, Ngoc | Nordsiek, Gabriele | Nyakatura, Gerald | O’Dell, Christopher N. | Okwuonu, Geoffery | Palmer, Sophie | Pandian, Richard | Parker, David | Parrish, Julia | Pasternak, Shiran | Patel, Dina | Pearce, Alex V. | Pearson, Danita M. | Pelan, Sarah E. | Perez, Lesette | Porter, Keith M. | Ramsey, Yvonne | Reichwald, Kathrin | Rhodes, Susan | Ridler, Kerry A. | Schlessinger, David | Schueler, Mary G. | Sehra, Harminder K. | Shaw-Smith, Charles | Shen, Hua | Sheridan, Elizabeth M. | Shownkeen, Ratna | Skuce, Carl D. | Smith, Michelle L. | Sotheran, Elizabeth C. | Steingruber, Helen E. | Steward, Charles A. | Storey, Roy | Swann, R. Mark | Swarbreck, David | Tabor, Paul E. | Taudien, Stefan | Taylor, Tineace | Teague, Brian | Thomas, Karen | Thorpe, Andrea | Timms, Kirsten | Tracey, Alan | Trevanion, Steve | Tromans, Anthony C. | d’Urso, Michele | Verduzco, Daniel | Villasana, Donna | Waldron, Lenee | Wall, Melanie | Wang, Qiaoyan | Warren, James | Warry, Georgina L. | Wei, Xuehong | West, Anthony | Whitehead, Siobhan L. | Whiteley, Mathew N. | Wilkinson, Jane E. | Willey, David L. | Williams, Gabrielle | Williams, Leanne | Williamson, Angela | Williamson, Helen | Wilming, Laurens | Woodmansey, Rebecca L. | Wray, Paul W. | Yen, Jennifer | Zhang, Jingkun | Zhou, Jianling | Zoghbi, Huda | Zorilla, Sara | Buck, David | Reinhardt, Richard | Poustka, Annemarie | Rosenthal, André | Lehrach, Hans | Meindl, Alfons | Minx, Patrick J. | Hillier, LaDeana W. | Willard, Huntington F. | Wilson, Richard K. | Waterston, Robert H. | Rice, Catherine M. | Vaudin, Mark | Coulson, Alan | Nelson, David L. | Weinstock, George | Sulston, John E. | Durbin, Richard | Hubbard, Tim | Gibbs, Richard A. | Beck, Stephan | Rogers, Jane | Bentley, David R.
Nature  2005;434(7031):325-337.
The human X chromosome has a unique biology that was shaped by its evolution as the sex chromosome shared by males and females. We have determined 99.3% of the euchromatic sequence of the X chromosome. Our analysis illustrates the autosomal origin of the mammalian sex chromosomes, the stepwise process that led to the progressive loss of recombination between X and Y, and the extent of subsequent degradation of the Y chromosome. LINE1 repeat elements cover one-third of the X chromosome, with a distribution that is consistent with their proposed role as way stations in the process of X-chromosome inactivation. We found 1,098 genes in the sequence, of which 99 encode proteins expressed in testis and in various tumour types. A disproportionately high number of mendelian diseases are documented for the X chromosome. Of this number, 168 have been explained by mutations in 113 X-linked genes, which in many cases were characterized with the aid of the DNA sequence.
PMCID: PMC2665286  PMID: 15772651
19.  The Early Kinetics of Cytomegalovirus-Specific CD8+ T-Cell Responses Are Not Affected by Antigen Load or the Absence of Perforin or Gamma Interferon▿  
Journal of Virology  2008;82(10):4931-4937.
Both innate and adaptive immune responses participate in the control of murine cytomegalovirus (mCMV) infection. In some mouse strains, like BALB/c, the control of infection relies on the activities of CD8+ T cells. mCMV-specific CD8+ T-cell responses are unusual in that, even after mCMV has been controlled in the periphery, the numbers of circulating virus-specific CD8+ T cells remain high compared to those observed in other viral infections. To better understand the generation and maintenance of mCMV-specific CD8+ T-cell responses, we evaluated how antigen load and effector molecules, such as perforin (Prf) and gamma interferon (IFN-γ), influence these responses during acute infection in vivo. Viral burden affected the magnitude, but not the early kinetics, of antigen-specific CD8+ T-cell responses. Similarly, the magnitude of virus-specific CD8+ T-cell expansion was affected by Prf and IFN-γ, but contraction of antigen-specific responses occurred normally in both Prf- and IFN-γ-deficient mice. These data indicate that control of mCMV-specific CD8+ T-cell expansion and contraction is more complex than anticipated and, despite the role of Prf or IFN-γ in controlling viral replication, a full program of T-cell expansion and contraction can occur in their absence.
PMCID: PMC2346769  PMID: 18337574
20.  Breaking the waves: improved detection of copy number variation from microarray-based comparative genomic hybridization 
Genome Biology  2007;8(10):R228.
Datasets used for detecting copy number variation (CNV) are shown to be affected by a technical artifact. A novel CNV calling algorithm is presented which removes this artifact and identifies regions of CNV better than existing methods.
Large-scale high throughput studies using microarray technology have established that copy number variation (CNV) throughout the genome is more frequent than previously thought. Such variation is known to play an important role in the presence and development of phenotypes such as HIV-1 infection and Alzheimer's disease. However, methods for analyzing the complex data produced and identifying regions of CNV are still being refined.
We describe the presence of a genome-wide technical artifact, spatial autocorrelation or 'wave', which occurs in a large dataset used to determine the location of CNV across the genome. By removing this artifact we are able to obtain both a more biologically meaningful clustering of the data and an increase in the number of CNVs identified by current calling methods without a major increase in the number of false positives detected. Moreover, removing this artifact is critical for the development of a novel model-based CNV calling algorithm - CNVmix - that uses cross-sample information to identify regions of the genome where CNVs occur. For regions of CNV that are identified by both CNVmix and current methods, we demonstrate that CNVmix is better able to categorize samples into groups that represent copy number gains or losses.
Removing artifactual 'waves' (which appear to be a general feature of array comparative genomic hybridization (aCGH) datasets) and using cross-sample information when identifying CNVs enables more biological information to be extracted from aCGH experiments designed to investigate copy number variation in normal individuals.
PMCID: PMC2246302  PMID: 17961237
21.  A novel checkpoint in the Bcl-2–regulated apoptotic pathway revealed by murine cytomegalovirus infection of dendritic cells 
The Journal of Cell Biology  2004;166(6):827-837.
Infection with murine cytomegalovirus (MCMV) has contributed to understanding many aspects of human infection and, additionally, has provided important insight to understanding complex cellular responses. Dendritic cells (DCs) are a major target for MCMV infection. Here, we analyze the effects of MCMV infection on DC viability, and show that infected DCs become resistant to apoptosis induced by growth factor deprivation. The precise contribution of changes in the expression of Bcl-2 family proteins has been assessed and a new checkpoint in the apoptotic pathway identified. Despite their resistance to apoptosis, MCMV-infected DCs showed Bax to be tightly associated with mitochondria and, together with Bak, forming high molecular weight oligomers, changes normally associated with apoptotic cell death. Exposure of a constitutively occluded Bax NH2-terminal epitope was blocked after infection. These results suggest that MCMV has evolved a novel strategy for inhibiting apoptosis and provide evidence that apoptosis can be regulated after translocation, integration, and oligomerization of Bax at the mitochondrial membrane.
PMCID: PMC2172116  PMID: 15353550
dendritic cell; cytomegalovirus; apoptosis; Bcl-2; Bax

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