Evidence of Xist RNA-independent initiation of mouse imprinted X-chromosome inactivation

Nature advance online publication 01 July 2009. doi:10.1038/nature08161

Authors: Sundeep Kalantry, Sonya Purushothaman, Randall Bryant Bowen, Joshua Starmer & Terry Magnuson

XX female mammals undergo transcriptional silencing of most genes on one of their two X chromosomes to equalize X-linked gene dosage with XY males in a process referred to as X-chromosome inactivation (XCI). XCI is an example of epigenetic regulation. Once enacted in individual cells of the early female embryo, XCI is stably transmitted such that most descendant cells maintain silencing of that X chromosome. In eutherian mammals, XCI is thought to be triggered by the expression of the non-coding Xist RNA from the future inactive X chromosome (Xi); Xist RNA in turn is proposed to recruit protein complexes that bring about heterochromatinization of the Xi. Here we test whether imprinted XCI, which results in preferential inactivation of the paternal X chromosome (Xp), occurs in mouse embryos inheriting an Xp lacking Xist. We find that silencing of Xp-linked genes can initiate in the absence of paternal Xist; Xist is, however, required to stabilize silencing along the Xp. Xp-linked gene silencing associated with mouse imprinted XCI, therefore, can initiate in the embryo independently of Xist RNA.

Nature advance online publication 01 July 2009. doi:10.1038/nature08185

Authors:

Schizophrenia is a severe mental disorder with a lifetime risk of about 1%, characterized by hallucinations, delusions and cognitive deficits, with heritability estimated at up to 80%. We performed a genome-wide association study of 3,322 European individuals with schizophrenia and 3,587 controls. Here we show, using two analytic approaches, the extent to which common genetic variation underlies the risk of schizophrenia. First, we implicate the major histocompatibility complex. Second, we provide molecular genetic evidence for a substantial polygenic component to the risk of schizophrenia involving thousands of common alleles of very small effect. We show that this component also contributes to the risk of bipolar disorder, but not to several non-psychiatric diseases.

Nature advance online publication 01 July 2009. doi:10.1038/nature08192

Authors: Jianxin Shi, Douglas F. Levinson, Jubao Duan, Alan R. Sanders, Yonglan Zheng, Itsik Pe’er, Frank Dudbridge, Peter A. Holmans, Alice S. Whittemore, Bryan J. Mowry, Ann Olincy, Farooq Amin, C. Robert Cloninger, Jeremy M. Silverman, Nancy G. Buccola, William F. Byerley, Donald W. Black, Raymond R. Crowe, Jorge R. Oksenberg, Daniel B. Mirel, Kenneth S. Kendler, Robert Freedman & Pablo V. Gejman

Schizophrenia, a devastating psychiatric disorder, has a prevalence of 0.5–1%, with high heritability (80–85%) and complex transmission. Recent studies implicate rare, large, high-penetrance copy number variants in some cases, but the genes or biological mechanisms that underlie susceptibility are not known. Here we show that schizophrenia is significantly associated with single nucleotide polymorphisms (SNPs) in the extended major histocompatibility complex region on chromosome 6. We carried out a genome-wide association study of common SNPs in the Molecular Genetics of Schizophrenia (MGS) case-control sample, and then a meta-analysis of data from the MGS, International Schizophrenia Consortium and SGENE data sets. No MGS finding achieved genome-wide statistical significance. In the meta-analysis of European-ancestry subjects (8,008 cases, 19,077 controls), significant association with schizophrenia was observed in a region of linkage disequilibrium on chromosome 6p22.1 (P = 9.54 × 10-9). This region includes a histone gene cluster and several immunity-related genes—possibly implicating aetiological mechanisms involving chromatin modification, transcriptional regulation, autoimmunity and/or infection. These results demonstrate that common schizophrenia susceptibility alleles can be detected. The characterization of these signals will suggest important directions for research on susceptibility mechanisms.

Nature advance online publication 01 July 2009. doi:10.1038/nature08186

Authors: Hreinn Stefansson, Roel A. Ophoff, Stacy Steinberg, Ole A. Andreassen, Sven Cichon, Dan Rujescu, Thomas Werge, Olli P. H. Pietiläinen, Ole Mors, Preben B. Mortensen, Engilbert Sigurdsson, Omar Gustafsson, Mette Nyegaard, Annamari Tuulio-Henriksson, Andres Ingason, Thomas Hansen, Jaana Suvisaari, Jouko Lonnqvist, Tiina Paunio, Anders D. Børglum, Annette Hartmann, Anders Fink-Jensen, Merete Nordentoft, David Hougaard, Bent Norgaard-Pedersen, Yvonne Böttcher, Jes Olesen, René Breuer, Hans-Jürgen Möller, Ina Giegling, Henrik B. Rasmussen, Sally Timm, Manuel Mattheisen, István Bitter, János M. Réthelyi, Brynja B. Magnusdottir, Thordur Sigmundsson, Pall Olason, Gisli Masson, Jeffrey R. Gulcher, Magnus Haraldsson, Ragnheidur Fossdal, Thorgeir E. Thorgeirsson, Unnur Thorsteinsdottir, Mirella Ruggeri, Sarah Tosato, Barbara Franke, Eric Strengman, Lambertus A. Kiemeney, GROUP†, Ingrid Melle, Srdjan Djurovic, Lilia Abramova, Vasily Kaleda, Julio Sanjuan, Rosa de Frutos, Elvira Bramon, Evangelos Vassos, Gillian Fraser, Ulrich Ettinger, Marco Picchioni, Nicholas Walker, Timi Toulopoulou, Anna C. Need, Dongliang Ge, Joeng Lim Yoon, Kevin V. Shianna, Nelson B. Freimer, Rita M. Cantor, Robin Murray, Augustine Kong, Vera Golimbet, Angel Carracedo, Celso Arango, Javier Costas, Erik G. Jönsson, Lars Terenius, Ingrid Agartz, Hannes Petursson, Markus M. Nöthen, Marcella Rietschel, Paul M. Matthews, Pierandrea Muglia, Leena Peltonen, David St Clair, David B. Goldstein, Kari Stefansson & David A. Collier

Schizophrenia is a complex disorder, caused by both genetic and environmental factors and their interactions. Research on pathogenesis has traditionally focused on neurotransmitter systems in the brain, particularly those involving dopamine. Schizophrenia has been considered a separate disease for over a century, but in the absence of clear biological markers, diagnosis has historically been based on signs and symptoms. A fundamental message emerging from genome-wide association studies of copy number variations (CNVs) associated with the disease is that its genetic basis does not necessarily conform to classical nosological disease boundaries. Certain CNVs confer not only high relative risk of schizophrenia but also of other psychiatric disorders. The structural variations associated with schizophrenia can involve several genes and the phenotypic syndromes, or the ‘genomic disorders’, have not yet been characterized. Single nucleotide polymorphism (SNP)-based genome-wide association studies with the potential to implicate individual genes in complex diseases may reveal underlying biological pathways. Here we combined SNP data from several large genome-wide scans and followed up the most significant association signals. We found significant association with several markers spanning the major histocompatibility complex (MHC) region on chromosome 6p21.3-22.1, a marker located upstream of the neurogranin gene (NRGN) on 11q24.2 and a marker in intron four of transcription factor 4 (TCF4) on 18q21.2. Our findings implicating the MHC region are consistent with an immune component to schizophrenia risk, whereas the association with NRGN and TCF4 points to perturbation of pathways involved in brain development, memory and cognition.

Nature advance online publication 01 July 2009. doi:10.1038/nature08156

Authors: Alexander Stein, Gert Weber, Markus C. Wahl & Reinhard Jahn

Neurotransmission relies on synaptic vesicles fusing with the membrane of nerve cells to release their neurotransmitter content into the synaptic cleft, a process requiring the assembly of several members of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family. SNAREs represent an evolutionarily conserved protein family that mediates membrane fusion in the secretory and endocytic pathways of eukaryotic cells. On membrane contact, these proteins assemble intrans between the membranes as a bundle of four α-helices, with the energy released during assembly being thought to drive fusion. However, it is unclear how the energy is transferred to the membranes and whether assembly is conformationally linked to fusion. Here, we report the X-ray structure of the neuronal SNARE complex, consisting of rat syntaxin 1A, SNAP-25 and synaptobrevin 2, with the carboxy-terminal linkers and transmembrane regions at 3.4 Å resolution. The structure shows that assembly proceeds beyond the already known core SNARE complex, resulting in a continuous helical bundle that is further stabilized by side-chain interactions in the linker region. Our results suggest that the final phase of SNARE assembly is directly coupled to membrane merger.

Nature advance online publication 28 June 2009. doi:10.1038/nature08121

Authors: L. DiCarlo, J. M. Chow, J. M. Gambetta, Lev S. Bishop, B. R. Johnson, D. I. Schuster, J. Majer, A. Blais, L. Frunzio, S. M. Girvin & R. J. Schoelkopf

Quantum computers, which harness the superposition and entanglement of physical states, could outperform their classical counterparts in solving problems with technological impact—such as factoring large numbers and searching databases. A quantum processor executes algorithms by applying a programmable sequence of gates to an initialized register of qubits, which coherently evolves into a final state containing the result of the computation. Building a quantum processor is challenging because of the need to meet simultaneously requirements that are in conflict: state preparation, long coherence times, universal gate operations and qubit readout. Processors based on a few qubits have been demonstrated using nuclear magnetic resonance, cold ion trap and optical systems, but a solid-state realization has remained an outstanding challenge. Here we demonstrate a two-qubit superconducting processor and the implementation of the Grover search and Deutsch–Jozsa quantum algorithms. We use a two-qubit interaction, tunable in strength by two orders of magnitude on nanosecond timescales, which is mediated by a cavity bus in a circuit quantum electrodynamics architecture. This interaction allows the generation of highly entangled states with concurrence up to 94 per cent. Although this processor constitutes an important step in quantum computing with integrated circuits, continuing efforts to increase qubit coherence times, gate performance and register size will be required to fulfil the promise of a scalable technology.

Nature advance online publication 28 June 2009. doi:10.1038/nature08138

Authors: Yasutaka Okabe, Teruyuki Sano & Shigekazu Nagata

Innate immunity is stimulated not only by viral or bacterial components, but also by non-microbial danger signals (damage-associated molecular patterns). One of the damage-associated molecular patterns is chromosomal DNA that escapes degradation. In programmed cell death and erythropoiesis, DNA from dead cells or nuclei expelled from erythroblasts is digested by DNase II in the macrophages after they are engulfed. DNase II-/- (also known as Dnase2a-/-) mice suffer from severe anaemia or chronic arthritis due to interferon-β (IFN-β) and tumour necrosis factor-α (TNF-α) produced from the macrophages carrying undigested DNA in a Toll-like receptor (TLR)-independent mechanism. Here we show that Eyes absent 4 (EYA4), originally identified as a co-transcription factor, stimulates the expression of IFN-β and CXCL10 in response to the undigested DNA of apoptotic cells. EYA4 enhanced the innate immune response against viruses (Newcastle disease virus and vesicular stomatitis virus), and could associate with signalling molecules (IPS-1 (also known as MAVS), STING (TMEM173) and NLRX1). Three groups have previously shown that EYA has phosphatase activity. We found that mouse EYA family members act as a phosphatase for both phosphotyrosine and phosphothreonine. The haloacid dehalogenase domain at the carboxy terminus contained the tyrosine-phosphatase, and the amino-terminal half carried the threonine-phosphatase. Mutations of the threonine-phosphatase, but not the tyrosine-phosphatase, abolished the ability of EYA4 to enhance the innate immune response, suggesting that EYA regulates the innate immune response by modulating the phosphorylation state of signal transducers for the intracellular pathogens.

Nature advance online publication 28 June 2009. doi:10.1038/nature08139

Authors: Andrew S. Yoo, Brett T. Staahl, Lei Chen & Gerald R. Crabtree

One of the most distinctive steps in the development of the vertebrate nervous system occurs at mitotic exit when cells lose multipotency and begin to develop stable connections that will persist for a lifetime. This transition is accompanied by a switch in ATP-dependent chromatin-remodelling mechanisms that appears to coincide with the final mitotic division of neurons. This switch involves the exchange of the BAF53a (also known as ACTL6a) and BAF45a (PHF10) subunits within Swi/Snf-like neural-progenitor-specific BAF (npBAF) complexes for the homologous BAF53b (ACTL6b) and BAF45b (DPF1) subunits within neuron-specific BAF (nBAF) complexes in post-mitotic neurons. The subunits of the npBAF complex are essential for neural-progenitor proliferation, and mice with reduced dosage for the genes encoding its subunits have defects in neural-tube closure similar to those in human spina bifida, one of the most serious congenital birth defects. In contrast, BAF53b and the nBAF complex are essential for an evolutionarily conserved program of post-mitotic neural development and dendritic morphogenesis. Here we show that this essential transition is mediated by repression of BAF53a by miR-9* and miR-124. We find that BAF53a repression is mediated by sequences in the 3′ untranslated region corresponding to the recognition sites for miR-9* and miR-124, which are selectively expressed in post-mitotic neurons. Mutation of these sites led to persistent expression of BAF53a and defective activity-dependent dendritic outgrowth in neurons. In addition, overexpression of miR-9* and miR-124 in neural progenitors caused reduced proliferation. Previous studies have indicated that miR-9* and miR-124 are repressed by the repressor-element-1-silencing transcription factor (REST, also known as NRSF). Indeed, expression of REST in post-mitotic neurons led to derepression of BAF53a, indicating that REST-mediated repression of microRNAs directs the essential switch of chromatin regulatory complexes.

Nature advance online publication 24 June 2009. doi:10.1038/nature08130

Authors: Andrea C. Carrano, Zheng Liu, Andrew Dillin & Tony Hunter

Dietary restriction extends longevity in diverse species, suggesting that there is a conserved mechanism for nutrient regulation and prosurvival responses. Here we show a role for the HECT (homologous to E6AP carboxy terminus) E3 ubiquitin ligase WWP-1 as a positive regulator of lifespan in Caenorhabditis elegans in response to dietary restriction. We find that overexpression of wwp-1 in worms extends lifespan by up to 20% under conditions of ad libitum feeding. This extension is dependent on the FOXA transcription factor pha-4, and independent of the FOXO transcription factor daf-16. Reduction of wwp-1 completely suppresses the extended longevity of diet-restricted animals. However, the loss of wwp-1 does not affect the long lifespan of animals with compromised mitochondrial function or reduced insulin/IGF-1 signalling. Overexpression of a mutant form of WWP-1 lacking catalytic activity suppresses the increased lifespan of diet-restricted animals, indicating that WWP-1 ubiquitin ligase activity is essential for longevity. Furthermore, we find that the E2 ubiquitin conjugating enzyme, UBC-18, is essential and specific for diet-restriction-induced longevity. UBC-18 interacts with WWP-1 and is required for the ubiquitin ligase activity of WWP-1 and the extended longevity of worms overexpressing wwp-1. Taken together, our results indicate that WWP-1 and UBC-18 function to ubiquitinate substrates that regulate diet-restriction-induced longevity.

Nature advance online publication 24 June 2009. doi:10.1038/nature08169

Authors: Nicholas J. Conard, Maria Malina & Susanne C. Münzel

Considerable debate surrounds claims for early evidence of music in the archaeological record. Researchers universally accept the existence of complex musical instruments as an indication of fully modern behaviour and advanced symbolic communication but, owing to the scarcity of finds, the archaeological record of the evolution and spread of music remains incomplete. Although arguments have been made for Neanderthal musical traditions and the presence of musical instruments in Middle Palaeolithic assemblages, concrete evidence to support these claims is lacking. Here we report the discovery of bone and ivory flutes from the early Aurignacian period of southwestern Germany. These finds demonstrate the presence of a well-established musical tradition at the time when modern humans colonized Europe, more than 35,000 calendar years ago. Other than the caves of the Swabian Jura, the earliest secure archaeological evidence for music comes from sites in France and Austria and post-date 30,000 years ago.

Nature advance online publication 24 June 2009. doi:10.1038/nature08117

Authors: Jenna L. Galloway, Irene Delgado, Maria A. Ros & Clifford J. Tabin

Phocomelia is a devastating, rare congenital limb malformation in which the long bones are shorter than normal, with the upper portion of the limb being most severely affected. In extreme cases, the hands or fingers are attached directly to the shoulder and the most proximal elements (those closest to the shoulder) are entirely missing. This disorder, previously known in both autosomal recessive and sporadic forms, showed a marked increase in incidence in the early 1960s due to the tragic toxicological effects of the drug thalidomide, which had been prescribed as a mild sedative. This human birth defect is mimicked in developing chick limb buds exposed to X-irradiation. Both X-irradiation and thalidomide-induced phocomelia have been interpreted as patterning defects in the context of the progress zone model, which states that a cell’s proximodistal identity is determined by the length of time spent in a distal limb region termed the ‘progress zone’. Indeed, studies of X-irradiation-induced phocomelia have served as one of the two major experimental lines of evidence supporting the validity of the progress zone model. Here, using a combination of molecular analysis and lineage tracing in chick, we show that X-irradiation-induced phocomelia is fundamentally not a patterning defect, but rather results from a time-dependent loss of skeletal progenitors. Because skeletal condensation proceeds from the shoulder to fingers (in a proximal to distal direction), the proximal elements are differentially affected in limb buds exposed to radiation at early stages. This conclusion changes the framework for considering the effect of thalidomide and other forms of phocomelia, suggesting the possibility that the aetiology lies not in a defect in the patterning process, but rather in progenitor cell survival and differentiation. Moreover, molecular evidence that proximodistal patterning is unaffected after X-irradiation does not support the predictions of the progress zone model.

Nature advance online publication 24 June 2009. doi:10.1038/nature08149

Authors: Jenny Tung, Alexander Primus, Andrew J. Bouley, Tonya F. Severson, Susan C. Alberts & Gregory A. Wray

The ecology, behaviour and genetics of our closest living relatives, the nonhuman primates, should help us to understand the evolution of our own lineage. Although a large amount of data has been amassed on primate ecology and behaviour, much less is known about the functional and evolutionary genetic aspects of primate biology, especially in wild primates. As a result, even in well-studied populations in which nongenetic factors that influence adaptively important characteristics have been identified, we have almost no understanding of the underlying genetic basis for such traits. Here, we report on the functional consequences of genetic variation at the malaria-related FY (DARC) gene in a well-studied population of yellow baboons (Papio cynocephalus) living in Amboseli National Park in Kenya. FY codes for a chemokine receptor normally expressed on the erythrocyte surface that is the known entry point for the malarial parasite Plasmodium vivax. We identified variation in the cis-regulatory region of the baboon FY gene that was associated with phenotypic variation in susceptibility to Hepatocystis, a malaria-like pathogen that is common in baboons. Genetic variation in this region also influenced gene expression in vivo in wild individuals, a result we confirmed using in vitro reporter gene assays. The patterns of genetic variation in and around this locus were also suggestive of non-neutral evolution, raising the possibility that the evolution of the FY cis-regulatory region in baboons has exhibited both mechanistic and selective parallels with the homologous region in humans. Together, our results represent the first reported association and functional characterization linking genetic variation and a complex trait in a natural population of nonhuman primates.

Nature advance online publication 21 June 2009. doi:10.1038/nature08111

Authors: Yiguo Wang, Liliana Vera, Wolfgang H. Fischer & Marc Montminy

In fasted mammals, circulating pancreatic glucagon stimulates hepatic gluconeogenesis in part through the CREB regulated transcription coactivator 2 (CRTC2, also referred to as TORC2). Hepatic glucose production is increased in obesity, reflecting chronic increases in endoplasmic reticulum (ER) stress that promote insulin resistance. Whether ER stress also modulates the gluconeogenic program directly, however, is unclear. Here we show that CRTC2 functions as a dual sensor for ER stress and fasting signals. Acute increases in ER stress triggered the dephosphorylation and nuclear entry of CRTC2, which in turn promoted the expression of ER quality control genes through an association with activating transcription factor 6 alpha (ATF6α, also known as ATF6)—an integral branch of the unfolded protein response. In addition to mediating CRTC2 recruitment to ER stress inducible promoters, ATF6α also reduced hepatic glucose output by disrupting the CREB–CRTC2 interaction and thereby inhibiting CRTC2 occupancy over gluconeogenic genes. Conversely, hepatic glucose output was upregulated when hepatic ATF6α protein amounts were reduced, either by RNA interference (RNAi)-mediated knockdown or as a result of persistent stress in obesity. Because ATF6α overexpression in the livers of obese mice reversed CRTC2 effects on the gluconeogenic program and lowered hepatic glucose output, our results demonstrate how cross-talk between ER stress and fasting pathways at the level of a transcriptional coactivator contributes to glucose homeostasis.

Nature advance online publication 17 June 2009. doi:10.1038/nature08181

Authors: A. McGarry Houghton, William O. Hartzell, Clinton S. Robbins, F. Xavier Gomis-Rüth & Steven D. Shapiro

Macrophages are aptly positioned to function as the primary line of defence against invading pathogens in many organs, including the lung and peritoneum. Their ability to phagocytose and clear microorganisms has been well documented. Macrophages possess several substances with which they can kill bacteria, including reactive oxygen species, nitric oxide, and antimicrobial proteins. We proposed that macrophage-derived proteinases may contribute to the antimicrobial properties of macrophages. Macrophage elastase (also known as matrix metalloproteinase 12 or MMP12) is an enzyme predominantly expressed in mature tissue macrophages and is implicated in several disease processes, including emphysema. Physiological functions for MMP12 have not been described. Here we show that Mmp12-/- mice exhibit impaired bacterial clearance and increased mortality when challenged with both Gram-negative and Gram-positive bacteria at macrophage-rich portals of entry, such as the peritoneum and lung. Intracellular stores of MMP12 are mobilized to macrophage phagolysosomes after the ingestion of bacterial pathogens. Once inside phagolysosomes, MMP12 adheres to bacterial cell walls where it disrupts cellular membranes resulting in bacterial death. The antimicrobial properties of MMP12 do not reside within its catalytic domain, but rather within the carboxy-terminal domain. This domain contains a unique four amino acid sequence on an exposed β loop of the protein that is required for the observed antimicrobial activity. The present study represents, to our knowledge, the first report of direct antimicrobial activity by a matrix metallopeptidase, and describes a new antimicrobial peptide that is sequentially and structurally unique in nature.

Nature advance online publication 17 June 2009. doi:10.1038/nature08102

Authors: Matthew Thomson & Jeremy Gunawardena

Reversible phosphorylation on serine, threonine and tyrosine is the most widely studied posttranslational modification of proteins. The number of phosphorylated sites on a protein (n) shows a significant increase from prokaryotes, with n ≤ 7 sites, to eukaryotes, with examples having n ≥ 150 sites. Multisite phosphorylation has many roles and site conservation indicates that increasing numbers of sites cannot be due merely to promiscuous phosphorylation. A substrate with n sites has an exponential number (2n) of phospho-forms and individual phospho-forms may have distinct biological effects. The distribution of these phospho-forms and how this distribution is regulated have remained unknown. Here we show that, when kinase and phosphatase act in opposition on a multisite substrate, the system can exhibit distinct stable phospho-form distributions at steady state and that the maximum number of such distributions increases with n. Whereas some stable distributions are focused on a single phospho-form, others are more diffuse, giving the phospho-proteome the potential to behave as a fluid regulatory network able to encode information and flexibly respond to varying demands. Such plasticity may underlie complex information processing in eukaryotic cells and suggests a functional advantage in having many sites. Our results follow from the unusual geometry of the steady-state phospho-form concentrations, which we show to constitute a rational algebraic curve, irrespective of n. We thereby reduce the complexity of calculating steady states from simulating 3 × 2n differential equations to solving two algebraic equations, while treating parameters symbolically. We anticipate that these methods can be extended to systems with multiple substrates and multiple enzymes catalysing different modifications, as found in posttranslational modification ‘codes’ such as the histone code. Whereas simulations struggle with exponentially increasing molecular complexity, mathematical methods of the kind developed here can provide a new language in which to articulate the principles of cellular information processing.

Nature advance online publication 14 June 2009. doi:10.1038/nature08118

Authors: Ivan Zanoni, Renato Ostuni, Giusy Capuano, Maddalena Collini, Michele Caccia, Antonella Ellena Ronchi, Marcella Rocchetti, Francesca Mingozzi, Maria Foti, Giuseppe Chirico, Barbara Costa, Antonio Zaza, Paola Ricciardi-Castagnoli & Francesca Granucci

Toll-like receptors (TLRs) are the best characterized pattern recognition receptors. Individual TLRs recruit diverse combinations of adaptor proteins, triggering signal transduction pathways and leading to the activation of various transcription factors, including nuclear factor κB, activation protein 1 and interferon regulatory factors. Interleukin-2 is one of the molecules produced by mouse dendritic cells after stimulation by different pattern recognition receptor agonists. By analogy with the events after T-cell receptor engagement leading to interleukin-2 production, it is therefore plausible that the stimulation of TLRs on dendritic cells may lead to activation of the Ca2+/calcineurin and NFAT (nuclear factor of activated T cells) pathway. Here we show that mouse dendritic cell stimulation with lipopolysaccharide (LPS) induces Src-family kinase and phospholipase Cγ2 activation, influx of extracellular Ca2+ and calcineurin-dependent nuclear NFAT translocation. The initiation of this pathway is independent of TLR4 engagement, and dependent exclusively on CD14. We also show that LPS-induced NFAT activation via CD14 is necessary to cause the apoptotic death of terminally differentiated dendritic cells, an event that is essential for maintaining self-tolerance and preventing autoimmunity. Consequently, blocking this pathway in vivo causes prolonged dendritic cell survival and an increase in T-cell priming capability. Our findings reveal novel aspects of molecular signalling triggered by LPS in dendritic cells, and identify a new role for CD14: the regulation of the dendritic cell life cycle through NFAT activation. Given the involvement of CD14 in disease, including sepsis and chronic heart failure, the discovery of signal transduction pathways activated exclusively via CD14 is an important step towards the development of potential treatments involving interference with CD14 functions.

Nature advance online publication 10 June 2009. doi:10.1038/nature08099

Authors: Olaia Naveiras, Valentina Nardi, Pamela L. Wenzel, Peter V. Hauschka, Frederic Fahey & George Q. Daley

Osteoblasts and endothelium constitute functional niches that support haematopoietic stem cells in mammalian bone marrow. Adult bone marrow also contains adipocytes, the number of which correlates inversely with the haematopoietic activity of the marrow. Fatty infiltration of haematopoietic red marrow follows irradiation or chemotherapy and is a diagnostic feature in biopsies from patients with marrow aplasia. To explore whether adipocytes influence haematopoiesis or simply fill marrow space, we compared the haematopoietic activity of distinct regions of the mouse skeleton that differ in adiposity. Here we show, by flow cytometry, colony-forming activity and competitive repopulation assay, that haematopoietic stem cells and short-term progenitors are reduced in frequency in the adipocyte-rich vertebrae of the mouse tail relative to the adipocyte-free vertebrae of the thorax. In lipoatrophic A-ZIP/F1 ‘fatless’ mice, which are genetically incapable of forming adipocytes, and in mice treated with the peroxisome proliferator-activated receptor-γ inhibitor bisphenol A diglycidyl ether, which inhibits adipogenesis, marrow engraftment after irradiation is accelerated relative to wild-type or untreated mice. These data implicate adipocytes as predominantly negative regulators of the bone-marrow microenvironment, and indicate that antagonizing marrow adipogenesis may enhance haematopoietic recovery in clinical bone-marrow transplantation.

Nature advance online publication 07 June 2009. doi:10.1038/nature08136

Authors: Neil J. Ganem, Susana A. Godinho & David Pellman

Chromosomal instability (CIN) is a hallmark of many tumours and correlates with the presence of extra centrosomes. However, a direct mechanistic link between extra centrosomes and CIN has not been established. It has been proposed that extra centrosomes generate CIN by promoting multipolar anaphase, a highly abnormal division that produces three or more aneuploid daughter cells. Here we use long-term live-cell imaging to demonstrate that cells with multiple centrosomes rarely undergo multipolar cell divisions, and the progeny of these divisions are typically inviable. Thus, multipolar divisions cannot explain observed rates of CIN. In contrast, we observe that CIN cells with extra centrosomes routinely undergo bipolar cell divisions, but display a significantly increased frequency of lagging chromosomes during anaphase. To define the mechanism underlying this mitotic defect, we generated cells that differ only in their centrosome number. We demonstrate that extra centrosomes alone are sufficient to promote chromosome missegregation during bipolar cell division. These segregation errors are a consequence of cells passing through a transient ‘multipolar spindle intermediate’ in which merotelic kinetochore–microtubule attachment errors accumulate before centrosome clustering and anaphase. These findings provide a direct mechanistic link between extra centrosomes and CIN, two common characteristics of solid tumours. We propose that this mechanism may be a common underlying cause of CIN in human cancer.

Nature advance online publication 03 June 2009. doi:10.1038/nature08100

Authors: Greta Guarda, Catherine Dostert, Francesco Staehli, Katrin Cabalzar, Rosa Castillo, Aubry Tardivel, Pascal Schneider & Jürg Tschopp

Inflammation is a protective attempt by the host to remove injurious stimuli and initiate the tissue healing process. The inflammatory response must be actively terminated, however, because failure to do so can result in ‘bystander’ damage to tissues and diseases such as arthritis or type-2 diabetes. Yet the mechanisms controlling excessive inflammatory responses are still poorly understood. Here we show that mouse effector and memory CD4+ T cells abolish macrophage inflammasome-mediated caspase-1 activation and subsequent interleukin 1β release in a cognate manner. Inflammasome inhibition is observed for all tested NLRP1 (commonly called NALP1) and NLRP3 (NALP3 or cryopyrin) activators, whereas NLRC4 (IPAF) inflammasome function and release of other inflammatory mediators such as CXCL2, interleukin 6 and tumour necrosis factor are not affected. Suppression of the NLRP3 inflammasome requires cell-to-cell contact and can be mimicked by macrophage stimulation with selected ligands of the tumour necrosis factor family, such as CD40L (also known as CD40LG). In a NLRP3-dependent peritonitis model, effector CD4+ T cells are responsible for decreasing neutrophil recruitment in an antigen-dependent manner. Our findings reveal an unexpected mechanism of inflammasome inhibition, whereby effector and memory T cells suppress potentially damaging inflammation, yet leave the primary inflammatory response, crucial for the onset of immunity, intact.

Nature advance online publication 03 June 2009. doi:10.1038/nature08084

Authors: Rebecca A. Mosher, Charles W. Melnyk, Krystyna A. Kelly, Ruth M. Dunn, David J. Studholme & David C. Baulcombe

Most eukaryotes produce small RNA (sRNA) mediators of gene silencing that bind to Argonaute proteins and guide them, by base pairing, to an RNA target. MicroRNAs (miRNAs) that normally target messenger RNAs for degradation or translational arrest are the best-understood class of sRNAs. However, in Arabidopsis thaliana flowers, miRNAs account for only 5% of the sRNA mass and less than 0.1% of the sequence complexity. The remaining sRNAs form a complex population of more than 100,000 different small interfering RNAs (siRNAs) transcribed from thousands of loci. The biogenesis of most of the siRNAs in Arabidopsis are dependent on RNA polymerase IV (PolIV), a homologue of DNA-dependent RNA polymerase II. A subset of these PolIV-dependent (p4)-siRNAs are involved in stress responses, and others are associated with epigenetic modifications to DNA or chromatin; however, the biological role is not known for most of them. Here we show that the predominant phase of p4-siRNA accumulation is initiated in the maternal gametophyte and continues during seed development. Expression of p4-siRNAs in developing endosperm is specifically from maternal chromosomes. Our results provide the first evidence for a link between genomic imprinting and RNA silencing in plants.

Nature advance online publication 31 May 2009. doi:10.1038/nature08086

Authors: Keisuke Nimura, Kiyoe Ura, Hidetaka Shiratori, Masato Ikawa, Masaru Okabe, Robert J. Schwartz & Yasufumi Kaneda

Diverse histone modifications are catalysed and recognized by various specific proteins, establishing unique modification patterns that act as transcription signals. In particular, histone H3 trimethylation at lysine 36 (H3K36me3) is associated with actively transcribed regions and has been proposed to provide landmarks for continuing transcription; however, the control mechanisms and functions of H3K36me3 in higher eukaryotes are unknown. Here we show that the H3K36me3-specific histone methyltransferase (HMTase) Wolf–Hirschhorn syndrome candidate 1 (WHSC1, also known as NSD2 or MMSET) functions in transcriptional regulation together with developmental transcription factors whose defects overlap with the human disease Wolf–Hirschhorn syndrome (WHS). We found that mouse Whsc1, one of five putative Set2 homologues, governed H3K36me3 along euchromatin by associating with the cell-type-specific transcription factors Sall1, Sall4 and Nanog in embryonic stem cells, and Nkx2-5 in embryonic hearts, regulating the expression of their target genes. Whsc1-deficient mice showed growth retardation and various WHS-like midline defects, including congenital cardiovascular anomalies. The effects of Whsc1 haploinsufficiency were increased in Nkx2-5 heterozygous mutant hearts, indicating their functional link. We propose that WHSC1 functions together with developmental transcription factors to prevent the inappropriate transcription that can lead to various pathophysiologies.

Nature advance online publication 20 May 2009. doi:10.1038/nature08079

Authors: Suzana Hadjur, Luke M. Williams, Natalie K. Ryan, Bradley S. Cobb, Tom Sexton, Peter Fraser, Amanda G. Fisher & Matthias Merkenschlager

Cohesin-mediated sister chromatid cohesion is essential for chromosome segregation and post-replicative DNA repair. In addition, evidence from model organisms and from human genetics suggests that cohesin is involved in the control of gene expression. This non-canonical role has recently been rationalized by the findings that mammalian cohesin complexes are recruited to a subset of DNase I hypersensitive sites and to conserved noncoding sequences by the DNA-binding protein CTCF. CTCF functions at insulators (which control interactions between enhancers and promoters) and at boundary elements (which demarcate regions of distinct chromatin structure), and cohesin contributes to its enhancer-blocking activity. The underlying mechanisms remain unknown, and the full spectrum of cohesin functions remains to be determined. Here we show that cohesin forms the topological and mechanistic basis for cell-type-specific long-range chromosomal interactions in cis at the developmentally regulated cytokine locus IFNG. Hence, the ability of cohesin to constrain chromosome topology is used not only for the purpose of sister chromatid cohesion, but also to dynamically define the spatial conformation of specific loci. This new aspect of cohesin function is probably important for normal development and disease.