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The discovery of part of a 55,000-year-old human skull in Israel will help to answer some questions about our species’ evolution — but it shows that the tale is complicated.
A few Republicans agreeing with basic climate research is not an environmental victory.
Technicians are often under appreciated, but without them there could be no research.
Drug cheats will not be tackled properly until anti-doping agencies do more to assess the scale of the problem scientifically, says Roger Pielke Jr.
Dams have altered 48% of all river flow worldwide. And if all dams planned for the next few decades are built, that proportion will nearly double.Günther Grill of McGill University in Montreal, Canada, and his team developed two ways to analyse how dams break
Mice live longer— and seem to age more slowly — if they express lower levels of a cancer-promoting protein called MYC.High levels of MYC favour tumour growth, but some expression of the protein is required for survival. John Sedivy of Brown University in
Researchers have measured the aerodynamic forces of a bird flying inside a box.Until now, the aerodynamic lift achieved by free-flying animals has only been estimated using models. David Lentink of Stanford University in California and his team built an enclosed device to directly measure
Chemists have scrutinized a classic piece of bench chemistry— the explosion that happens when sodium metal hits water — and revised the thinking of how it works.On contact with water, the metal produces sodium hydroxide, hydrogen and heat, which was thought to ignite
Weather extremes could become more common as the climate warms this century, because extreme cooling events in the Pacific Ocean are predicted to occur more often.La Niña events occur when the equatorial Pacific cools, causing droughts and floods worldwide. Wenju Cai of the Commonwealth
The hands of hominins that lived about 3 million years ago were capable of clutching tools.The first tool-using hominin is widely believed to have been Homo habilis— known as the handyman — in part because its appearance in the fossil record 2.4
Female pumas that live near human populations hunt more often but spend less time eating their prey than do those in less populated areas.Humans can cause declines in wildlife populations, but their effect on animal behaviour is less well understood. Justine Smith and her
The ageing pipeline infrastructure of Boston, Massachusetts, is leaking natural gas— mostly methane, a potent greenhouse gas — at more than double the rate of previous estimates.Atmospheric methane levels had plateaued but have been growing worldwide since 2007, for reasons that are unclear.
A genetic mutation in single-celled yeast turns it into a multicellular organism— hinting at how multicellularity might have evolved.William Ratcliff at the Georgia Institute of Technology in Atlanta and his co-workers studied a strain of yeast (Saccharomyces cerevisiae) in which the
A blog post urging scientists to write elegantly triggers online discussion.
The week in science: Obama announces precision-medicine push, marijuana research endorsed by paediatric group, and Israel frees jailed Palestinian physicist.
US president defies hostile Congress and takes action on global warming.
Rosetta mission would have to sacrifice other science to search for comet lander.
Study on decision-making stokes controversy over power of distracted mind.
Major report calls for cuts to infrastructure, including fledgling Ocean Observatories Initiative, to increase spending on science.
Announcement by President Obama comes amidst growing interest in targeted therapies.
Cranium discovery shows that Homo sapiens was living in Middle East 55,000 years ago.
Research relies on unsung heroes working behind the scenes— and some of them have rather unusual jobs.
Until violence eases and fieldwork can resume, fund research in labs, museums and on computers, urges Savino di Lernia.
Herbert Gintis applauds two books that powerfully enrich the dialogue on behavioural science.
Barbara Kiser reviews five of the week's best science picks.
The American Association for the Advancement of Science (AAAS) has chosen Rush Holt, who was a Democratic congressman for eight terms, as its new chief executive. Daniel Sarewitz attacks this choice as“political” (Nature516, 9;10.1038/516009a2014), but it is
You claim that the most recent estimates of future output for shale gas in the United States have become more conservative, but in our view this is a red herring (Nature516, 28–30;10.1038/516028a2014).State-of-the-art projections for the
We believe that your comparison of US fracking forecasts creates a false dichotomy between modelling results from the US Energy Information Administration (EIA) and the Bureau of Economic Geology at the University of Texas at Austin (Nature516, 28–30;10.1038/516028a
Some 500 years after Leonardo da Vinci declared that more was known about celestial bodies than about the soil underfoot, the United Nations has proclaimed 2015 the International Year of Soils. This offers a unique opportunity to address the crisis in soil sustainability (see www.fao.org/soils-2015
Discoverer of hormones that regulate blood-cell proliferation.
Where to go to get advice on finding a job.
University of California strives to make database of job options
Granting agency distributes the wealth
Poor mentoring holds back minorities
Is this the real life?
The News Feature‘Laser focus’ (Nature517, 430–432; 2015) gave the wrong amount for the funding behind the Center for Adaptive Optics. The grant was in fact for around US$40 million over 10 years.
The 'West Asia' article in the Nature Index (Nature515, S88–S89; 2014) stated that King Abdullah University of Science and Technology had an article count of 121 and a weighted fractional count of 9.96. In fact, it was King
Influenza virus severely damages the epithelial tissue that lines the lung. Findings suggest that, in mice, activation of a back-up population of stem cells mediates effective repair of the injured lung. See Letters p.616 aamp; p.621
A link between rotation and age for Sun-like stars has long been known, but a stringent test of it for older stars has been lacking. The Kepler mission helps to fill this gap with observations of an old star cluster. See Letter p.589
The identification of the gene regulatory network that controls the formation of xylem— the major component of wood — opens up new avenues for manipulating plant biomass. See Article p.571
Splitting and recombining an electron wave packet has been used to test relativity at a record sensitivity. The result heralds an era of precision measurements of relativity using quantum-information methods. See Letter p.592
The repurposing of a bacterial defence system known as CRISPR into a potent activator of gene expression in human cells enables powerful studies of gene function, as exemplified in cancer cells. See Article p.583
A combination of simulations and data shows that short-term climate trends are dominated by natural internal variations, providing a basis for climate forecasting, but not for assessing sensitivity to forced changes. See Article p.565
The discovery that the estimated number of stem-cell divisions in a tissue correlates with cancer incidence suggests that the varying probability of developing cancer in different tissues is mostly down to random mutations.
Most present-generation climate models simulate an increase in global-mean surface temperature (GMST) since 1998, whereas observations suggest a warming hiatus. It is unclear to what extent this mismatch is caused by incorrect model forcing, by incorrect model response to forcing or by random factors. Here
The plant cell wall is an important factor for determining cell shape, function and response to the environment. Secondary cell walls, such as those found in xylem, are composed of cellulose, hemicelluloses and lignin and account for the bulk of plant biomass. The coordination between
The Cancer Genome Atlas profiled 279 head and neck squamous cell carcinomas (HNSCCs) to provide a comprehensive landscape of somatic genomic alterations. Here we show that human-papillomavirus-associated tumours are dominated by helical domain mutations of the oncogene PIK3CA, novel alterations involving loss of TRAF3
Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate
The ages of the most common stars—low-mass (cool) stars like the Sun, and smaller—are difficult to derive because traditional dating methods use stellar properties that either change little as the stars age or are hard to measure. The rotation rates of all cool stars decrease substantially with time as the stars steadily lose their angular momenta. If properly calibrated, rotation therefore can act as a reliable determinant of their ages based on the method of gyrochronology. To calibrate gyrochronology, the relationship between rotation period and age must be determined for cool stars of different masses, which is best accomplished with rotation period measurements for stars in clusters with well-known ages. Hitherto, such measurements have been possible only in clusters with ages of less than about one billion years, and gyrochronology ages for older stars have been inferred from model predictions. Here we reportrotation period measurements for 30 cool stars in the 2.5-billion-year-old cluster NGC 6819. The periods reveal a well-defined relationship between rotation period and stellar mass at the cluster age, suggesting that ages with a precision of order 10 per cent can be derived for large numbers of cool Galactic field stars.
All evidence so far suggests that the absolute spatial orientation of an experiment never affects its outcome. This is reflected in the standard model of particle physics by requiring all particles and fields to be invariant under Lorentz transformations. The best-known tests of this important cornerstone of physics are Michelson–Morley-type experiments verifying the isotropy of the speed of light. For matter, Hughes–Drever-type experiments test whether the kinetic energy of particles is independent of the direction of their velocity, that is, whether their dispersion relations are isotropic. To provide more guidance for physics beyond the standard model, refined experimental verifications of Lorentz symmetry are desirable. Here we search for violation of Lorentz symmetry for electrons by performing an electronic analogue of a Michelson–Morley experiment. We split an electron wave packet bound inside a calcium ion into two parts with different orientations and recombine them after a time evolution of 95 milliseconds. As the Earth rotates, the absolute spatial orientation of the two parts of the wave packet changes, and anisotropies in the electron dispersion will modify the phase of the interference signal. To remove noise, we prepare a pair of calcium ions in a superposition of two decoherence-free states, thereby rejecting magnetic field fluctuations common to both ions. After a 23-hour measurement, we find a limit of h × 11 millihertz (h is Planck’s constant) on the energy variations, verifying the isotropy of the electron’s dispersion relation at the level of one part in 1018, a 100-fold improvement on previous work. Alternatively, we can interpret our result as testing the rotational invariance of the Coulomb potential. Assuming that Lorentz symmetry holds for electrons and that the photon dispersion relation governs the Coulomb force, we obtain a fivefold-improved limit on anisotropies in the speed of light. Our result probes Lorentz symmetry violation at levels comparable to the ratio between the electroweak and Planck energy scales. Our experiment demonstrates the potential of quantum information techniques in the search for physics beyond the standard model.
Hydrophobic particles in water and hydrophilic particles in oil aggregate, but can form colloidal dispersions if their surfaces are chemically camouflaged with surfactants, organic tethers, adsorbed polymers or other particles that impart affinity for the solvent and increase interparticle repulsion. A different strategy for modulating the interaction between a solid and a liquid uses surface corrugation, which gives rise to unique wetting behaviour. Here we show that this topographical effect can also be used to disperse particles in a wide range of solvents without recourse to chemicals to camouflage the particles’ surfaces: we produce micrometre-sized particles that are coated with stiff, nanoscale spikes and exhibit long-term colloidal stability in both hydrophilic and hydrophobic media. We find that these ‘hedgehog’ particles do not interpenetrate each other with their spikes, which markedly decreases the contact area between the particles and, therefore, the attractive forces between them. The trapping of air in aqueous dispersions, solvent autoionization at highly developed interfaces, and long-range electrostatic repulsion in organic media also contribute to the colloidal stability of our particles. The unusual dispersion behaviour of our hedgehog particles, overturning the notion that like dissolves like, might help to mitigate adverse environmental effects of the use of surfactants and volatile organic solvents, and deepens our understanding of interparticle interactions and nanoscale colloidal chemistry.
Many enzymes oxidize unactivated aliphatic C–H bonds selectively to form alcohols; however, biological systems do not possess enzymes that catalyse the analogous aminations of C–H bonds. The absence of such enzymes limits the discovery of potential medicinal candidates because nitrogen-containing groups are crucial to the biological activity of therapeutic agents and clinically useful natural products. In one prominent example illustrating the importance of incorporating nitrogen-based functionality, the conversion of the ketone of erythromycin to the –N(Me)CH2– group in azithromycin leads to a compound that can be dosed once daily with a shorter treatment time. For such reasons, synthetic chemists have sought catalysts that directly convert C–H bonds to C–N bonds. Most currently used catalysts for C–H bond amination are ill suited to the intermolecular functionalization of complex molecules because they require excess substrate or directing groups, harsh reaction conditions, weak or acidic C–H bonds, or reagents containing specialized groups on the nitrogen atom. Among C–H bond amination reactions, those forming a C–N bond at a tertiary alkyl group would be particularly valuable, because this linkage isdifficult to form from ketones or alcohols that might be created in a biosynthetic pathway by oxidation. Here we report a mild, selective, iron-catalysed azidation of tertiary C–H bonds that occurs without excess of the valuable substrate. The reaction tolerates aqueous environments and is suitable for the functionalization of complex structures in the late stages of a multistep synthesis. Moreover, this azidation makes it possible to install a range of nitrogen-based functional groups, including those from Huisgen ‘click’ cycloadditions and the Staudinger ligation. We anticipate that these reactions will create opportunities to modify natural products, their precursors and their derivatives to produce analogues that contain different polarity and charge as a result of nitrogen-containing groups. It could also be used to help identify targets of biologically active molecules by creating a point of attachment—for example, to fluorescent tags or ‘handles’ for affinity chromatography—directly on complex molecular structures.
Earth’s magnetic field has been thought to arise from thermal convection of molten iron alloy in the outer core, but recent density functional theory calculations have suggested that the conductivity of iron is too high to support thermal convection, resulting in the investigation of chemically drivenconvection. These calculations for resistivity were based on electron–phonon scattering. Here we apply self-consistent density functional theory plus dynamical mean-field theory (DFT + DMFT) to iron and find that at high temperatures electron–electron scattering is comparable to the electron–phonon scattering, bringing theory into agreement with experiments and solving the transport problem in Earth’s core. The conventional thermal dynamo picture is safe. We find that electron–electron scattering of d electrons is important at high temperatures in transition metals, in contrast to textbook analyses since Mott, and that 4s electron contributions to transport are negligible, in contrast to numerous models used for over fifty years. The DFT+DMFT method should be applicable to other high-temperature systems where electron correlations are important.
The human genome is arguably the most complete mammalian reference assembly, yet more than 160 euchromatic gaps remain and aspects of its structural variation remain poorly understood ten years after its completion. To identify missing sequence and genetic variation, here we sequence and analyse a haploid human genome (CHM1) using single-molecule, real-time DNA sequencing. We close or extend 55% of the remaining interstitial gaps in the human GRCh37 reference genome—78% of which carried long runs of degenerate short tandem repeats, often several kilobases in length, embedded within (G+C)-rich genomic regions. We resolve the complete sequence of 26,079 euchromatic structural variants at the base-pair level, including inversions, complex insertions and long tracts of tandem repeats. Most have not been previously reported, with the greatest increases in sensitivity occurring for events less than 5 kilobases in size. Compared to the human reference, we find a significant insertional bias (3:1) in regions corresponding to complex insertions and long shorttandem repeats. Our results suggest a greater complexity of the human genome in the form of variation of longer and more complex repetitive DNA that can now be largely resolved with the application of this longer-read sequencing technology.
Pathogenic mycobacteria induce the formation of complex cellular aggregates called granulomas that are the hallmark of tuberculosis. Here we examine the development and consequences of vascularization of the tuberculous granuloma in the zebrafish–Mycobacterium marinum infection model, which is characterized by organized granulomas with necrotic cores that bear striking resemblance to those of human tuberculosis. Using intravital microscopy in the transparent larval zebrafish, we show that granuloma formation is intimately associated withangiogenesis. The initiation of angiogenesis in turn coincides with the generation of local hypoxia and transcriptional induction of the canonical pro-angiogenic molecule Vegfaa. Pharmacological inhibition of the Vegf pathway suppresses granuloma-associated angiogenesis, reduces infection burden and limits dissemination. Moreover, anti-angiogenic therapies synergize with the first-line anti-tubercular antibiotic rifampicin, as well as with the antibiotic metronidazole, which targets hypoxic bacterial populations. Our data indicate that mycobacteria induce granuloma-associated angiogenesis, which promotes mycobacterial growth and increases spread of infection to new tissue sites. We propose the use of anti-angiogenic agents, now being used in cancer regimens, as a host-targeting tuberculosis therapy, particularly in extensively drug-resistant disease for which current antibiotic regimensare largely ineffective.
Lung diseases such as chronic obstructive pulmonary disease and pulmonary fibrosis involve the progressive and inexorable destruction of oxygen exchange surfaces and airways, and have emerged as a leading cause of death worldwide. Mitigating therapies, aside from impractical organ transplantation, remain limited and the possibility of regenerative medicine has lacked empirical support. However, it is clinically known that patients who survive sudden, massive loss of lung tissue from necrotizing pneumonia or acute respiratory distress syndrome often recover full pulmonary function within six months. Correspondingly, we recently demonstrated lung regeneration in mice following H1N1 influenza virus infection, and linked distal airway stem cells expressing Trp63 (p63) and keratin 5, called DASCp63/Krt5, to this process. Here we show that pre-existing, intrinsically committed DASCp63/Krt5 undergo a proliferative expansion in response to influenza-induced lung damage, and assemble into nascent alveoli at sites of interstitial lung inflammation. We also show that the selective ablation of DASCp63/Krt5in vivo prevents this regeneration, leading to pre-fibrotic lesions and deficient oxygen exchange. Finally, we demonstrate that single DASCp63/Krt5-derived pedigrees differentiate to type I and type II pneumocytes as well as bronchiolar secretory cells following transplantation to infected lung and also minimize the structural consequences of endogenous stem cell loss on this process. The ability to propagate these cells in culture while maintaining their intrinsic lineage commitment suggests their potential in stem cell-based therapies for acute and chronic lung diseases.
Broadly, tissue regeneration is achieved in two ways: by proliferation of common differentiated cells and/or by deployment of specialized stem/progenitor cells. Which of these pathways applies is both organ- and injury-specific. Current models in the lung posit that epithelial repair can be attributed to cells expressing mature lineage markers. By contrast, here we define the regenerative role of previously uncharacterized, rare lineage-negative epithelial stem/progenitor (LNEP) cells present within normal distal lung. Quiescent LNEPs activate aΔNp63 (a p63 splice variant) and cytokeratin 5 remodelling program after influenza or bleomycin injury in mice. Activated cells proliferate and migrate widely to occupy heavily injured areas depleted of mature lineages, at which point they differentiate towards mature epithelium. Lineage tracing revealed scant contribution of pre-existing mature epithelial cells in such repair, whereas orthotopic transplantation of LNEPs, isolated by a definitive surface profile identified through single-cell sequencing, directly demonstrated the proliferative capacity and multipotency of this population. LNEPs require Notch signalling to activate the ΔNp63 and cytokeratin 5 program, and subsequent Notch blockade promotes an alveolar cell fate. Persistent Notch signalling after injury led to parenchymal ‘micro-honeycombing’ (alveolar cysts), indicative of failed regeneration. Lungs from patients with fibrosis show analogous honeycomb cysts with evidence of hyperactive Notch signalling. Our findings indicate that distinct stem/progenitor cell pools repopulate injured tissue depending on the extent of the injury, and the outcomes of regeneration or fibrosis may depend in part on the dynamics of LNEP Notch signalling.
TP53 is commonly altered in human cancer, and Tp53 reactivation suppresses tumours in vivo in mice (TP53 and Tp53 are also known as p53). This strategy has proven difficult to implement therapeutically, and here we examine an alternative strategy by manipulating the p53 family members, Tp63 and Tp73 (also known as p63 and p73, respectively). The acidic transactivation-domain-bearing (TA) isoforms of p63 and p73 structurally and functionally resemble p53, whereas theΔN isoforms (lacking the acidic transactivation domain) of p63 and p73 are frequently overexpressed in cancer and act primarily in a dominant-negative fashion against p53, TAp63 and TAp73 to inhibit their tumour-suppressive functions. The p53 family interacts extensively in cellular processes thatpromote tumour suppression, such as apoptosis and autophagy, thus a clear understanding of this interplay in cancer is needed to treat tumours with alterations in the p53 pathway. Here we show that deletion of the ΔN isoforms of p63 or p73 leads to metabolic reprogramming and regression of p53-deficient tumours through upregulation of IAPP, the gene that encodes amylin, a 37-amino-acid peptide co-secreted with insulin by the β cells of the pancreas. We found that IAPP is causally involved in this tumour regression and that amylin functions through the calcitonin receptor (CalcR) and receptor activity modifying protein 3 (RAMP3) to inhibit glycolysis and induce reactive oxygen species and apoptosis. Pramlintide, a synthetic analogue of amylin that is currently used to treat type 1 and type 2 diabetes, caused rapid tumour regression in p53-deficient thymic lymphomas, representing a novel strategy to target p53-deficient cancers.
The spindle assembly checkpoint (SAC) maintains genomic stability by delaying chromosome segregation until the last chromosome has attached to the mitotic spindle. The SAC prevents the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase from recognizing cyclin B and securin by catalysing the incorporation of the APC/C co-activator, CDC20, into a complex called the mitotic checkpoint complex (MCC). The SAC works through unattached kinetochores generating a diffusible‘wait anaphase’ signal that inhibits the APC/C in the cytoplasm, but the nature of this signal remains a key unsolved problem. Moreover, the SAC and the APC/C are highly responsive to each other: the APC/C quickly targets cyclin B and securin once all the chromosomes attach in metaphase, but israpidly inhibited should kinetochore attachment be perturbed. How this is achieved is also unknown. Here, we show that the MCC can inhibit a second CDC20 that has already bound and activated the APC/C. We show how the MCC inhibits active APC/C and that this is essential for the SAC. Moreover, this mechanism can prevent anaphase in the absence of kinetochore signalling. Thus, we propose that the diffusible ‘wait anaphase’ signal could be the MCC itself, and explain how reactivating the SAC can rapidly inhibit active APC/C.
Oxidative stress promotes genomic instability and human diseases. A common oxidized nucleoside is 8-oxo-7,8-dihydro-2′-deoxyguanosine, which is found both in DNA (8-oxo-G) and as a free nucleotide (8-oxo-dGTP). Nucleotide pools are especially vulnerable to oxidative damage. Therefore cells encode an enzyme (MutT/MTH1) that removes free oxidized nucleotides. This cleansing function is required for cancer cell survival and to modulate Escherichia coli antibiotic sensitivity in a DNA polymerase (pol)-dependent manner. How polymerases discriminate between damaged and non-damaged nucleotides is not well understood. This analysis is essential given the role of oxidized nucleotides in mutagenesis, cancer therapeutics, and bacterial antibiotics. Even with cellular sanitizing activities, nucleotide pools contain enough 8-oxo-dGTP to promote mutagenesis. This arises from the dual coding potential where 8-oxo-dGTP(anti) base pairs with cytosine and 8-oxo-dGTP(syn) uses its Hoogsteen edge to base pair with adenine. Here we use time-lapse crystallography to follow 8-oxo-dGTP insertion opposite adenine or cytosine with human pol β, to reveal that insertion is accommodated in either the syn- or anti-conformation, respectively. For 8-oxo-dGTP(anti) insertion, a novel divalent metal relieves repulsive interactions between the adducted guanine base and the triphosphate of the oxidized nucleotide. With either templating base, hydrogen-bonding interactions between the bases are lost as the enzyme reopens after catalysis, leading to a cytotoxic nicked DNA repair intermediate. Combining structural snapshots with kinetic and computational analysis reveals how 8-oxo-dGTP uses charge modulation during insertion that can lead to a blocked DNA repair intermediate.
DNA methylation is an important epigenetic modification that is essential for various developmental processes through regulating gene expression, genomic imprinting, and epigenetic inheritance. Mammalian genomic DNA methylation is established during embryogenesis by de novo DNA methyltransferases, DNMT3A and DNMT3B, and the methylation patterns vary with developmental stages and cell types. DNA methyltransferase 3-like protein (DNMT3L) is a catalytically inactive paralogue of DNMT3 enzymes, which stimulates the enzymatic activity of Dnmt3a. Recent studies have established a connection between DNA methylation and histone modifications, and revealed a histone-guided mechanism for the establishment of DNA methylation. The ATRX–DNMT3–DNMT3L (ADD) domain of Dnmt3a recognizes unmethylated histone H3 (H3K4me0). The histone H3 tail stimulates the enzymatic activity of Dnmt3a in vitro, whereas the molecular mechanism remains elusive. Here we show that DNMT3A exists in an autoinhibitory form and that the histone H3 tail stimulates its activity in a DNMT3L-independent manner. We determine the crystal structures of DNMT3A–DNMT3L (autoinhibitory form) and DNMT3A–DNMT3L-H3 (active form) complexes at 3.82 and 2.90 Å resolution, respectively. Structural and biochemical analyses indicate that the ADD domain of DNMT3A interacts with and inhibits enzymatic activity of the catalytic domain (CD) through blocking its DNA-binding affinity. Histone H3 (but not H3K4me3) disrupts ADD–CD interaction, induces a large movement of the ADD domain, and thus releases the autoinhibition of DNMT3A. The finding adds another layer of regulation of DNA methylation to ensure that the enzyme is mainly activated at proper targeting loci when unmethylated H3K4 is present, and strongly supports a negative correlation between H3K4me3 and DNA methylation across the mammalian genome. Our study provides a new insight into an unexpected autoinhibition and histone H3-induced activation of the de novo DNA methyltransferase after its initial genomic positioning.
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Non-small-cell lung cancer is the leading cause of cancer-related death worldwide. Chemotherapies such as the topoisomerase II (TopoII) inhibitor etoposide effectively reduce disease in a minority of patients with this cancer; therefore, alternative drug targets, including epigenetic enzymes, are under consideration for therapeutic intervention. A promising potential epigenetic target is the methyltransferase EZH2, which in the context of the polycomb repressive complex 2 (PRC2) is well known to tri-methylate histone H3 at lysine 27 (H3K27me3) and elicit gene silencing. Here we demonstrate that EZH2 inhibition has differential effects on the TopoII inhibitor response of non-small-cell lung cancers in vitro and in vivo. EGFR and BRG1 mutations are genetic biomarkers that predict enhanced sensitivity to TopoII inhibitor in response to EZH2 inhibition. BRG1 loss-of-function mutant tumours respond to EZH2 inhibition with increased S phase, anaphase bridging, apoptosis and TopoII inhibitor sensitivity. Conversely, EGFR and BRG1 wild-type tumours upregulate BRG1 in response to EZH2 inhibition and ultimately become more resistant to TopoII inhibitor. EGFR gain-of-function mutant tumours are also sensitive to dual EZH2 inhibition and TopoII inhibitor, because of genetic antagonism between EGFR and BRG1. These findings suggest an opportunity for precision medicine in the genetically complex disease of non-small-cell lung cancer.
A key event in human evolution is the expansion of modern humans of African origin across Eurasia between 60 and 40 thousand years (kyr) before present (bp), replacing all other forms of hominins. Owing to the scarcity of human fossils from this period, these ancestors of all present-day non-African modern populations remain largely enigmatic. Here we describe a partial calvaria, recently discovered at Manot Cave (Western Galilee, Israel) and dated to 54.7 ± 5.5 kyr bp (arithmetic mean ± 2 standard deviations) by uranium–thorium dating, that sheds light on this crucial event. The overall shape and discrete morphological features of the Manot 1 calvaria demonstrate that this partial skull is unequivocally modern. It is similar in shape to recent African skulls as well as to European skulls from the Upper Palaeolithic period, but different from most other early anatomically modern humans in the Levant. This suggests that the Manot people could be closely related to the first modern humans who later successfully colonized Europe. Thus, the anatomical features used to support the ‘assimilation model’ in Europe might not have been inherited from European Neanderthals, but rather from earlier Levantine populations. Moreover, at present, Manot 1 is the only modern human specimen to provide evidence that during the Middle to Upper Palaeolithic interface, both modern humans and Neanderthals contemporaneously inhabited the southern Levant, close in time to the likely interbreeding event with Neanderthals.
The emRiboSeq sequencing method is used to track polymerase activity genome-wide in vivo; despite Okazaki fragment processing, DNA synthesized by error-prone polymerase-α (Pol-α) is retained in vivo and comprises ∼1.5% of the genome, establishing Pol-α as an important source of genomic variability and providing a mechanism for site-specific variation in nucleotide substitution rates.
Non-ribosomal peptide synthetases are giant enzymes composed of modules that house repeated sets of functional domains, which select, activate and couple amino acids drawn from a pool of nearly 500 potential building blocks. The structurally and stereochemically diverse peptides generated in this manner underlie the biosynthesis of a large sector of natural products. Many of their derived metabolites are bioactive such as the antibiotics vancomycin, bacitracin, daptomycin and theβ-lactam-containing penicillins, cephalosporins and nocardicins. Penicillins and cephalosporins are synthesized from a classically derived non-ribosomal peptide synthetase tripeptide (from δ-(l-α-aminoadipyl)–l-cysteinyl–d-valine synthetase). Here we report an unprecedented non-ribosomal peptide synthetase activity that both assembles a serine-containing peptide and mediates its cyclization to the critical β-lactam ring of the nocardicin family of antibiotics. A histidine-rich condensation domain, which typically performs peptide bond formation during product assembly, also synthesizes the embedded four-membered ring. We propose a mechanism, and describe supporting experiments, that is distinct from the pathways that have evolved to the three other β-lactam antibiotic families: penicillin/cephalosporins, clavams and carbapenems. These findings raise the possibility that β-lactam rings can be regio- and stereospecifically integrated into engineered peptides for application as, for example, targeted protease inactivators.
Infectious agents develop intricate mechanisms to interact with host cell pathways and hijack their genetic and epigenetic machinery to change host cell phenotypic states. Among the Apicomplexa phylum of obligate intracellular parasites, which cause veterinary and human diseases, Theileria is the only genus that transforms its mammalian host cells. Theileria infection of bovine leukocytes induces proliferative and invasive phenotypes associated with activated signalling pathways, notably JNK and AP-1 (ref. 2). The transformed phenotypes are reversed by treatment with the theilericidal drug buparvaquone. We used comparative genomics to identify a homologue of the peptidyl-prolyl isomerase PIN1 in T. annulata (TaPIN1) that is secreted into the host cell and modulates oncogenic signalling pathways. Here we show that TaPIN1 is a bona fide prolyl isomerase and that it interacts with the host ubiquitin ligase FBW7, leading to its degradation and subsequent stabilization of c-JUN, which promotes transformation. We performed in vitro and in silico analysis and in vivo zebrafish xenograft experiments to demonstrate that TaPIN1 is directly inhibited by the anti-parasite drug buparvaquone (and other known PIN1 inhibitors) and is mutated in a drug-resistant strain. Prolyl isomerization is thus a conserved mechanism that is important in cancer and is used by Theileria parasites to manipulate host oncogenic signalling.
The enzyme hydrogenase reversibly converts dihydrogen to protons and electrons at a metal catalyst. The location of the abundant hydrogens is of key importance for understanding structure and function of the protein. However, in protein X-ray crystallography the detection of hydrogen atoms is one of the major problems, since they display only weak contributions to diffraction and the quality of the single crystals is often insufficient to obtain sub-ångström resolution. Here we report the crystal structure of a standard [NiFe] hydrogenase (∼91.3 kDa molecular mass) at 0.89 Å resolution. The strictly anoxically isolated hydrogenase has been obtained in a specific spectroscopic state, the active reduced Ni-R (subform Ni-R1) state. The high resolution, proper refinement strategy and careful modelling allow the positioning of a large part of the hydrogen atoms in the structure. This has led to the direct detection of the products of the heterolytic splitting of dihydrogen into a hydride (H−) bridging the Ni and Fe and a proton (H+) attached to the sulphur of a cysteine ligand. The Ni–H− and Fe–H− bond lengths are 1.58 Å and 1.78Å, respectively. Furthermore, we can assign the Fe–CO and Fe–CN− ligands at the active site, and can obtain the hydrogen-bond networks and the preferred proton transfer pathway in the hydrogenase. Our results demonstrate the precise comprehensive information available from ultra-high-resolution structures of proteins as an alternative to neutron diffraction and other methods such as NMR structural analysis.
Thirst is the basic instinct to drink water. Previously, it was shown that neurons in several circumventricular organs of the hypothalamus are activated by thirst-inducing conditions. Here we identify two distinct, genetically separable neural populations in the subfornical organ that trigger or suppress thirst. We show that optogenetic activation of subfornical organ excitatory neurons, marked by the expression of the transcription factor ETV-1, evokes intense drinking behaviour, and does so even in fully water-satiated animals. The light-induced response is highly specific for water, immediate and strictly locked to the laser stimulus. In contrast, activation of a second population of subfornical organ neurons, marked by expression of the vesicular GABA transporter VGAT, drastically suppresses drinking, even in water-craving thirsty animals. These results reveal an innate brain circuit that can turn an animal’s water-drinking behaviour on and off, and probably functions as a centre for thirst control in the mammalian brain.
Comets are composed of dust and frozen gases. The ices are mixed with the refractory material either as an icy conglomerate, or as an aggregate of pre-solar grains (grains that existed prior to the formation of the Solar System), mantled by an ice layer. The presence of water-ice grains in periodic comets is now well established. Modelling of infrared spectra obtained about ten kilometres from the nucleus of comet Hartley 2 suggests that larger dust particles are being physically decoupled from fine-grained water-ice particles that may be aggregates, which supports the icy-conglomerate model. It is known that comets build up crusts of dust that are subsequently shed as they approach perihelion. Micrometre-sized interplanetary dust particles collected in the Earth’s stratosphere and certain micrometeorites are assumed to be of cometary origin. Here we report that grains collected from the Jupiter-family comet 67P/Churyumov-Gerasimenko come from a dusty crust that quenches the material outflow activity at the comet surface. The larger grains (exceeding 50 micrometres across) are fluffy (with porosity over 50 per cent), and many shattered when collected on the target plate, suggesting that they are agglomerates of entities in the size range of interplanetary dust particles. Their surfaces are generally rich in sodium, which explains the high sodium abundance in cometary meteoroids. The particles collected to date therefore probably represent parent material of interplanetary dust particles. This argues against comet dust being composed of a silicate core mantled by organic refractory material and then by a mixture of water-dominated ices. At its previous recurrence (orbital period 6.5 years), the comet’s dust production doubled when it was between 2.7 and 2.5 astronomical units from the Sun, indicating that this was when the nucleus shed its mantle. Once the mantle is shed, unprocessed material starts to supply the developing coma, radically changing its dust component, which then also contains icy grains, as detected during encounters with other comets closer to the Sun.
The most powerful oxidant found in nature is compound Q, an enzymatic intermediate that oxidizes methane. New spectroscopic data have resolved the long-running controversy about Q's chemical structure.
An analysis of dying cells reveals that they play an active part in modifying tissue shape by pulling on neighbouring cells. This induces neighbouring cells to contract at their apices, which results in tissue folding.
Essential enzymes in genetically modified organisms are computationally redesigned to functionally depend on non-standard amino acids, thereby achieving biocontainment with unprecedented resistance to escape by evolution or by supplementation with environmental metabolites.
Methane monooxygenase (MMO) catalyses the O2-dependent conversion of methane to methanol in methanotrophic bacteria, thereby preventing the atmospheric egress of approximately one billion tons of this potent greenhouse gas annually. The key reaction cycle intermediate of the soluble form of MMO (sMMO) is termed compound Q (Q). Q contains a unique dinuclear FeIV cluster that reacts with methane to break an exceptionally strong 105 kcal mol−1 C-H bond and insert one oxygen atom. No other biological oxidant, except that found in the particulate form of MMO, is capable of such catalysis. The structure of Q remains controversial despite numerous spectroscopic, computational and synthetic model studies. A definitive structural assignment can be made from resonance Raman vibrational spectroscopy but, despite efforts over the past two decades, no vibrational spectrum of Q has yet been obtained. Here we report the core structures of Q and the following product complex, compound T, using time-resolved resonance Raman spectroscopy (TR3). TR3 permits fingerprinting of intermediates by their unique vibrational signatures through extended signal averaging for short-lived species. We report unambiguous evidence that Q possesses a bis-μ-oxo diamond core structure and show that both bridging oxygens originate from O2. Thisobservation strongly supports a homolytic mechanism for O-O bond cleavage. We also show that T retains a single oxygen atom from O2 as a bridging ligand, while the other oxygen atom is incorporated into the product. Capture of the extreme oxidizing potential of Q is of great contemporary interest for bioremediation and the development of synthetic approaches to methane-based alternative fuels and chemical industry feedstocks. Insight into the formation and reactivity of Q from the structure reported here is an important step towards harnessing this potential.
DNA methylation is an epigenetic modification associated with transcriptional repression of promoters and is essential for mammalian development. Establishment of DNA methylation is mediated by the de novo DNA methyltransferases DNMT3A and DNMT3B, whereas DNMT1 ensures maintenance of methylation through replication. Absence of these enzymes is lethal, and somatic mutations in these genes have been associated with several human diseases. How genomic DNA methylation patterns are regulated remains poorly understood, as the mechanisms that guide recruitment and activity of DNMTs in vivo are largely unknown. To gain insights into this matter we determined genomic binding and site-specific activity of the mammalian de novo DNA methyltransferases DNMT3A and DNMT3B. We show that both enzymes localize to methylated, CpG-dense regions in mouse stem cells, yet are excluded from active promoters and enhancers. By specifically measuring sites of de novo methylation, we observe that enzymatic activity reflects binding. De novo methylation increases with CpG density, yet is excluded from nucleosomes. Notably, we observed selective binding of DNMT3B to the bodies of transcribed genes, which leads to their preferential methylation. This targeting to transcribed sequences requires SETD2-mediated methylation of lysine 36 on histone H3 and a functional PWWP domain of DNMT3B. Together these findings reveal how sequence and chromatin cues guide de novo methyltransferase activity to ensure methylome integrity.
Neurobiological models of long-term memory propose a mechanism by which initially weak memories are strengthened through subsequent activation that engages common neural pathways minutes to hours later. This synaptic tag-and-capture model has been hypothesized to explain how inconsequential information is selectively consolidated following salient experiences. Behavioural evidence for tag-and-capture is provided by rodent studies in which weak early memories are strengthened by future behavioural training. Whether a process of behavioural tagging occurs in humans to transform weak episodic memories into stable long-term memories is unknown. Here we show, in humans, that information is selectively consolidated if conceptually related information, putatively represented in a common neural substrate, is made salient through an emotional learning experience. Memory for neutral objects was selectively enhanced if other objects from the same category were paired with shock. Retroactive enhancements as a result of emotional learning were observed following a period of consolidation, but were not observed in an immediate memory test or for items strongly encoded before fear conditioning. These findings provide new evidence for a generalized retroactive memory enhancement, whereby inconsequential information can be retroactively credited as relevant, and therefore selectively remembered, if conceptually related information acquires salience in the future.
The highly complex structure of the human brain is strongly shaped by genetic influences. Subcortical brain regions form circuits with cortical areas to coordinate movement, learning, memory and motivation, and altered circuits can lead to abnormal behaviour and disease. To investigate how common genetic variants affect the structure of these brain regions, here we conduct genome-wide association studies of the volumes of seven subcortical regions and the intracranial volume derived from magnetic resonance images of 30,717 individuals from 50 cohorts. We identify five novel genetic variants influencing the volumes of the putamen and caudate nucleus. We also find stronger evidence for three loci with previously established influences on hippocampal volume and intracranial volume. These variants show specific volumetric effects on brain structures rather than global effects across structures. The strongest effects were found for the putamen, where a novel intergenic locus with replicable influence on volume (rs945270; P = 1.08 × 10−33; 0.52% variance explained) showed evidence of altering the expression of the KTN1 gene in both brain and blood tissue. Variants influencing putamen volume clustered near developmental genes that regulate apoptosis, axon guidance and vesicle transport. Identification of these geneticvariants provides insight into the causes of variability in human brain development, and may help to determine mechanisms of neuropsychiatric dysfunction.
Epithelium folding is a basic morphogenetic event that is essential in transforming simple two-dimensional epithelial sheets into three-dimensional structures in both vertebrates and invertebrates. Folding has been shown to rely on apical constriction. The resulting cell-shape changes depend either on adherens junction basal shift or on a redistribution of myosin II, which could be driven by mechanical signals. Yet the initial cellular mechanisms that trigger and coordinate cell remodelling remain largely unknown. Here we unravel the active role of apoptotic cells in initiating morphogenesis, thus revealing a novel mechanism of epithelium folding. We show that, in a live developing tissue, apoptotic cells exert a transient pulling force upon the apical surface of the epithelium through a highly dynamic apico-basal myosin II cable. The apoptotic cells then induce a non-autonomous increase in tissue tension together with cortical myosin II apical stabilization in the surrounding tissue, eventually resulting in epithelium folding. Together our results, supported by a theoretical biophysical three-dimensional model, identify an apoptotic myosin-II-dependent signal as the initial signal leading to cell reorganization and tissue folding. This work further reveals that, far from being passively eliminated as generally assumed (for example, during digit individualization), apoptotic cells actively influence their surroundings and trigger tissue remodelling through regulation of tissue tension.
Genetically modified organisms (GMOs) are increasingly used in research and industrial systems to produce high-value pharmaceuticals, fuels and chemicals. Genetic isolation and intrinsic biocontainment would provide essential biosafety measures to secure these closed systems and enable safe applications of GMOs in open systems, which include bioremediation and probiotics. Although safeguards have been designed to control cell growth by essential gene regulation, inducible toxin switches and engineered auxotrophies, these approaches are compromised by cross-feeding of essential metabolites, leaked expression of essential genes, or genetic mutations. Here we describe the construction of a series of genomically recoded organisms (GROs) whose growth is restricted by the expression of multiple essential genes that depend on exogenously supplied synthetic amino acids (sAAs). We introduced a Methanocaldococcus jannaschii tRNA:aminoacyl-tRNA synthetase pair into the chromosome of a GRO derived from Escherichia coli that lacks all TAG codons and release factor 1, endowing this organism with the orthogonal translational components to convert TAG into a dedicated sense codon for sAAs. Using multiplex automated genome engineering, we introduced in-frame TAG codons into 22 essential genes, linking their expression to the incorporation of synthetic phenylalanine-derived amino acids. Of the 60 sAA-dependent variants isolated, a notable strain harbouring three TAG codons in conserved functional residues of MurG, DnaA and SerS and containing targeted tRNA deletions maintained robust growth and exhibited undetectable escape frequencies upon culturing∼1011 cells on solid media for 7 days or in liquid media for 20 days. This is a significant improvement over existing biocontainment approaches. We constructed synthetic auxotrophs dependent on sAAs that were not rescued by cross-feeding in environmental growth assays. These auxotrophic GROs possess alternative genetic codes that impart genetic isolation by impeding horizontal gene transfer and now depend on the use of synthetic biochemical building blocks, advancing orthogonal barriers between engineered organisms and the environment.
In a warming climate, surface meltwater production on large ice sheets is expected to increase. If this water is delivered to the ice sheet base it may have important consequences for ice dynamics. For example, basal water distributed in a diffuse network can decrease basal friction and accelerate ice flow, whereas channelized basal water can move quickly to the ice margin, where it can alter fjord circulation and submarine melt rates. Less certain is whether surface meltwater can be trapped and stored in subglacial lakes beneath large ice sheets. Here we show that a subglacial lake in Greenland drained quickly, as seen in the collapse of the ice surface, and then refilled from surface meltwater input. We use digital elevation models from stereo satellite imagery and airborne measurements to resolve elevation changes during the evolution of the surface and basal hydrologic systems at the Flade Isblink ice cap in northeast Greenland. During the autumn of 2011, a collapse basin about 70 metres deep and about 0.4 cubic kilometres in volume formed near the southern summit of the ice cap as a subglacial lake drained into a nearby fjord. Over the next two years, rapid uplift of the floor of the basin (which is approximately 8.4 square kilometres in area) occurred as surface meltwater flowed into crevasses around the basin margin and refilled the subglacial lake. Our observations show that surface meltwater can be trapped and stored at the bed of an ice sheet. Sensible and latent heat released by this trapped meltwater could soften nearby colder basal ice and alter downstream ice dynamics. Heat transport associated with meltwater trapped in subglacial lakes should be considered when predicting how ice sheet behaviour will change in a warming climate.
Fear memories allow animals to avoid danger, thereby increasing their chances of survival. Fear memories can be retrieved long after learning, but little is known about how retrieval circuits change with time. Here we show that the dorsal midline thalamus of rats is required for the retrieval of auditory conditioned fear at late (24 hours, 7 days, 28 days), but not early (0.5 hours, 6 hours) time points after learning. Consistent with this, the paraventricular nucleus of the thalamus (PVT), a subregion of the dorsal midline thalamus, showed increased c-Fos expression only at late time points, indicating that the PVT is gradually recruited for fear retrieval. Accordingly, the conditioned tone responses of PVT neurons increased with time after training. The prelimbic (PL) prefrontal cortex, which is necessary for fear retrieval, sends dense projections to the PVT. Retrieval at late time points activated PL neurons projecting to the PVT, and optogenetic silencing of these projections impaired retrieval at late, but not early, time points. In contrast, silencing of PL inputs to the basolateral amygdala impaired retrieval at early, but not late, time points, indicating a time-dependent shift in retrieval circuits.Retrieval at late time points also activated PVT neurons projecting to the central nucleus of the amygdala, and silencing these projections at late, but not early, time points induced a persistent attenuation of fear. Thus, the PVT may act as a crucial thalamic node recruited into cortico-amygdalarnetworks for retrieval and maintenance of long-term fear memories.
The regulated release of anorexigenicα-melanocyte stimulating hormone (α-MSH) and orexigenic Agouti-related protein (AgRP) from discrete hypothalamic arcuate neurons onto common target sites in the central nervous system has a fundamental role in the regulation of energy homeostasis. Both peptides bind with high affinity to the melanocortin-4 receptor (MC4R); existing data show that α-MSH is an agonist that couples the receptor to the Gαs signalling pathway, while AgRP binds competitively to block α-MSH binding and blocks the constitutive activity mediated by the ligand-mimetic amino-terminal domain of the receptor. Here weshow that, in mice, regulation of firing activity of neurons from the paraventricular nucleus of the hypothalamus (PVN) by α-MSH and AgRP can be mediated independently of Gαs signalling by ligand-induced coupling of MC4R to closure of inwardly rectifying potassium channel, Kir7.1. Furthermore, AgRP is a biased agonist that hyperpolarizes neurons by binding to MC4R and opening Kir7.1, independently of its inhibition of α-MSH binding. Consequently, Kir7.1 signalling appears to be central to melanocortin-mediated regulation of energy homeostasis within the PVN. Coupling of MC4R to Kir7.1 may explain unusual aspects of the control of energy homeostasis by melanocortin signalling, including the gene dosage effect of MC4R and the sustained effects of AgRP on food intake.
Gradual accumulation of evidence is thought to be fundamental for decision-making, and its neural correlates have been found in several brain regions. Here we develop a generalizable method to measure tuning curves that specify the relationship between neural responses and mentally accumulated evidence, and apply it to distinguish the encoding of decision variables in posterior parietal cortex and prefrontal cortex (frontal orienting fields, FOF). We recorded the firing rates of neurons in posterior parietal cortex and FOF from rats performing a perceptual decision-making task. Classical analyses uncovered correlates of accumulating evidence, similar to previous observations in primates and also similar across the two regions. However, tuning curve assays revealed that while the posterior parietal cortex encodes a graded value of the accumulating evidence, the FOF has a more categorical encoding that indicates, throughout the trial, the decision provisionally favoured by the evidence accumulated so far. Contrary to current views, this suggests that premotor activity in the frontal cortex does not have a role in the accumulation process, but instead has a more categorical function, such as transforming accumulated evidence into a discrete choice. To probe causally the role of FOF activity, we optogenetically silenced it during different time points of the trial. Consistent with a role in committing to a categorical choice at the end of the evidence accumulation process, but not consistent with a role during the accumulation itself, a behavioural effect was observed only when FOF silencing occurred at the end of the perceptual stimulus. Our results place important constraints on the circuit logic of brain regions involved in decision-making.
Appropriate responses to an imminent threat brace us for adversities. The ability to sense and predict threatening or stressful events is essential for such adaptive behaviour. In the mammalian brain, one putative stress sensor is the paraventricular nucleus of the thalamus (PVT), an area that is readily activated by both physical and psychological stressors. However, the role of the PVT in the establishment of adaptive behavioural responses remains unclear. Here we show in mice that the PVT regulates fear processing in the lateral division of the central amygdala (CeL), a structure that orchestrates fear learning and expression. Selective inactivation of CeL-projecting PVT neurons prevented fear conditioning, an effect that can be accounted for by an impairment in fear-conditioning-induced synaptic potentiation onto somatostatin-expressing (SOM+) CeL neurons, which has previously been shown to store fear memory. Consistently, we found that PVT neurons preferentially innervate SOM+ neurons in the CeL, and stimulation of PVT afferents facilitated SOM+ neuron activity and promoted intra-CeL inhibition, two processes that are critical for fear learning and expression. Notably, PVT modulation of SOM+ CeL neurons was mediated by activation of the brain-derived neurotrophic factor (BDNF) receptor tropomysin-related kinase B (TrkB). As a result, selective deletion of either Bdnf in the PVT or Trkb in SOM+ CeL neurons impaired fear conditioning, while infusion of BDNF into the CeL enhanced fear learning and elicited unconditioned fear responses. Our results demonstrate that the PVT–CeL pathway constitutes a novel circuit essential for both the establishment of fear memory and the expression of fear responses, and uncover mechanisms linking stress detection in PVT with the emergence of adaptive behaviour.
The gut microbiota plays a crucial role in the maturation of the intestinal mucosal immune system of its host. Within the thousand bacterial species present in the intestine, the symbiont segmented filamentous bacterium (SFB) is unique in its ability to potently stimulate the post-natal maturation of the B- and T-cell compartments and induce a striking increase in the small-intestinal Th17 responses. Unlike other commensals, SFB intimately attaches to absorptive epithelial cells in the ileum and cells overlying Peyer’s patches. This colonization does not result in pathology; rather, it protects the host from pathogens. Yet, little is known about the SFB–host interaction that underlies the important immunostimulatory properties of SFB, because SFB have resisted in vitro culturing for more than 50 years. Here we grow mouse SFB outside their host in an SFB–host cell co-culturing system. Single-celled SFB isolated from monocolonized mice undergo filamentation, segmentation, and differentiation to release viable infectious particles, the intracellular offspring, which can colonize mice to induce signature immune responses. In vitro, intracellular offspring can attach to mouse and human host cells and recruit actin. In addition, SFB can potently stimulate the upregulation of host innate defence genes, inflammatory cytokines, and chemokines. In vitro culturing thereby mimics the in vivo niche, provides new insights into SFB growth requirements and their immunostimulatory potential, and makes possible the investigation of the complex developmental stages of SFB and the detailed dissection of the unique SFB–host interaction at the cellular and molecular levels.
A protein released during hypothermia has been found to affect the progression of neurodegenerative disease in mice by sparing neurons from death and preserving the connections between them.
The metabolic origin of the sulfur atom in the naturally occurring antibiotic lincomycin A has been obscure— until now. The biosynthetic steps involved reveal surprising roles for two sulfur-containing metabolites.
Mass coral bleaching events can drive reefs from being the domains of corals to becoming dominated by seaweed. But longitudinal data show that more than half of the reefs studied rebound to their former glory.
Climate-induced coral bleaching is among the greatest current threats to coral reefs, causing widespread loss of live coral cover. Conditions under which reefs bounce back from bleaching events or shift from coral to algal dominance are unknown, making it difficult to predict and plan for differing reef responses under climate change. Here we document and predict long-term reef responses to a major climate-induced coral bleaching event that caused unprecedented region-wide mortality of Indo-Pacific corals. Following loss of ggt;90% live coral cover, 12 of 21 reefs recovered towards pre-disturbance live coral states, while nine reefs underwent regime shifts to fleshy macroalgae. Functional diversity of associated reef fish communities shifted substantially following bleaching, returning towards pre-disturbance structure on recovering reefs, while becoming progressively altered on regime shifting reefs. We identified threshold values for a range of factors that accurately predicted ecosystem response to the bleaching event. Recovery was favoured when reefs were structurally complex and in deeper water, when density of juvenile corals and herbivorous fishes was relatively high and when nutrient loads were low. Whether reefs were inside no-take marine reserves had no bearing on ecosystem trajectory. Although conditions governing regime shift or recovery dynamics were diverse, pre-disturbance quantification of simple factors such as structural complexity and water depth accurately predicted ecosystem trajectories. These findings foreshadow the likely divergent but predictable outcomes for reef ecosystems in response to climate change, thus guiding improved management and adaptation.
In the healthy adult brain synapses are continuously remodelled through a process of elimination and formation known as structural plasticity. Reduction in synapse number is a consistent early feature of neurodegenerative diseases, suggesting deficient compensatory mechanisms. Although much is known about toxic processes leading to synaptic dysfunction and loss in these disorders, how synaptic regeneration is affected is unknown. In hibernating mammals, cooling induces loss of synaptic contacts, which are reformed on rewarming, a form of structural plasticity. We have found that similar changes occur in artificially cooled laboratory rodents. Cooling and hibernation also induce a number of cold-shock proteins in the brain, including the RNA binding protein, RBM3 (ref. 6). The relationship of such proteins to structural plasticity is unknown. Here we show that synapse regeneration is impaired in mouse models of neurodegenerative disease, in association with the failure to induce RBM3. In both prion-infected and 5XFAD (Alzheimer-type) mice, the capacity to regenerate synapses after cooling declined in parallel with the loss of induction of RBM3. Enhanced expression of RBM3 in the hippocampus prevented this deficit and restored the capacity for synapse reassembly after cooling. RBM3 overexpression, achieved either by boosting endogenous levels through hypothermia before the loss of the RBM3 response or by lentiviral delivery, resulted in sustained synaptic protection in 5XFAD mice and throughout the course of prion disease, preventing behavioural deficits and neuronal loss and significantly prolonging survival. In contrast, knockdown of RBM3 exacerbated synapse loss in both models and accelerated disease and prevented the neuroprotective effects of cooling. Thus, deficient synapse regeneration, mediated at least in part by failure of the RBM3 stress response, contributes to synapse loss throughout the course of neurodegenerative disease. The data support enhancing cold-shock pathways as potential protective therapies in neurodegenerative disorders.
Low-molecular-mass thiols in organisms are well known for their redox-relevant role in protection against various endogenous and exogenous stresses. In eukaryotes and Gram-negative bacteria, the primary thiol is glutathione (GSH), a cysteinyl-containing tripeptide. In contrast, mycothiol (MSH), a cysteinyl pseudo-disaccharide, is dominant in Gram-positive actinobacteria, including antibiotic-producing actinomycetes and pathogenic mycobacteria. MSH is equivalent to GSH, either as a cofactor or as a substrate, in numerous biochemical processes, most of which have not been characterized, largely due to the dearth of information concerning MSH-dependent proteins. Actinomycetes are able to produce another thiol, ergothioneine (EGT), a histidine betaine derivative that is widely assimilated by plants and animals for variable physiological activities. The involvement of EGT in enzymatic reactions, however, lacks any precedent. Here we report that the unprecedented coupling of two bacterial thiols, MSH and EGT, has a constructive role in the biosynthesis of lincomycin A, a sulfur-containing lincosamide (C8 sugar) antibiotic that has been widely used for half a century to treat Gram-positive bacterial infections. EGT acts as a carrier to template the molecular assembly, and MSH is the sulfur donor for lincomycin maturation after thiol exchange. These thiols function through two unusual S-glycosylations that program lincosamide transfer, activation and modification, providing the first paradigm for EGT-associated biochemical processes and for the poorly understood MSH-dependent biotransformations, a newly described model that is potentially common in the incorporation of sulfur, an element essential for life and ubiquitous in living systems.
Using single-particle electron cryomicroscopy, several structures are reported which illuminate the mechanisms of action of the ATPase NSF that disassembles the SNARE complex into individual protein components.
The phylogeny of Silurian and Devonian (443–358 million years (Myr) ago) fishes remains the foremost problem in the study of the origin of modern gnathostomes (jawed vertebrates). A central question concerns the morphology of the last common ancestor of living jawed vertebrates, with competing hypotheses advancing either a chondrichthyan-or osteichthyan-like model. Here we present Janusiscus schultzei gen. et sp. nov., an Early Devonian (approximately 415 Myr ago) gnathostome from Siberia previously interpreted as a ray-finned fish, which provides important new information about cranial anatomy near the last common ancestor of chondrichthyans and osteichthyans. The skull roof of Janusiscus resembles that of early osteichthyans, with large plates bearing vermiform ridges and partially enclosed sensory canals. High-resolution computed tomography (CT) reveals a braincase bearing characters typically associated with either chondrichthyans (large hypophyseal opening accommodating the internal carotid arteries) or osteichthyans (facial nerve exiting through jugular canal, endolymphatic ducts exiting posterior to the skull roof) but lacking a ventral cranial fissure, the presence of which is considered a derived feature of crown gnathostomes. A conjunction of well-developed cranial processes in Janusiscus helps unify the comparative anatomy of early jawed vertebrate neurocrania, clarifying primary homologies in ‘placoderms’, osteichthyans and chondrichthyans. Phylogenetic analysis further supports the chondrichthyanaffinities of ‘acanthodians’, and places Janusiscus and the enigmatic Ramirosuarezia in a polytomy with crown gnathostomes. The close correspondence between the skull roof of Janusiscus and that of osteichthyans suggests that an extensive dermal skeleton was present in the last common ancestor of jawed vertebrates, but ambiguities arise from uncertainties in the anatomy of Ramirosuarezia. The unexpected contrast between endoskeletal structure in Janusiscus and its superficially osteichthyan-like dermal skeleton highlights the potential importance of other incompletely known Siluro-Devonian ‘bony fishes’ for reconstructing patterns of trait evolution near the origin of modern gnathostomes.
Dengue disease is caused by four different flavivirus serotypes, which infect 390 million people yearly with 25% symptomatic cases and for which no licensed vaccine is available. Recent phase III vaccine trials showed partial protection, and in particular no protection for dengue virus serotype 2 (refs 3, 4). Structural studies so far have characterized only epitopes recognized by serotype-specific human antibodies. We recently isolated human antibodies potently neutralizing all four dengue virus serotypes. Here we describe the X-ray structures of four of these broadly neutralizing antibodies in complex with the envelope glycoprotein E from dengue virus serotype 2, revealing that the recognition determinants are at a serotype-invariant site at the E-dimer interface, including the exposed main chain of the E fusion loop and the two conserved glycan chains. This ‘E-dimer-dependent epitope’ is also the binding site for the viral glycoprotein prM during virus maturation in the secretory pathway of the infected cell, explaining its conservation across serotypes and highlighting an Achilles’ heel of the virus with respect to antibody neutralization. These findings will be instrumental for devising novel immunogens to protect simultaneously against all four serotypes of dengue virus.
Optogenetic techniques enable light-activated control of protein–protein interactions in the cell. This approach has now been used to alter membrane dynamics and induce cellular reorganization.
Quasars have long been known to be variable sources at all wavelengths. Their optical variability is stochastic and can be due to a variety of physical mechanisms; it is also well-described statistically in terms of a damped random walk model. The recent availability of large collections of astronomical time series of flux measurements (light curves) offers new data sets for a systematic exploration of quasar variability. Here we report the detection of a strong, smooth periodic signal in the optical variability of the quasar PG 1302−102 with a mean observed period of 1,884 ± 88 days. It was identified in a search for periodic variability in a data set of light curves for 247,000 known, spectroscopically confirmed quasars with a temporal baseline of about 9 years. Although the interpretation of this phenomenon is still uncertain, the most plausible mechanisms involve a binary system of two supermassive black holes with a subparsec separation. Such systems are an expected consequence of galaxy mergers and can provide important constraints on models of galaxy formation and evolution.
Cell growth and proliferation are tightly linked to nutrient availability. The mechanistic target of rapamycin complex 1 (mTORC1) integrates the presence of growth factors, energy levels, glucose and amino acids to modulate metabolic status and cellular responses. mTORC1 is activated at the surface of lysosomes by the RAG GTPases and the Ragulator complex through a not fully understood mechanism monitoring amino acid availability in the lysosomal lumen and involving the vacuolar H+-ATPase. Here we describe the uncharacterized human member 9 of the solute carrier family 38 (SLC38A9) as a lysosomal membrane-resident protein competent in amino acid transport. Extensive functional proteomic analysis established SLC38A9 as an integral part of the Ragulator–RAG GTPases machinery. Gain of SLC38A9 function rendered cells resistant to amino acid withdrawal, whereas loss of SLC38A9 expression impaired amino-acid-induced mTORC1 activation. Thus SLC38A9 is a physical and functional component of the amino acid sensing machinery that controls the activation of mTOR.
Proper positioning of organelles by cytoskeleton-based motor proteins underlies cellular events such as signalling, polarization and growth. For many organelles, however, the precise connection between position and function has remained unclear, because strategies to control intracellular organelle positioning with spatiotemporal precision are lacking. Here we establish optical control of intracellular transport by using light-sensitive heterodimerization to recruit specific cytoskeletal motor proteins (kinesin, dynein or myosin) to selected cargoes. We demonstrate that the motility of peroxisomes, recycling endosomes and mitochondria can be locally and repeatedly induced or stopped, allowing rapid organelle repositioning. We applied this approach in primary rat hippocampal neurons to test how local positioning of recycling endosomes contributes to axon outgrowth and found that dynein-driven removal of endosomes from axonal growth cones reversibly suppressed axon growth, whereas kinesin-driven endosome enrichment enhanced growth. Our strategy for optogenetic control of organelle positioning will be widely applicable to explore site-specific organelle functions in different model systems.
Meiotic recombination is a critical step in gametogenesis for many organisms, enabling the creation of genetically diverse haploid gametes. In each meiotic cell, recombination is initiated by numerous DNA double-strand breaks (DSBs) created by Spo11, the evolutionarily conserved topoisomerase-like protein, but how these DSBs are distributed relatively uniformly across the four chromatids that make up each chromosome pair is poorly understood. Here we employ Saccharomyces cerevisiae to demonstrate distance-dependent DSB interference in cis (in which the occurrence of a DSB suppresses adjacent DSB formation)—a process that is mediated by the conserved DNA damage response kinase, Tel1ATM. The inhibitory function of Tel1 acts on a relatively local scale, while over large distances DSBs have a tendency to form independently of one another even in the presence of Tel1. Notably, over very short distances, loss of Tel1 activity causes DSBs to cluster within discrete zones of concerted DSB activity. Our observations support a hierarchical view of recombination initiation where Tel1ATM prevents clusters of DSBs, and further suppresses DSBs within the surrounding chromosomal region. Such collective negative regulation will help to ensure that recombination events are dispersed evenly and arranged optimally for genetic exchange and efficient chromosome segregation.
Hox genes regulate regionalization of the axial skeleton in vertebrates, and changes in their expression have been proposed to be a fundamental mechanism driving the evolution of new body forms. The origin of the snake-like body form, with its deregionalized pre-cloacal axial skeleton, has been explained as either homogenization of Hox gene expression domains, or retention of standard vertebrate Hox domains with alteration of downstream expression that suppresses development of distinct regions. Both models assume a highly regionalized ancestor, but the extent of deregionalization of the primaxial domain (vertebrae, dorsal ribs) of the skeleton in snake-like body forms has never been analysed. Here we combine geometric morphometrics and maximum-likelihood analysis to show that the pre-cloacal primaxial domain of elongate, limb-reduced lizards and snakes is not deregionalized compared with limbed taxa, and that the phylogenetic structure of primaxial morphology in reptiles does not support a loss of regionalization in the evolution of snakes. We demonstrate that morphometric regional boundaries correspond to mapped gene expression domains in snakes, suggesting that their primaxial domain is patterned by a normally functional Hox code. Comparison of primaxial osteology in fossil and modern amniotes with Hox gene distributions within Amniota indicates that a functional, sequentially expressed Hox code patterned a subtle morphological gradient along the anterior–posterior axis in stem members of amniote clades and extant lizards, including snakes. The highly regionalized skeletons of extant archosaurs and mammals result from independent evolution in the Hox code and do not represent ancestral conditions for clades with snake-like body forms. The developmental origin of snakes is best explained by decoupling of the primaxial and abaxial domains and by increases in somite number, not by changes in the function of primaxial Hox genes.
The skin represents the primary interface between the host and the environment. This organ is also home to trillions of microorganisms that play an important role in tissue homeostasis and local immunity. Skin microbial communities are highly diverse and can be remodelled over time or in response to environmental challenges. How, in the context of this complexity, individual commensal microorganisms may differentially modulate skin immunity and the consequences of these responses for tissue physiology remains unclear. Here we show that defined commensals dominantly affect skin immunity and identify the cellular mediators involved in this specification. In particular, colonization with Staphylococcus epidermidis induces IL-17A+ CD8+ T cells that home to the epidermis, enhance innate barrier immunity and limit pathogen invasion. Commensal-specific T-cell responses result from the coordinated action of skin-resident dendritic cell subsets and are not associated with inflammation, revealing that tissue-resident cells are poised to sense and respond to alterations in microbial communities. This interaction may represent an evolutionary means by which the skin immune system uses fluctuating commensal signals to calibrate barrier immunity and provide heterologous protection against invasive pathogens. These findings reveal that the skin immune landscape is a highly dynamic environment that can be rapidly and specifically remodelled by encounters with defined commensals, findings that have profound implications for our understanding of tissue-specific immunity and pathologies.
Despite three decades of successful, predominantly phenotype-driven discovery of the genetic causes of monogenic disorders, up to half of children with severe developmental disorders of probable genetic origin remain without a genetic diagnosis. Particularly challenging are those disorders rare enough to have eluded recognition as a discrete clinical entity, those with highly variable clinical manifestations, and those that are difficult to distinguish from other, very similar, disorders. Here we demonstrate the power of using an unbiased genotype-driven approach to identify subsets of patients with similar disorders. By studying 1,133 children with severe, undiagnosed developmental disorders, and their parents, using a combination of exome sequencing and array-based detection of chromosomal rearrangements, we discovered 12 novel genes associated with developmental disorders. These newly implicated genes increase by 10% (from 28% to 31%) the proportion of children that could be diagnosed. Clustering of missense mutations in six of these newly implicated genes suggests that normal development is being perturbed by an activating or dominant-negative mechanism. Our findings demonstrate the value of adopting a comprehensive strategy, both genome-wide and nationwide, to elucidate the underlying causes of rare genetic disorders.
Collective behaviour enhances environmental sensing and decision-making in groups of animals. Experimental and theoretical investigations of schooling fish, flocking birds and human crowds have demonstrated that simple interactions between individuals can explain emergent group dynamics. These findings indicate the existence of neural circuits that support distributed behaviours, but the molecular and cellular identities of relevant sensory pathways are unknown. Here we show that Drosophila melanogaster exhibits collective responses to an aversive odour: individual flies weakly avoid the stimulus, but groups show enhanced escape reactions. Using high-resolution behavioural tracking, computational simulations, genetic perturbations, neural silencing and optogenetic activation we demonstrate that this collective odour avoidance arises from cascades of appendage touch interactions between pairs of flies. Inter-fly touch sensing and collective behaviour require the activity of distal leg mechanosensory sensilla neurons and the mechanosensory channel NOMPC. Remarkably, through these inter-fly encounters, wild-type flies can elicit avoidance behaviour in mutant animals that cannot sense the odour—a basic form of communication. Our data highlight the unexpected importance of social context in the sensory responses of a solitary species and open the door to a neural-circuit-level understanding of collective behaviour in animal groups.
Models derived from human pluripotent stem cells that accurately recapitulate neural development in vitro and allow for the generation of specific neuronal subtypes are of major interest to the stem cell and biomedical community. Notch signalling, particularly through the Notch effector HES5, is a major pathway critical for the onset and maintenance of neural progenitor cells in the embryonic and adult nervous system. Here we report the transcriptional and epigenomic analysis of six consecutive neural progenitor cell stages derived from a HES5::eGFP reporter human embryonic stem cell line. Using this system, we aimed to model cell-fate decisions including specification, expansion and patterning during the ontogeny of cortical neural stem and progenitor cells. In order to dissect regulatory mechanisms that orchestrate the stage-specific differentiation process, we developed a computational framework to infer key regulators of each cell-state transition based on the progressive remodelling of the epigenetic landscape and then validated these through a pooled short hairpin RNA screen. We were also able to refine our previous observations on epigenetic priming at transcription factor binding sites and suggest here that they are mediated by combinations of core and stage-specific factors. Taken together, we demonstrate the utility of our system and outline a general framework, not limited to the context of the neural lineage, to dissect regulatory circuits of differentiation.
Intrinsically disordered proteins play important roles in cell signalling, transcription, translation and cell cycle regulation. Although they lack stable tertiary structure, many intrinsically disordered proteins undergo disorder-to-order transitions upon binding to partners. Similarly, several folded proteins use regulated order-to-disorder transitions to mediate biological function. In principle, the function of intrinsically disordered proteins may be controlled by post-translational modifications that lead to structural changes such as folding, although this has not been observed. Here we show that multisite phosphorylation induces folding of the intrinsically disordered 4E-BP2, the major neural isoform of the family of three mammalian proteins that bind eIF4E and suppress cap-dependent translation initiation. In its non-phosphorylated state, 4E-BP2 interacts tightly with eIF4E using both a canonical YXXXXLΦ motif (starting at Y54) that undergoes a disorder-to-helix transition upon binding and a dynamic secondary binding site. We demonstrate that phosphorylation at T37 and T46 induces folding of residues P18–R62 of 4E-BP2 into a four-stranded β-domain that sequesters the helical YXXXXLΦ motif into a partly buried β-strand, blocking its accessibility to eIF4E. The folded state of pT37pT46 4E-BP2 is weakly stable, decreasing affinity by 100-fold and leading to an order-to-disorder transition upon binding to eIF4E, whereas fully phosphorylated 4E-BP2 is more stable, decreasing affinity by a factor of approximately 4,000. These results highlight stabilization of a phosphorylation-induced fold as the essential mechanism for phospho-regulation of the 4E-BP:eIF4E interaction and exemplify a new mode of biological regulation mediated by intrinsically disordered proteins.
Obesity is an increasingly prevalent disease regulated by genetic and environmental factors. Emerging studies indicate that immune cells, including monocytes, granulocytes and lymphocytes, regulate metabolic homeostasis and are dysregulated in obesity. Group 2 innate lymphoid cells (ILC2s) can regulate adaptive immunity and eosinophil and alternatively activated macrophage responses, and were recently identified in murine white adipose tissue (WAT) where they may act to limit the development of obesity. However, ILC2s have not been identified in human adipose tissue, and the mechanisms by which ILC2s regulate metabolic homeostasis remain unknown. Here we identify ILC2s in human WAT and demonstrate that decreased ILC2 responses in WAT are a conserved characteristic of obesity in humans and mice. Interleukin (IL)-33 was found to be critical for the maintenance of ILC2s in WAT and in limiting adiposity in mice by increasing caloric expenditure. This was associated with recruitment of uncoupling protein 1 (UCP1)+ beige adipocytes in WAT, a process known as beiging or browning that regulates caloric expenditure. IL-33-induced beiging was dependent on ILC2s, and IL-33 treatment or transfer of IL-33-elicited ILC2s was sufficient to drive beiging independently of the adaptive immune system, eosinophils or IL-4 receptor signalling. We found that ILC2s produce methionine-enkephalin peptides that can act directly on adipocytes to upregulate Ucp1 expression in vitro and that promote beiging in vivo. Collectively, these studies indicate that, in addition to responding to infection or tissue damage, ILC2s can regulate adipose function and metabolic homeostasis in part via production of enkephalin peptides that elicit beiging.
A distinctive feature of prokaryotic gene expression is the absence of 5′-capped RNA. In eukaryotes, 5′,5′-triphosphate-linked 7-methylguanosine protects messenger RNA from degradation and modulates maturation, localization and translation. Recently, the cofactor nicotinamide adenine dinucleotide (NAD) was reported as a covalent modification of bacterial RNA. Given the central role of NAD in redox biochemistry, posttranslational protein modification and signalling, its attachment to RNA indicates that there are unknown functions of RNA in these processes and undiscovered pathways in RNA metabolism and regulation. The unknown identity of NAD-modified RNAs has so far precluded functional analyses. Here we identify NAD-linked RNAs from bacteria by chemo-enzymatic capture and next-generation sequencing (NAD captureSeq). Among those identified, specific regulatory small RNAs (sRNAs) and sRNA-like 5′-terminal fragments of certain mRNAs are particularly abundant. Analogous to a eukaryotic cap, 5′-NAD modification is shown in vitro to stabilize RNA against 5′-processing by the RNA-pyrophosphohydrolase RppH and against endonucleolytic cleavage by ribonuclease (RNase) E. The nudix phosphohydrolase NudC decaps NAD-RNA and thereby triggers RNase-E-mediated RNA decay, while being inactive against triphosphate-RNA. In vivo, ∼13% of the abundant sRNA RNAI is NAD-capped in the presence, and ∼26% in the absence, of functional NudC. To our knowledge, this is the first description of a cap-like structure and a decapping machinery in bacteria.
Tubulin is a major component of the eukaryotic cytoskeleton, controlling cell shape, structure and dynamics, whereas its bacterial homologue FtsZ establishes the cytokinetic ring that constricts during cell division. How such different roles of tubulin and FtsZ evolved is unknown. Studying Archaea may provide clues as these organisms share characteristics with Eukarya and Bacteria. Here we report the structure and function of proteins from a distinct family related to tubulin and FtsZ, named CetZ, which co-exists with FtsZ in many archaea. CetZ X-ray crystal structures showed the FtsZ/tubulin superfamily fold, and one crystal form contained sheets of protofilaments, suggesting a structural role. However, inactivation of CetZ proteins in Haloferax volcanii did not affect cell division. Instead, CetZ1 was required for differentiation of the irregular plate-shaped cells into a rod-shaped cell type that was essential for normal swimming motility. CetZ1 formed dynamic cytoskeletal structures in vivo, relating to its capacity to remodel the cell envelope and direct rod formation. CetZ2 was also implicated in H. volcanii cell shape control. Our findings expand the known roles of the FtsZ/tubulin superfamily to include archaeal cell shape dynamics, suggesting that a cytoskeletal role might predate eukaryotic cell evolution, and they support the premise that a major function of the microbial rod shape is to facilitate swimming.
Resveratrol is reported to extend lifespan and provide cardio-neuro-protective, anti-diabetic, and anti-cancer effects by initiating a stress response that induces survival genes. Because human tyrosyl transfer-RNA (tRNA) synthetase (TyrRS) translocates to the nucleus under stress conditions, we considered the possibility that the tyrosine-like phenolic ring of resveratrol might fit into the active site pocket to effect a nuclear role. Here we present a 2.1 Å co-crystal structure of resveratrol bound to the active site of TyrRS. Resveratrol nullifies the catalytic activity and redirects TyrRS to a nuclear function, stimulating NAD+-dependent auto-poly-ADP-ribosylation of poly(ADP-ribose) polymerase 1 (PARP1). Downstream activation of key stress signalling pathways are causally connected to TyrRS–PARP1–NAD+ collaboration. This collaboration is also demonstrated in the mouse, and is specifically blocked in vivo by a resveratrol-displacing tyrosyl adenylate analogue. In contrast to functionally diverse tRNA synthetase catalytic nulls createdby alternative splicing events that ablate active sites, here a non-spliced TyrRS catalytic null reveals a new PARP1- and NAD+-dependent dimension to the physiological mechanism of resveratrol.
The orexin (also known as hypocretin) G protein-coupled receptors (GPCRs) respond to orexin neuropeptides in the central nervous system to regulate sleep and other behavioural functions in humans. Defects in orexin signalling are responsible for the human diseases of narcolepsy and cataplexy; inhibition of orexin receptors is an effective therapy for insomnia. The human OX2 receptor (OX2R) belongs to theβ branch of the rhodopsin family of GPCRs, and can bind to diverse compounds including the native agonist peptides orexin-A and orexin-B and the potent therapeutic inhibitor suvorexant. Here, using lipid-mediated crystallization and protein engineering with a novel fusion chimaera, we solved the structure of the human OX2R bound to suvorexant at 2.5 Å resolution. The structure reveals how suvorexant adopts a π-stacked horseshoe-like conformation and binds to the receptor deep in the orthosteric pocket, stabilizing a network of extracellular salt bridges and blocking transmembrane helix motions necessary for activation. Computational docking suggests how other classes of synthetic antagonists may interact with the receptor at a similar position in an analogous π-stacked fashion. Elucidation of the molecular architecture of the human OX2R expands our understanding of peptidergic GPCRligand recognition and will aid further efforts to modulate orexin signalling for therapeutic ends.
More than twenty types of retinal ganglion cells conduct visual information from the eye to the rest of the brain. Each retinal ganglion cell type tessellates the retina in a regular mosaic, so that every point in visual space is processed for visual primitives such as contrast and motion. This information flows to two principal brain centres: the visual cortex and the superior colliculus. The superior colliculus plays an evolutionarily conserved role in visual behaviours, but its functional architecture is poorly understood. Here we report on population recordings of visual responses from neurons in the mouse superior colliculus. Many neurons respond preferentially to lines of a certain orientation or movement axis. We show that cells with similar orientation preferences form large patches that span the vertical thickness of the retinorecipient layers. This organization is strikingly different from the randomly interspersed orientation preferences in the mouse’s visual cortex; instead, it resembles the orientation columns observed in the visual cortices of large mammals. Notably, adjacent superior colliculus orientation columns have only limited receptive field overlap. This is in contrast to the organization of visual cortex, where each point in the visual field activates neurons with all preferred orientations. Instead, the superior colliculus favours specific contour orientations within ∼30° regions of the visual field, a finding with implications for behavioural responses mediated by this brain centre.
Mast cells are primary effectors in allergic reactions, and may have important roles in disease by secreting histamine and various inflammatory and immunomodulatory substances. Although they are classically activated by immunoglobulin (Ig)E antibodies, a unique property of mast cells is their antibody-independent responsiveness to a range of cationic substances, collectively called basic secretagogues, including inflammatory peptides and drugs associated with allergic-type reactions. The pathogenic roles of these substances have prompted a decades-long search for their receptor(s). Here we report that basic secretagogues activate mouse mast cells in vitro and in vivo through a single receptor, Mrgprb2, the orthologue of the human G-protein-coupled receptor MRGPRX2. Secretagogue-induced histamine release, inflammation and airway contraction are abolished in Mrgprb2-null mutant mice. Furthermore, we show that most classes of US Food and Drug Administration (FDA)-approved peptidergic drugs associated with allergic-type injection-site reactions also activate Mrgprb2 and MRGPRX2, and that injection-site inflammation is absent in mutant mice. Finally, we determine that Mrgprb2 and MRGPRX2 are targets of many small-molecule drugs associated with systemic pseudo-allergic, or anaphylactoid, reactions; we show that drug-induced symptoms of anaphylactoid responses are significantly reduced in knockout mice; and we identify a common chemical motif in several of these molecules that may help predict side effects of other compounds. These discoveries introduce a mouse model to study mast cell activation by basic secretagogues and identify MRGPRX2 as a potential therapeutic target to reduce a subset of drug-induced adverse effects.
Interventions that delay ageing mobilize mechanisms that protect and repair cellular components, but it is unknown how these interventions might slow the functional decline of extracellular matrices, which are also damaged during ageing. Reduced insulin/IGF-1 signalling (rIIS) extends lifespan across the evolutionary spectrum, and in juvenile Caenorhabditis elegans also allows the transcription factor DAF-16/FOXO to induce development into dauer, a diapause that withstands harsh conditions. It has been suggested that rIIS delays C. elegans ageing through activation of dauer-related processes during adulthood, but some rIIS conditions confer robust lifespan extension unaccompanied by any dauer-like traits. Here we show that rIIS can promote C. elegans longevity through a program that is genetically distinct from the dauer pathway, and requires the Nrf (NF-E2-related factor) orthologue SKN-1 acting in parallel to DAF-16. SKN-1 is inhibited by IIS and has been broadly implicated in longevity, but is rendered dispensable for rIIS lifespan extension by even mild activity of dauer-related processes. When IIS is decreased under conditions that do not induce dauer traits, SKN-1 most prominently increases expression of collagens and other extracellular matrix genes. Diverse genetic, nutritional, and pharmacological pro-longevity interventions delay an age-related decline in collagen expression. These collagens mediate adulthood extracellular matrix remodelling, and are needed for ageing to be delayed by interventions that do not involve dauer traits. By genetically delineating a dauer-independent rIIS ageing pathway, our results show that IIS controls a broad set of protective mechanisms during C. elegans adulthood, and may facilitate elucidation of processes of general importance for longevity. The importance of collagen production in diverse anti-ageing interventions implies that extracellular matrix remodelling is a generally essential signature of longevity assurance, and that agents promoting extracellular matrix youthfulness may have systemic benefit.
Gene transcription in animals involves the assembly of RNA polymerase II at core promoters and its cell-type-specific activation by enhancers that can be located more distally. However, how ubiquitous expression of housekeeping genes is achieved has been less clear. In particular, it is unknown whether ubiquitously active enhancers exist and how developmental and housekeeping gene regulation is separated. An attractive hypothesis is that different core promoters might exhibit an intrinsic specificity to certain enhancers. This is conceivable, as various core promoter sequence elements are differentially distributed between genes of different functions, including elements that are predominantly found at either developmentally regulated or at housekeeping genes. Here we show that thousands of enhancers in Drosophila melanogaster S2 and ovarian somatic cells (OSCs) exhibit a marked specificity to one of two core promoters—one derived from a ubiquitously expressed ribosomal protein gene and another from a developmentally regulated transcription factor—and confirm the existence of these two classes for five additional core promoters from genes with diverse functions. Housekeeping enhancers are active across the two cell types, while developmental enhancers exhibit strong cell-type specificity. Both enhancer classes differ in their genomic distribution, the functions of neighbouring genes, and the core promoter elements of these neighbouring genes. In addition, we identify two transcription factors—Dref and Trl—that bind and activate housekeeping versus developmental enhancers, respectively. Our results provide evidence for a sequence-encoded enhancer–core-promoter specificity that separates developmental and housekeeping gene regulatory programs for thousands of enhancers and their target genes across the entire genome.
The role of cellular metabolism in regulating cell proliferation and differentiation remains poorly understood. For example, most mammalian cells cannot proliferate without exogenous glutamine supplementation even though glutamine is a non-essential amino acid. Here we show that mouse embryonic stem (ES) cells grown under conditions that maintain naive pluripotency are capable of proliferation in the absence of exogenous glutamine. Despite this, ES cells consume high levels of exogenous glutamine when the metabolite is available. In comparison to more differentiated cells, naive ES cells utilize both glucose and glutamine catabolism to maintain a high level of intracellularα-ketoglutarate (αKG). Consequently, naive ES cells exhibit an elevated αKG to succinate ratio that promotes histone/DNA demethylation and maintains pluripotency. Direct manipulation of the intracellular αKG/succinate ratio is sufficient to regulate multiple chromatin modifications, including H3K27me3 and ten-eleven translocation (Tet)-dependent DNA demethylation, which contribute to the regulation of pluripotency-associated gene expression. In vitro, supplementation with cell-permeable αKG directly supports ES-cell self-renewal while cell-permeable succinate promotes differentiation. This work reveals that intracellular αKG/succinate levels can contribute to the maintenance of cellular identity and have a mechanistic role in the transcriptional and epigenetic state of stem cells.
Myocardial infarction (MI), a leading cause of death around the world, displays a complex pattern of inheritance. When MI occurs early in life, genetic inheritance is a major component to risk. Previously, rare mutations in low-density lipoprotein (LDL) genes have been shown to contribute to MI risk in individual families, whereas common variants at more than 45 loci have been associated with MI risk in the population. Here we evaluate how rare mutations contribute to early-onset MI risk in the population. We sequenced the protein-coding regions of 9,793 genomes from patients with MI at an early age (≤50 years in males and ≤60 years in females) along with MI-free controls. We identified two genes in which rare coding-sequence mutations were more frequent in MI cases versus controls at exome-wide significance. At low-density lipoprotein receptor (LDLR), carriers of rare non-synonymous mutations were at 4.2-fold increased risk for MI; carriers of null alleles at LDLR were at even higher risk (13-fold difference). Approximately 2% of early MI cases harbour a rare, damaging mutation in LDLR; this estimate is similar to one made more than 40 years ago using an analysis of total cholesterol. Among controls, about 1 in 217 carried an LDLR coding-sequence mutation and had plasma LDL cholesterol ggt; 190 mg dl−1. At apolipoprotein A-V (APOA5), carriers of rare non-synonymous mutations were at 2.2-fold increased risk for MI. When compared with non-carriers, LDLR mutation carriers had higher plasma LDL cholesterol, whereas APOA5 mutation carriers had higher plasma triglycerides. Recent evidence has connected MI risk with coding-sequence mutations at two genes functionally related to APOA5, namely lipoprotein lipase and apolipoprotein C-III (refs 18, 19). Combined, these observations suggest that, as well as LDL cholesterol, disordered metabolism of triglyceride-rich lipoproteins contributes to MI risk.
The concept of germ layers has been one of the foremost organizing principles in developmental biology, classification, systematics and evolution for 150 years (refs 1, 2, 3). Of the three germ layers, the mesoderm is found in bilaterian animals but is absent in species in the phyla Cnidaria and Ctenophora, which has been taken as evidence that the mesoderm was the final germ layer to evolve. The origin of the ectoderm and endoderm germ layers, however, remains unclear, with models supporting the antecedence of each as well as a simultaneous origin. Here we determine the temporal and spatial components of gene expression spanning embryonic development for all Caenorhabditis elegans genes and use it to determine the evolutionary ages of the germ layers. The gene expression program of the mesoderm is induced after those of the ectoderm and endoderm, thus making it the last germ layer both to evolve and to develop. Strikingly, the C. elegans endoderm and ectoderm expression programs do not co-induce; rather the endoderm activates earlier, and this is also observed in the expression of endoderm orthologues during the embryology of the frog Xenopus tropicalis, the sea anemone Nematostella vectensis and the sponge Amphimedon queenslandica. Querying the phylogenetic ages of specifically expressed genes reveals that the endoderm comprises older genes. Taken together, we propose that the endoderm program dates back to the origin of multicellularity, whereas the ectoderm originated as a secondary germ layer freed from ancestral feeding functions.
A defining feature of vertebrates (craniates) is a pronounced head that is supported and protected by a robust cellular endoskeleton. In the first vertebrates, this skeleton probably consisted of collagenous cellular cartilage, which forms the embryonic skeleton of all vertebrates and the adult skeleton of modern jawless and cartilaginous fish. In the head, most cellular cartilage is derived from a migratory cell population called the neural crest, which arises from the edges of the central nervous system. Because collagenous cellular cartilage and neural crest cells have not been described in invertebrates, the appearance of cellular cartilage derived from neural crest cells is considered a turning point in vertebrate evolution. Here we show that a tissue with many of the defining features of vertebrate cellular cartilage transiently forms in the larvae of the invertebrate chordate Branchiostoma floridae (Florida amphioxus). We also present evidence that during evolution, a key regulator of vertebrate cartilage development, SoxE, gained new cis-regulatory sequences that subsequently directed its novel expression in neural crest cells. Together, these results suggest that the origin of the vertebrate head skeleton did not depend on the evolution of a new skeletal tissue, as is commonly thought, but on the spread of this tissue throughout the head. We further propose that the evolution of cis-regulatory elements near an ancient regulator of cartilage differentiation was a major factor in the evolution of the vertebrate head skeleton.
Therapy-related acute myeloid leukaemia (t-AML) and therapy-related myelodysplastic syndrome (t-MDS) are well-recognized complications of cytotoxic chemotherapy and/or radiotherapy. There are several features that distinguish t-AML from de novo AML, including a higher incidence of TP53 mutations, abnormalities of chromosomes 5 or 7, complex cytogenetics and a reduced response to chemotherapy. However, it is not clear how prior exposure to cytotoxic therapy influences leukaemogenesis. In particular, the mechanism by which TP53 mutations are selectively enriched in t-AML/t-MDS is unknown. Here, by sequencing the genomes of 22 patients with t-AML, we show that the total number of somatic single-nucleotide variants and the percentage of chemotherapy-related transversions are similar in t-AML and de novo AML, indicating that previous chemotherapy does not induce genome-wide DNA damage. We identified four cases of t-AML/t-MDS in which the exact TP53 mutation found at diagnosis was also present at low frequencies (0.003–0.7%) in mobilized blood leukocytes or bone marrow 3–6 years before the development of t-AML/t-MDS, including two cases in which the relevant TP53 mutation was detected before any chemotherapy. Moreover, functional TP53 mutations were identified in small populations of peripheral blood cells of healthy chemotherapy-naive elderly individuals. Finally, in mouse bone marrow chimaeras containing both wild-type and Tp53+/− haematopoietic stem/progenitor cells (HSPCs), the Tp53+/− HSPCs preferentially expanded after exposure to chemotherapy. These data suggest that cytotoxic therapy does not directly induce TP53 mutations. Rather, they support a model in which rare HSPCs carrying age-related TP53 mutations are resistant to chemotherapy and expand preferentially after treatment. The early acquisition of TP53 mutations in the founding HSPC clone probably contributes to the frequent cytogenetic abnormalities and poor responses to chemotherapy that are typical of patients with t-AML/t-MDS.
In human societies, cultural norms arise when behaviours are transmitted through social networks via high-fidelity social learning. However, a paucity of experimental studies has meant that there is no comparable understanding of the process by which socially transmitted behaviours might spread and persist in animal populations. Here we show experimental evidence of the establishment of foraging traditions in a wild bird population. We introduced alternative novel foraging techniques into replicated wild sub-populations of great tits (Parus major) and used automated tracking to map the diffusion, establishment and long-term persistence of the seeded innovations. Furthermore, we used social network analysis to examine the social factors that influenced diffusion dynamics. From only two trained birds in each sub-population, the information spread rapidly through social network ties, to reach an average of 75% of individuals, with a total of 414 knowledgeable individuals performing 57,909 solutions over all replicates. The sub-populations were heavily biased towards using the technique that was originally introduced, resulting in established local traditions that were stable over two generations, despite a high population turnover. Finally, we demonstrate a strong effect of social conformity, with individuals disproportionately adopting the most frequent local variant when first acquiring an innovation, and continuing to favour social information over personal information. Cultural conformity is thought to be a key factor in the evolution of complex culture in humans. In providing the first experimental demonstration of conformity in a wild non-primate, and of cultural norms in foraging techniques in any wild animal, our results suggest a much broader taxonomic occurrence of such an apparently complex cultural behaviour.
The manufacture of geometric engravings is generally interpreted as indicative of modern cognition and behaviour. Key questions in the debate on the origin of such behaviour are whether this innovation is restricted to Homo sapiens, and whether it has a uniquely African origin. Here we report on a fossil freshwater shell assemblage from the Hauptknochenschicht (‘main bone layer’) of Trinil (Java, Indonesia), the type locality of Homo erectus discovered by Eugène Dubois in 1891 (refs 2 and 3). In the Dubois collection (in the Naturalis museum, Leiden, The Netherlands) we found evidence for freshwater shellfish consumption by hominins, one unambiguous shell tool, and a shell with a geometric engraving. We dated sediment contained in the shells with 40Ar/39Ar and luminescence dating methods, obtaining a maximum age of 0.54 ± 0.10 million years and a minimum age of 0.43 ± 0.05 million years. This implies that the Trinil Hauptknochenschicht isyounger than previously estimated. Together, our data indicate that the engraving was made by Homo erectus, and that it is considerably older than the oldest geometric engravings described so far. Although it is at present not possible to assess the function or meaning of the engraved shell, this discovery suggests that engraving abstract patterns was in the realm of Asian Homo erectus cognition and neuromotor control.
Contusive spinal cord injury leads to a variety of disabilities owing to limited neuronal regeneration and functional plasticity. It is well established that an upregulation of glial-derived chondroitin sulphate proteoglycans (CSPGs) within the glial scar and perineuronal net creates a barrier to axonal regrowth and sprouting. Protein tyrosine phosphataseσ (PTPσ), along with its sister phosphatase leukocyte common antigen-related (LAR) and the nogo receptors 1 and 3 (NgR), have recently been identified as receptors for the inhibitory glycosylated side chains of CSPGs. Here we find in rats that PTPσ has a critical role in converting growth cones into a dystrophic state by tightly stabilizing them within CSPG-rich substrates. We generated a membrane-permeable peptide mimetic of the PTPσ wedge domain that binds to PTPσ and relieves CSPG-mediated inhibition. Systemic delivery of this peptide over weeks restored substantial serotonergic innervation to the spinal cord below the level of injury and facilitated functional recovery of both locomotor and urinary systems. Our results add a new layer of understanding to the critical role of PTPσ in mediating the growth-inhibited state of neurons due to CSPGs within the injured adult spinal cord.
Most haematopoietic cells renew from adult haematopoietic stem cells (HSCs), however, macrophages in adult tissues can self-maintain independently of HSCs. Progenitors with macrophage potential in vitro have been described in the yolk sac before emergence of HSCs, and fetal macrophages can develop independently of Myb, a transcription factor required for HSC, and can persist in adult tissues. Nevertheless, the origin of adult macrophages and the qualitative and quantitative contributions of HSC and putative non-HSC-derived progenitors are still unclear. Here we show in mice that the vast majority of adult tissue-resident macrophages in liver (Kupffer cells), brain (microglia), epidermis (Langerhans cells) and lung (alveolar macrophages) originate from a Tie2+ (also known as Tek) cellular pathway generating Csf1r+ erythro-myeloid progenitors (EMPs) distinct from HSCs. EMPs develop in the yolk sac at embryonic day (E) 8.5, migrate and colonize the nascent fetal liver before E10.5, and give rise to fetal erythrocytes, macrophages, granulocytes and monocytes until at least E16.5. Subsequently, HSC-derived cells replace erythrocytes, granulocytes and monocytes. Kupffer cells, microglia and Langerhans cells are only marginally replaced in one-year-old mice, whereas alveolar macrophages may be progressively replaced in ageing mice. Our fate-mapping experiments identify, in the fetal liver, a sequence of yolk sac EMP-derived and HSC-derived haematopoiesis, and identify yolk sac EMPs as a common origin for tissue macrophages.
T-helper type 17 (TH17) cells that produce the cytokines interleukin-17A (IL-17A) and IL-17F are implicated in the pathogenesis of several autoimmune diseases. The differentiation of TH17 cells is regulated by transcription factors such as RORγt, but post-translational mechanisms preventing the rampant production of pro-inflammatory IL-17A have received less attention. Here we show that the deubiquitylating enzyme DUBA is a negative regulator of IL-17A production in T cells. Mice with DUBA-deficient T cells developed exacerbated inflammation in the small intestine after challenge with anti-CD3 antibodies. DUBA interacted with the ubiquitin ligase UBR5, which suppressed DUBA abundance in naive T cells. DUBA accumulated in activated T cells and stabilized UBR5, which then ubiquitylated RORγt in response to TGF-β signalling. Our data identify DUBA as a cell-intrinsic suppressor of IL-17 production.
Angelman syndrome is a single-gene disorder characterized by intellectual disability, developmental delay, behavioural uniqueness, speech impairment, seizures and ataxia. It is caused by maternal deficiency of the imprinted gene UBE3A, encoding an E3 ubiquitin ligase. All patients carry at least one copy of paternal UBE3A, which is intact but silenced by a nuclear-localized long non-coding RNA, UBE3A antisense transcript (UBE3A-ATS). Murine Ube3a-ATS reduction by either transcription termination or topoisomerase I inhibition has been shown to increase paternal Ube3a expression. Despite a clear understanding of the disease-causing event in Angelman syndrome and the potential to harness the intact paternal allele to correct the disease, no gene-specific treatment exists for patients. Here we developed a potential therapeutic intervention for Angelman syndrome by reducing Ube3a-ATS with antisense oligonucleotides (ASOs). ASO treatment achieved specific reduction of Ube3a-ATS and sustained unsilencing of paternal Ube3a in neurons in vitro and in vivo. Partial restoration of UBE3A protein in an Angelman syndrome mouse model ameliorated some cognitive deficits associated with the disease. Although additional studies of phenotypic correction are needed, we have developed a sequence-specific and clinically feasible method to activate expression of the paternal Ube3a allele.
Members of the dynein family, consisting of cytoplasmic and axonemal isoforms, are motors that move towards the minus ends of microtubules. Cytoplasmic dynein-1 (dynein-1) plays roles in mitosis and cellular cargo transport, and is implicated in viral infections and neurodegenerative diseases. Cytoplasmic dynein-2 (dynein-2) performs intraflagellar transport and is associated with human skeletal ciliopathies. Dyneins share a conserved motor domain that couples cycles of ATP hydrolysis with conformational changes to produce movement. Here we present the crystal structure of the human cytoplasmic dynein-2 motor bound to the ATP-hydrolysis transition state analogue ADP.vanadate. The structure reveals a closure of the motor’s ring of six AAA+ domains (ATPases associated with various cellular activites: AAA1–AAA6). This induces a steric clash with the linker, the key element for the generation of movement, driving it into a conformation that is primed to produce force. Ring closure also changes the interface between the stalk and buttress coiled-coil extensions of the motor domain. This drives helix sliding in the stalk which causes the microtubule binding domain at its tip to release from the microtubule. Our structure answers the key questions of how ATP hydrolysis leads to linker remodelling and microtubule affinity regulation.
The emergence of catalysis in early genetic polymers such as RNA is considered a key transition in the origin of life, pre-dating the appearance of protein enzymes. DNA also demonstrates the capacity to fold into three-dimensional structures and form catalysts in vitro. However, to what degree these natural biopolymers comprise functionally privileged chemical scaffolds for folding or the evolution of catalysis is not known. The ability of synthetic genetic polymers (XNAs) with alternative backbone chemistries not found in nature to fold into defined structures and bind ligands raises the possibility that these too might be capable of forming catalysts (XNAzymes). Here we report the discovery of such XNAzymes, elaborated in four different chemistries (arabino nucleic acids, ANA; 2′-fluoroarabino nucleic acids, FANA; hexitol nucleic acids, HNA; and cyclohexene nucleic acids, CeNA) directly from random XNA oligomer pools, exhibiting in trans RNA endonuclease and ligase activities. We also describe an XNA–XNA ligase metalloenzyme in the FANA framework, establishing catalysis in an entirely synthetic system and enabling the synthesis of FANA oligomers and an active RNA endonuclease FANAzyme from its constituent parts. These results extend catalysis beyond biopolymers and establish technologies for the discovery of catalysts in a wide range of polymer scaffolds not found in nature. Evolution of catalysis independent of any natural polymer has implications for the definition of chemical boundary conditions for the emergence of life on Earth and elsewhere in the Universe.
Filamentous actin (F-actin) is the major protein of muscle thin filaments, and actin microfilaments are the main component of the eukaryotic cytoskeleton. Mutations in different actin isoforms lead to early-onset autosomal dominant non-syndromic hearing loss, familial thoracic aortic aneurysms and dissections, and multiple variations of myopathies. In striated muscle fibres, the binding of myosin motors to actin filaments is mainly regulated by tropomyosin and troponin. Tropomyosin also binds to F-actin in smooth muscle and in non-muscle cells and stabilizes and regulates the filaments there in the absence of troponin. Although crystal structures for monomeric actin (G-actin) are available, a high-resolution structure of F-actin is still missing, hampering our understanding of how disease-causing mutations affect the function of thin muscle filaments and microfilaments. Here we report the three-dimensional structure of F-actin at a resolution of 3.7 Å in complex with tropomyosin at a resolution of 6.5 Å, determined by electron cryomicroscopy. The structure reveals that the D-loop is ordered and acts as a central region for hydrophobic and electrostatic interactions that stabilize the F-actin filament. We clearly identify map density corresponding to ADP and Mg2+ and explain the possible effect of prominent disease-causing mutants. A comparison of F-actin with G-actin reveals the conformational changes during filament formation and identifies the D-loop as their key mediator. We also confirm that negatively charged tropomyosin interacts with a positively charged groove on F-actin. Comparison of the position of tropomyosin in F-actin–tropomyosin with its position in our previously determined F-actin–tropomyosin–myosin structure reveals a myosin-induced transition of tropomyosin. Our results allow us to understand the role of individual mutations in the genesis of actin- and tropomyosin-related diseases and will serve as a strong foundation for the targeted development of drugs.
Human cancers, including breast cancers, comprise clones differing in mutation content. Clones evolve dynamically in space and time following principles of Darwinian evolution, underpinning important emergent features such as drug resistance and metastasis. Human breast cancer xenoengraftment is used as a means of capturing and studying tumour biology, and breast tumour xenografts are generally assumed to be reasonable models of the originating tumours. However, the consequences and reproducibility of engraftment and propagation on the genomic clonal architecture of tumours have not been systematically examined at single-cell resolution. Here we show, using deep-genome and single-cell sequencing methods, the clonal dynamics of initial engraftment and subsequent serial propagation of primary and metastatic human breast cancers in immunodeficient mice. In all 15 cases examined, clonal selection on engraftment was observed in both primary and metastatic breast tumours, varying in degree from extreme selective engraftment of minor (llt;5% of starting population) clones to moderate, polyclonal engraftment. Furthermore, ongoing clonal dynamics during serial passaging is a feature of tumours experiencing modest initial selection. Through single-cell sequencing, we show that major mutation clusters estimated from tumour population sequencing relate predictably to the most abundant clonal genotypes, even in clonally complex and rapidly evolving cases. Finally, we show that similar clonal expansion patterns can emerge in independent grafts of the same starting tumour population, indicating that genomic aberrations can be reproducible determinants of evolutionary trajectories. Our results show that measurement of genomically defined clonal population dynamics will be highly informative for functional studies using patient-derived breast cancer xenoengraftment.
Safe and powerful energy storage devices are becoming increasingly important. Charging times of seconds to minutes, with power densities exceeding those of batteries, can in principle be provided by electrochemical capacitors—in particular, pseudocapacitors. Recent research has focused mainly on improving the gravimetric performance of the electrodes of such systems, but for portable electronics and vehicles volume is at a premium. The best volumetric capacitances of carbon-based electrodes are around 300 farads per cubic centimetre; hydrated ruthenium oxide can reach capacitances of 1,000 to 1,500 farads per cubic centimetre with great cyclability, but only in thin films. Recently, electrodes made of two-dimensional titanium carbide (Ti3C2, a member of the ‘MXene’ family), produced by etching aluminium from titanium aluminium carbide (Ti3AlC2, a ‘MAX’ phase) in concentrated hydrofluoric acid, have been shown to have volumetric capacitances of over 300 farads per cubic centimetre. Here we report a method of producing this material using a solution of lithium fluoride and hydrochloric acid. The resulting hydrophilic material swells in volume when hydrated, and can be shaped like clay and dried into a highly conductive solid or rolled into films tens of micrometres thick. Additive-free films of this titanium carbide ‘clay’ have volumetric capacitances of up to 900 farads per cubic centimetre, with excellent cyclability and rate performances. This capacitance is almost twice that of our previous report, and our synthetic method also offers a much faster route to film production as well as the avoidance of handling hazardous concentrated hydrofluoric acid.
The TRIM37 (also known as MUL) gene is located in the 17q23 chromosomal region, which is amplified in up to∼40% of breast cancers. TRIM37 contains a RING finger domain, a hallmark of E3 ubiquitin ligases, but its protein substrate(s) is unknown. Here we report that TRIM37 mono-ubiquitinates histone H2A, a chromatin modification associated with transcriptional repression. We find that in human breast cancer cell lines containing amplified 17q23, TRIM37 is upregulated and, reciprocally, the major H2A ubiquitin ligase RNF2 (also known as RING1B) is downregulated. Genome-wide chromatin immunoprecipitation (ChIP)-chip experiments in 17q23-amplified breast cancer cells identified many genes, includingmultiple tumour suppressors, whose promoters were bound by TRIM37 and enriched for ubiquitinated H2A. However, unlike RNF2, which is a subunit of polycomb repressive complex 1 (PRC1), we find that TRIM37 associates with polycomb repressive complex 2 (PRC2). TRIM37, PRC2 and PRC1 are co-bound to specific target genes, resulting in their transcriptional silencing. RNA-interference-mediated knockdown of TRIM37 results in loss of ubiquitinated H2A, dissociation of PRC1 and PRC2 from target promoters, and transcriptional reactivation of silenced genes. Knockdown of TRIM37 in human breast cancer cells containing amplified 17q23 substantially decreases tumour growth in mouse xenografts. Conversely, ectopic expression of TRIM37 renders non-transformed cells tumorigenic. Collectively, our results reveal TRIM37 as an oncogenic H2A ubiquitin ligase that is overexpressed in a subset of breast cancers and promotes transformation by facilitating silencing of tumour suppressors and other genes.
DEAD-box RNA helicases are vital for the regulation of various aspects of the RNA life cycle, but the molecular underpinnings of their involvement, particularly in mammalian cells, remain poorly understood. Here we show that the DEAD-box RNA helicase DDX21 can sense the transcriptional status of both RNA polymerase (Pol) I and II to control multiple steps of ribosome biogenesis in human cells. We demonstrate that DDX21 widely associates with Pol I- and Pol II-transcribed genes and with diverse species of RNA, most prominently with non-coding RNAs involved in the formation of ribonucleoprotein complexes, including ribosomal RNA, small nucleolar RNAs (snoRNAs) and 7SK RNA. Although broad, these molecular interactions, both at the chromatin and RNA level, exhibit remarkable specificity for the regulation of ribosomal genes. In the nucleolus, DDX21 occupies the transcribed rDNA locus, directly contacts both rRNA and snoRNAs, and promotes rRNA transcription, processing and modification. In the nucleoplasm, DDX21 binds 7SK RNA and, as a component of the 7SK small nuclear ribonucleoprotein (snRNP) complex, is recruited to the promoters of Pol II-transcribed genes encoding ribosomal proteins and snoRNAs. Promoter-bound DDX21 facilitates the release of the positive transcription elongation factor b (P-TEFb) from the 7SK snRNP in a manner that is dependent on its helicase activity, thereby promoting transcription of its target genes. Our results uncover the multifaceted role of DDX21 in multiple steps of ribosome biogenesis, and provide evidence implicating a mammalian RNA helicase in RNA modification and Pol II elongation control.
Horizontal gene transfer allows organisms to rapidly acquire adaptive traits. Although documented instances of horizontal gene transfer from bacteria to eukaryotes remain rare, bacteria represent a rich source of new functions potentially available for co-option. One benefit that genes of bacterial origin could provide to eukaryotes is the capacity to produce antibacterials, which have evolved in prokaryotes as the result of eons of interbacterial competition. The type VI secretion amidase effector (Tae) proteins are potent bacteriocidal enzymes that degrade the cell wall when delivered into competing bacterial cells by the type VI secretion system. Here we show that tae genes have been transferred to eukaryotes on at least six occasions, and that the resulting domesticated amidase effector (dae) genes have been preserved for hundreds of millions of years through purifying selection. We show that the dae genes acquired eukaryotic secretion signals, are expressed within recipient organisms, and encode active antibacterial toxins that possess substrate specificity matching extant Tae proteins of the same lineage. Finally, we show that a dae gene in the deer tick Ixodes scapularis limits proliferation of Borrelia burgdorferi, the aetiologic agent of Lyme disease. Our work demonstrates that a family of horizontally acquired toxins honed to mediate interbacterial antagonism confers previously undescribed antibacterial capacity to eukaryotes. We speculate that the selective pressure imposed by competition between bacteria has produced a reservoir of genes encoding diverse antimicrobial functions that are tailored for co-option by eukaryotic innate immune systems.
A new class of fatty acid— found in food and synthesized by mammalian tissues — enhances glucose uptake from the blood and reduces inflammation, suggesting that these fats might be used to treat diabetes.
An experiment shows that although bank employees behave honestly on average, their dishonesty increases when they make decisions after having been primed to think about their professional identity.
The finding that intestinal viruses can substitute for intestinal bacteria to promote the health of their mammalian hosts raises the possibility that viruses in the gut may be beneficial in some circumstances.
Trust in others’ honesty is a key component of the long-term performance of firms, industries, and even whole countries. However, in recent years, numerous scandals involving fraud have undermined confidence in the financial industry. Contemporary commentators have attributed these scandals to the financial sector’s business culture, but no scientific evidence supports this claim. Here we show that employees of a large, international bank behave, on average, honestly in a control condition. However, when their professional identity as bank employees is rendered salient, a significant proportion of them become dishonest. This effect is specific to bank employees because control experiments with employees from other industries and with students show that they do not become more dishonest when their professional identity or bank-related items are rendered salient. Our results thus suggest that the prevailing business culture in the banking industry weakens and undermines the honesty norm, implying that measures to re-establish an honest culture are very important.
Intestinal microbial communities have profound effects on host physiology. Whereas the symbiotic contribution of commensal bacteria is well established, the role of eukaryotic viruses that are present in the gastrointestinal tract under homeostatic conditions is undefined. Here we demonstrate that a common enteric RNA virus can replace the beneficial function of commensal bacteria in the intestine. Murine norovirus (MNV) infection of germ-free or antibiotic-treated mice restored intestinal morphology and lymphocyte function without inducing overt inflammation and disease. The presence of MNV also suppressed an expansion of group 2 innate lymphoid cells observed in the absence of bacteria, and induced transcriptional changes in the intestine associated with immune development and type I interferon (IFN) signalling. Consistent with this observation, the IFN-α receptor was essential for the ability of MNV to compensate for bacterial depletion. Importantly, MNV infection offset the deleterious effect of treatment with antibiotics in models of intestinal injury and pathogenic bacterial infection. These data indicate that eukaryotic viruses have the capacity to support intestinal homeostasis and shape mucosal immunity, similarly to commensal bacteria.
The prominent and evolutionarily ancient role of the plant hormone auxin is the regulation of cell expansion. Cell expansion requires ordered arrangement of the cytoskeleton but molecular mechanisms underlying its regulation by signalling molecules including auxin are unknown. Here we show in the model plant Arabidopsis thaliana that in elongating cells exogenous application of auxin or redistribution of endogenous auxin induces very rapid microtubule re-orientation from transverse to longitudinal, coherent with the inhibition of cell expansion. This fast auxin effect requires auxin binding protein 1 (ABP1) and involves a contribution of downstream signalling components such as ROP6 GTPase, ROP-interactive protein RIC1 and the microtubule-severing protein katanin. These components are required for rapid auxin- and ABP1-mediated re-orientation of microtubules to regulate cell elongation in roots and dark-grown hypocotyls as well as asymmetric growth during gravitropic responses.
MicroRNAs are short non-coding RNAs expressed in different tissue and cell types that suppress the expression of target genes. As such, microRNAs are critical cogs in numerous biological processes, and dysregulated microRNA expression is correlated with many human diseases. Certain microRNAs, called oncomiRs, play a causal role in the onset and maintenance of cancer when overexpressed. Tumours that depend on these microRNAs are said to display oncomiR addiction. Some of the most effective anticancer therapies target oncogenes such as EGFR and HER2; similarly, inhibition of oncomiRs using antisense oligomers (that is, antimiRs) is an evolving therapeutic strategy. However, the in vivo efficacy of current antimiR technologies is hindered by physiological and cellular barriers to delivery into targeted cells. Here we introduce a novel antimiR delivery platform that targets the acidic tumour microenvironment, evades systemic clearance by the liver, and facilitates cell entry via a non-endocytic pathway. We find that the attachment of peptide nucleic acid antimiRs to a peptide with a low pH-induced transmembrane structure (pHLIP) produces a novel construct that could target the tumour microenvironment, transport antimiRs across plasma membranes under acidic conditions such as those found in solid tumours (pH approximately 6), and effectively inhibit the miR-155 oncomiR in a mouse model of lymphoma. This study introduces a new model for using antimiRs as anti-cancer drugs, which can have broad impacts on the field of targeted drug delivery.
Broad and deep tumour genome sequencing has shed new light on tumour heterogeneity and provided important insights into the evolution of metastases arising from different clones. There is an additional layer of complexity, in that tumour evolution may be influenced by selective pressure provided by therapy, in a similar fashion to that occurring in infectious diseases. Here we studied tumour genomic evolution in a patient (index patient) with metastatic breast cancer bearing an activating PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha, PI(3)Kα) mutation. The patient was treated with the PI(3)Kα inhibitor BYL719, which achieved a lasting clinical response, but the patient eventually became resistant to this drug (emergence of lung metastases) and died shortly thereafter. A rapid autopsy was performed and material from a total of 14 metastatic sites was collected and sequenced. All metastatic lesions, when compared to the pre-treatment tumour, had a copy loss of PTEN (phosphatase and tensin homolog) and those lesions that became refractory to BYL719 had additional and different PTEN genetic alterations, resulting in the loss of PTEN expression. To put these results in context, we examined six other patients also treated with BYL719. Acquired bi-allelic loss of PTEN was found in one of these patients, whereas in two others PIK3CA mutations present in the primary tumour were no longer detected at the time of progression. To characterize our findings functionally, we examined the effects of PTEN knockdown in several preclinical models (both in cell lines intrinsically sensitive to BYL719 and in PTEN-null xenografts derived from our index patient), which we found resulted in resistance to BYL719, whereas simultaneous PI(3)K p110β blockade reverted this resistance phenotype. We conclude that parallel genetic evolution of separate metastatic sites with different PTEN genomic alterations leads to a convergent PTEN-null phenotype resistant to PI(3)Kα inhibition.
Chronic stress can cause depression in some individuals, but leaves others untouched. Engagement of a molecular pathway controlling the production of tiny RNA snippets might help to explain the difference.
Two studies find that an intracellular quality-control mechanism called autophagy is regulated by nuclear receptor proteins that govern the expression of autophagy genes.
Hereβ-catenin, which has been implicated in neurological and psychiatric diseases, including depression, is shown to mediate resilience to chronic stress in mice through induction of Dicer and microRNAs in nucleus accumbens, a key brain reward region.
Autophagy is an evolutionarily conserved catabolic process that recycles nutrients upon starvation and maintains cellular energy homeostasis. Its acute regulation by nutrient-sensing signalling pathways is well described, but its longer-term transcriptional regulation is not. The nuclear receptors peroxisome proliferator-activated receptor-α (PPARα) and farnesoid X receptor (FXR) are activated in the fasted and fed liver, respectively. Here we show that both PPARα and FXR regulate hepatic autophagy in mice. Pharmacological activation of PPARα reverses the normal suppression of autophagy in the fed state, inducing autophagic lipiddegradation, or lipophagy. This response is lost in PPARα knockout (Ppara−/−, also known as Nr1c1−/−) mice, which are partially defective in the induction of autophagy by fasting. Pharmacological activation of the bile acid receptor FXR strongly suppresses the induction of autophagy in thefasting state, and this response is absent in FXR knockout (Fxr−/−, also known as Nr1h4−/−) mice, which show a partial defect in suppression of hepatic autophagy in the fed state. PPARα and FXR compete for binding to shared sites in autophagic gene promoters, with opposite transcriptional outputs. These results reveal complementary, interlocking mechanisms for regulation of autophagy by nutrient status.
Lysosomal degradation of cytoplasmic components by autophagy is essential for cellular survival and homeostasis under nutrient-deprived conditions. Acute regulation of autophagy by nutrient-sensing kinases is well defined, but longer-term transcriptional regulation is relatively unknown. Here we show that the fed-state sensing nuclear receptor farnesoid X receptor (FXR) and the fasting transcriptional activator cAMP response element-binding protein (CREB) coordinately regulate the hepatic autophagy gene network. Pharmacological activation of FXR repressed many autophagy genes and inhibited autophagy even in fasted mice, and feeding-mediated inhibition of macroautophagy was attenuated in FXR-knockout mice. From mouse liver chromatin immunoprecipitation and high-throughput sequencing data, FXR and CREB binding peaks were detected at 178 and 112 genes, respectively, out of 230 autophagy-related genes, and 78 genes showed shared binding, mostly in their promoter regions. CREB promoted autophagic degradation of lipids, or lipophagy, under nutrient-deprived conditions, and FXR inhibited this response. Mechanistically, CREB upregulated autophagy genes, including Atg7, Ulk1 and Tfeb, by recruiting the coactivator CRTC2. After feeding or pharmacological activation, FXR trans-repressed these genes by disrupting the functional CREB–CRTC2 complex. This study identifies the new FXR–CREB axis as a key physiological switch regulating autophagy, resulting in sustained nutrient regulation of autophagy during feeding/fasting cycles.
Biotin-dependent carboxylases are widely distributed in nature and have important functions in the metabolism of fatty acids, amino acids, carbohydrates, cholesterol and other compounds. Defective mutations in several of these enzymes have been linked to serious metabolic diseases in humans, and acetyl-CoA carboxylase is a target for drug discovery in the treatment of diabetes, cancer and other diseases. Here we report the identification and biochemical, structural and functional characterizations of a novel single-chain (120 kDa), multi-domain biotin-dependent carboxylase in bacteria. It has preference for long-chain acyl-CoA substrates, although it is also active towards short-chain and medium-chain acyl-CoAs, and we have named it long-chain acyl-CoA carboxylase. The holoenzyme is a homo-hexamer with molecular mass of 720 kDa. The 3.0 Å crystal structure of the long-chain acyl-CoA carboxylase holoenzyme from Mycobacterium avium subspecies paratuberculosis revealed an architecture that is strikingly different from those of related biotin-dependent carboxylases. In addition, the domains of each monomer have no direct contact with each other. They are instead extensively swapped in the holoenzyme, such that one cycle of catalysis involves the participation of four monomers. Functional studies in Pseudomonas aeruginosa suggest that the enzyme is involved in the utilization of selected carbon and nitrogen sources.
Genome-wide association studies combined with data from epigenomic maps for immune cells have been used to fine-map causal variants for 21 autoimmune diseases; disease risk tends to be linked to single nucleotide polymorphisms in cell-type-specific enhancers, often in regions adjacent to transcription factor binding motifs.
The semi-conservative centrosome duplication in cycling cells gives rise to a centrosome composed of a mother and a newly formed daughter centriole. Both centrioles are regarded as equivalent in their ability to form new centrioles and their symmetric duplication is crucial for cell division homeostasis. Multiciliated cells do not use the archetypal duplication program and instead form more than a hundred centrioles that are required for the growth of motile cilia and the efficient propelling of physiological fluids. The majority of these new centrioles are thought to appear de novo, that is, independently from the centrosome, around electron-dense structures called deuterosomes. Their origin remains unknown. Using live imaging combined with correlative super-resolution light and electron microscopy, we show that all new centrioles derive from the pre-existing progenitor cell centrosome through multiple rounds of procentriole seeding. Moreover, we establish that only the daughter centrosomal centriole contributes to deuterosome formation, and thus to over ninety per cent of the final centriole population. This unexpected centriolar asymmetry grants new perspectives when studying cilia-related diseases and pathological centriole amplification observed in cycling cells and associated with microcephaly and cancer.
The adoption of a new form of tool use has been observed to spread along social-network pathways in a chimpanzee community. The finding offers the first direct evidence of cultural diffusion in these animals in the wild.
The spliceosome enzyme complex removes intron sequences from RNA transcripts to form messenger RNA. The crystal structure of a lasso-shaped RNA suggests a mechanism for this splicing process.
This study determines the structure of a branched lariat RNA, providing insights into rearrangement of the intron between the two steps of RNA splicing.
The response of the terrestrial carbon cycle to climate change is among the largest uncertainties affecting future climate change projections. The feedback between the terrestrial carbon cycle and climate is partly determined by changes in the turnover time of carbon in land ecosystems, which in turn is an ecosystem property that emerges from the interplay between climate, soil and vegetation type. Here we present a global, spatially explicit and observation-based assessment of whole-ecosystem carbon turnover times that combines new estimates of vegetation and soil organic carbon stocks and fluxes. We find that the overall mean global carbon turnover time is years (95 per cent confidence interval). On average, carbon resides in the vegetation and soil near the Equator for a shorter time than at latitudes north of 75° north (mean turnover times of 15 and 255 years, respectively). We identify a clear dependence of the turnover time on temperature, asexpected from our present understanding of temperature controls on ecosystem dynamics. Surprisingly, our analysis also reveals a similarly strong association between turnover time and precipitation. Moreover, we find that the ecosystem carbon turnover times simulated by state-of-the-art coupled climate/carbon-cycle models vary widely and that numerical simulations, on average, tend to underestimate the global carbon turnover time by 36 per cent. The models show stronger spatial relationships with temperature than do observation-based estimates, but generally do not reproduce the strong relationships with precipitation and predict faster carbon turnover in many semi-arid regions. Our findings suggest that future climate/carbon-cycle feedbacks may depend more strongly on changes in the hydrological cycle than is expected at present and is considered in Earth system models.
Analyses in mice and humans indicate that non-caloric artificial sweeteners may promote obesity-associated metabolic changes by changing the function of the bacteria that colonize the gut.
Non-caloric artificial sweeteners (NAS), widely used food additives considered to be safe and beneficial alternatives to sugars, are shown here to lead to the development of glucose intolerance through compositional and functional changes in the gut microbiota of mice, and the deleterious metabolic effects are transferred to germ-free mice by faecal transplant; NAS-induced dysbiosis and glucose intolerance are also demonstrated in healthy human subjects.
Rapid industrialization and urbanization in developing countries has led to an increase in air pollution, along a similar trajectory to that previously experienced by the developed nations. In China, particulate pollution is a serious environmental problem that is influencing air quality, regional and global climates, and human health. In response to the extremely severe and persistent haze pollution experienced by about 800 million people during the first quarter of 2013 (refs 4, 5), the Chinese State Council announced its aim to reduce concentrations of PM2.5 (particulate matter with an aerodynamic diameter less than 2.5 micrometres) by up to 25 per cent relative to 2012 levels by 2017 (ref. 6). Such efforts however require elucidation of the factors governing the abundance and composition of PM2.5, which remain poorly constrained in China. Here we combine a comprehensive set of novel and state-of-the-art offlineanalytical approaches and statistical techniques to investigate the chemical nature and sources of particulate matter at urban locations in Beijing, Shanghai, Guangzhou and Xi’an during January 2013. We find that the severe haze pollution event was driven to a large extent by secondary aerosol formation, which contributed 30–77 per cent and 44–71 per cent (average for all four cities) of PM2.5 and of organic aerosol, respectively. On average, the contribution of secondary organic aerosol (SOA) and secondary inorganic aerosol (SIA) are found to be of similar importance (SOA/SIA ratios range from 0.6 to 1.4). Our results suggest that, in addition to mitigating primary particulate emissions, reducing the emissions of secondary aerosol precursors from, for example, fossil fuel combustion and biomass burning is likely to be important for controlling China’s PM2.5 levels and for reducing the environmental, economic and health impacts resulting from particulate pollution.
Magnetoresistance is the change in a material’s electrical resistance in response to an applied magnetic field. Materials with large magnetoresistance have found use as magnetic sensors, in magnetic memory, and in hard drives at room temperature, and their rarity has motivated many fundamental studies in materials physics at low temperatures. Here we report the observation of an extremely large positive magnetoresistance at low temperatures in the non-magnetic layered transition-metal dichalcogenide WTe2: 452,700 per cent at 4.5 kelvins in a magnetic field of 14.7 teslas, and 13 million per cent at 0.53 kelvins in a magnetic field of 60 teslas. In contrast with other materials, there is no saturation of the magnetoresistance value even at very high applied fields. Determination of the origin and consequences of this effect, and the fabrication of thin films, nanostructures and devices based on the extremely large positive magnetoresistance of WTe2, will represent a significant new direction in the study of magnetoresistivity.
β-Thalassaemia major (β-TM) is an inherited haemoglobinopathy caused by a quantitative defect in the synthesis of β-globin chains of haemoglobin, leading to the accumulation of free α-globin chains that form toxic aggregates. Despite extensive knowledge of the molecular defects causing β-TM, little is known of the mechanisms responsible for the ineffective erythropoiesis observed in the condition, which is characterized by accelerated erythroid differentiation, maturation arrest and apoptosis at the polychromatophilic stage. We have previously demonstrated that normal human erythroid maturation requires a transient activation of caspase-3 at the later stages of maturation. Although erythroid transcription factor GATA-1, the master transcriptional factor of erythropoiesis, is a caspase-3 target, it is not cleaved during erythroid differentiation. We have shown that, in human erythroblasts, the chaperone heat shock protein70 (HSP70) is constitutively expressed and, at later stages of maturation, translocates into the nucleus and protects GATA-1 from caspase-3 cleavage. The primary role of this ubiquitous chaperone is to participate in the refolding of proteins denatured by cytoplasmic stress, thus preventing their aggregation. Here we show in vitro that during the maturation of human β-TM erythroblasts, HSP70 interacts directly with free α-globin chains. As a consequence, HSP70 is sequestrated in the cytoplasm and GATA-1 is no longer protected, resulting in end-stage maturation arrest and apoptosis. Transduction of a nuclear-targeted HSP70 mutant or a caspase-3-uncleavable GATA-1 mutant restores terminal maturation of β-TM erythroblasts, which may provide a rationale for new targeted therapies of β-TM.
The polycomb repressive complex 2 (PRC2) exerts oncogenic effects in many tumour types. However, loss-of-function mutations in PRC2 components occur in a subset of haematopoietic malignancies, suggesting that this complex plays a dichotomous and poorly understood role in cancer. Here we provide genomic, cellular, and mouse modelling data demonstrating that the polycomb group gene SUZ12 functions as tumour suppressor in PNS tumours, high-grade gliomas and melanomas by cooperating with mutations in NF1. NF1 encodes a Ras GTPase-activating protein (RasGAP) and its loss drives cancer by activating Ras. We show that SUZ12 loss potentiates the effects of NF1 mutations by amplifying Ras-driven transcription through effects on chromatin. Importantly, however, SUZ12 inactivation also triggers an epigenetic switch that sensitizes these cancers to bromodomain inhibitors. Collectively, these studies not only reveal an unexpected connection between the PRC2 complex, NF1 and Ras, but also identify a promising epigenetic-based therapeutic strategy that may be exploited for a variety of cancers.
Caspase-4 and caspase-11 are shown to be the direct sensors for cytoplasmic lipopolysaccharide in humans and mice, respectively, mediating inflammatory cell death in intracellular bacterial infection.
The balance between stem cell self-renewal and differentiation is controlled by intrinsic factors and niche signals. In the Drosophila melanogaster ovary, some intrinsic factors promote germline stem cell (GSC) self-renewal, whereas others stimulate differentiation. However, it remains poorly understood how the balance between self-renewal and differentiation is controlled. Here we use D. melanogaster ovarian GSCs to demonstrate that the differentiation factor Bam controls the functional switch of the COP9 complex from self-renewal to differentiation via protein competition. The COP9 complex is composed of eight Csn subunits, Csn1–8, and removes Nedd8 modifications from target proteins. Genetic results indicated that the COP9 complex is required intrinsically for GSC self-renewal, whereas other Csn proteins, with the exception of Csn4, were also required for GSC progeny differentiation. Bam-mediated Csn4 sequestration from the COP9 complex via protein competition inactivated the self-renewing function of COP9 and allowed other Csn proteins to promote GSC differentiation. Therefore, this study reveals a protein-competition-based mechanism for controlling the balance between stem cell self-renewal and differentiation.Because numerous self-renewal factors are ubiquitously expressed throughout the stem cell lineage in various systems, protein competition may function as an important mechanism for controlling the self-renewal-to-differentiation switch.
The connection between an altered gut microbiota and metabolic disorders such as obesity, diabetes, and cardiovascular disease is well established. Defects in preserving the integrity of the mucosal barriers can result in systemic endotoxaemia that contributes to chronic low-grade inflammation, which further promotes the development of metabolic syndrome. Interleukin (IL)-22 exerts essential roles in eliciting antimicrobial immunity and maintaining mucosal barrier integrity within the intestine. Here we investigate the connection between IL-22 and metabolic disorders. We find that the induction of IL-22 from innate lymphoid cells and CD4+ T cells is impaired in obese mice under various immune challenges, especially in the colon during infection with Citrobacter rodentium. While innate lymphoid cell populations are largely intact in obese mice, the upregulation of IL-23, a cytokine upstream of IL-22, is compromised during the infection. Consequently, these mice are susceptible to C. rodentium infection, and both exogenous IL-22 and IL-23 are able to restore the mucosal host defence. Importantly, we further unveil unexpected functions of IL-22 in regulating metabolism. Mice deficient in IL-22 receptor and fed with high-fat diet are prone to developing metabolic disorders. Strikingly, administration of exogenous IL-22 in genetically obese leptin-receptor-deficient (db/db) mice and mice fed with high-fat diet reverses many of the metabolic symptoms, including hyperglycaemia and insulin resistance. IL-22 shows diverse metabolic benefits, as it improves insulin sensitivity, preserves gut mucosal barrier and endocrine functions, decreases endotoxaemia and chronic inflammation, and regulates lipid metabolism in liver and adipose tissues. In summary, we identify the IL-22 pathway as a novel target for therapeutic intervention in metabolic diseases.
The pluripotency factor Lin28 inhibits the biogenesis of the let-7 family of mammalian microRNAs. Lin28 is highly expressed in embryonic stem cells and has a fundamental role in regulation of development, glucose metabolism and tissue regeneration. Overexpression of Lin28 is correlated with the onset of numerous cancers, whereas let-7, a tumour suppressor, silences several human oncogenes. Lin28 binds to precursor let-7 (pre-let-7) hairpins, triggering the 3′ oligo-uridylation activity of TUT4 and TUT7 (refs 10, 11, 12). The oligoU tail added to pre-let-7 serves as a decay signal, as it is rapidly degraded by Dis3l2 (refs 13, 14), a homologue of the catalytic subunit of the RNA exosome. The molecular basis of Lin28-mediated recruitment of TUT4 and TUT7 to pre-let-7 and its subsequent degradation by Dis3l2 is largely unknown. To examine the mechanism of Dis3l2 substrate recognition we determined the structure of mouse Dis3l2 in complex with an oligoU RNA to mimic the uridylated tail of pre-let-7. Three RNA-binding domains form an open funnel onone face of the catalytic domain that allows RNA to navigate a path to the active site different from that of its exosome counterpart. The resulting path reveals an extensive network of uracil-specific interactions spanning the first 12 nucleotides of an oligoU-tailed RNA. We identify three U-specificity zones that explain how Dis3l2 recognizes, binds and processes uridylated pre-let-7 in the final step of the Lin28–let-7 pathway.
Homeodomain proteins, described 30 years ago, exert essential roles in development as regulators of target gene expression; however, the molecular mechanisms underlying transcriptional activity of homeodomain factors remain poorly understood. Here investigation of a developmentally required POU-homeodomain transcription factor, Pit1 (also known as Pou1f1), has revealed that, unexpectedly, binding of Pit1-occupied enhancers to a nuclear matrin-3-rich network/architecture is a key event in effective activation of the Pit1-regulated enhancer/coding gene transcriptional program. Pit1 association with Satb1 (ref. 8) andβ-catenin is required for this tethering event. A naturally occurring, dominant negative, point mutation in human PIT1(R271W), causing combined pituitary hormone deficiency, results in loss of Pit1 association with β-catenin and Satb1 and therefore the matrin-3-rich network, blocking Pit1-dependent enhancer/coding target gene activation. This defective activation can be rescued by artificial tethering of the mutant R271W Pit1 protein to the matrin-3 network, bypassing the pre-requisite association with β-catenin and Satb1 otherwise required. The matrin-3 network-tethered R271W Pit1 mutant,but not the untethered protein, restores Pit1-dependent activation of the enhancers and recruitment of co-activators, exemplified by p300, causing both enhancer RNA transcription and target gene activation. These studies have thus revealed an unanticipated homeodomain factor/β-catenin/Satb1-dependent localization of target gene regulatory enhancer regions to a subnuclear architectural structure that serves as an underlying mechanism by which an enhancer-bound homeodomain factor effectively activates developmental gene transcriptional programs.
CHARGE syndrome is a multiple anomaly disorder in which patients present with a variety of phenotypes, including ocular coloboma, heart defects, choanal atresia, retarded growth and development, genitourinary hypoplasia and ear abnormalities. Despite 70–90% of CHARGE syndrome cases resulting from mutations in the gene CHD7, which encodes an ATP-dependent chromatin remodeller, the pathways underlying the diverse phenotypes remain poorly understood. Surprisingly, our studies of a knock-in mutant mouse strain that expresses a stabilized and transcriptionally dead variant of the tumour-suppressor protein p53 (p5325,26,53,54), along with a wild-type allele of p53 (also known as Trp53), revealed late-gestational embryonic lethality associated with a host of phenotypes that are characteristic of CHARGE syndrome, including coloboma, inner and outer ear malformations, heart outflow tract defects and craniofacial defects. We found that the p5325,26,53,54 mutant protein stabilized and hyperactivated wild-type p53, which then inappropriately induced its target genes and triggered cell-cycle arrest or apoptosis during development. Importantly, these phenotypes were only observed with a wild-type p53 allele, as p5325,26,53,54/− embryos were fully viable. Furthermore, we found that CHD7 can bind to the p53 promoter, thereby negatively regulating p53 expression, and that CHD7 loss in mouse neural crest cells or samples from patients with CHARGE syndrome results in p53 activation. Strikingly, we found that p53 heterozygosity partially rescued the phenotypes in Chd7-null mouse embryos, demonstrating that p53 contributes to the phenotypes that result from CHD7 loss. Thus, inappropriate p53 activation during development can promote CHARGEphenotypes, supporting the idea that p53 has a critical role in developmental syndromes and providing important insight into the mechanisms underlying CHARGE syndrome.
Mice deficient in the EDA protein lack normal tooth features. Restoring EDA in embryonic teeth at increasing doses has now been found to recover these dental features in a stepwise pattern that mimics evolution.
Gradual changes that occur to mammalian tooth morphology across evolutionary time were modelled in vitro and in vivo by modulation of signalling pathways in the mouse, and computer modelling was used to provide further analysis of the parameters influencing tooth morphology.
A comparison of colorectal cancer and normal cells from 103 patients identifies dozens of genes that are differently expressed in tumour cells as a result of altered regulation of transcription.
The cis-regulatory effects responsible for cancer development have not been as extensively studied as the perturbations of the protein coding genome in tumorigenesis. To better characterize colorectal cancer (CRC) development we conducted an RNA-sequencing experiment of 103 matched tumour and normal colon mucosa samples from Danish CRC patients, 90 of which were germline-genotyped. By investigating allele-specific expression (ASE) we show that the germline genotypes remain important determinants of allelic gene expression in tumours. Using the changes in ASE in matched pairs of samples we discover 71 genes with excess of somatic cis-regulatory effects in CRC, suggesting a cancer driver role. We correlate genotypes and gene expression to identify expression quantitative trait loci (eQTLs) and find 1,693 and 948 eQTLs in normal samples and tumours, respectively. We estimate that 36% of the tumour eQTLs are exclusive to CRC and show that this specificity is partially driven by increased expression of specific transcription factors and changes in methylation patterns. We show that tumour-specific eQTLs are more enriched for low CRC genome-wide association study (GWAS) P values than shared eQTLs, which suggests that some of the GWAS variants are tumour specific regulatory variants. Importantly, tumour-specific eQTL genes also accumulate more somatic mutations when compared to the shared eQTL genes, raising the possibility that they constitute germline-derived cancer regulatory drivers. Collectively the integration of genome and the transcriptome reveals a substantial number of putative somatic and germline cis-regulatory cancer changes that may have a role in tumorigenesis.
Giving monkeys antiretroviral therapy from just three days after exposure to simian immunodeficiency virus does not prevent a subsequent rebound of viral replication, suggesting that viral reservoirs are established early.
Net primary production is affected by temperature and precipitation, but whether this is a direct kinetic effect on plant metabolism or an indirect ecological effect mediated by changes in plant age, plant biomass or growing season length is unclear— this study develops metabolic scaling theory to be able to answer this question and applies it to a global data set of plant productivity, concluding that it is indirect effects that explain the influence of climate on productivity, which is characterized by a common scaling relationship acrossclimate gradients.
The viral reservoir represents a critical challenge for human immunodeficiency virus type 1 (HIV-1) eradication strategies. However, it remains unclear when and where the viral reservoir is seeded during acute infection and the extent to which it is susceptible to early antiretroviral therapy (ART). Here we show that the viral reservoir is seeded rapidly after mucosal simian immunodeficiency virus (SIV) infection of rhesus monkeys and before systemic viraemia. We initiated suppressive ART in groups of monkeys on days 3, 7, 10 and 14 after intrarectal SIVMAC251 infection. Treatment with ART on day 3 blocked the emergence of viral RNA and proviral DNA in peripheral blood and also substantially reduced levels of proviral DNA in lymph nodes and gastrointestinal mucosa as compared with treatment at later time points. In addition, treatment on day 3 abrogated the induction of SIV-specific humoral and cellular immune responses. Nevertheless, after discontinuation of ART following 24 weeks of fully suppressive therapy, virus rebounded in all animals, although the monkeys that were treated on day 3 exhibited a delayed viral rebound as compared with those treated on days 7, 10 and 14. The time to viral rebound correlated with total viraemia during acute infection and with proviral DNA at the time of ART discontinuation. These data demonstrate that the viral reservoir is seeded rapidly after intrarectal SIV infection of rhesus monkeys, during the‘eclipse’ phase, and before detectable viraemia. This strikingly early seeding of the refractory viral reservoir raises important new challenges for HIV-1 eradication strategies.
The crystal structures of thalidomide and its derivatives bound to the E3 ligase subcomplex DDB1–CRBN are shown; these drugs are found to have dual functions, interfering with the binding of certain cellular substrates to the E3 ligase but promoting the binding of others, thereby modulating the degradation of cellular proteins.
Developmental enhancers initiate transcription and are fundamental to our understanding of developmental networks, evolution and disease. Despite their importance, the properties governing enhancer–promoter interactions and their dynamics during embryogenesis remain unclear. At the β-globin locus, enhancer–promoter interactions appear dynamic and cell-type specific, whereas at the HoxD locus they are stable and ubiquitous, being present in tissues where the target genes are not expressed. The extent to which preformed enhancer–promoter conformations exist at other, more typical, loci and how transcription is eventually triggered is unclear. Here we generated a high-resolution map of enhancer three-dimensional contacts during Drosophila embryogenesis, covering two developmental stages and tissue contexts, at unprecedented resolution. Although local regulatory interactions are common, long-range interactions are highly prevalent within the compact Drosophila genome. Each enhancer contacts multiple enhancers, and promoters with similar expression, suggesting a role in their co-regulation. Notably, most interactions appear unchanged between tissue context and across development, arising before gene activation, and are frequently associated with paused RNA polymerase. Our results indicate that the general topology governing enhancer contacts is conserved from flies to humans and suggest that transcription initiates from preformed enhancer–promoter loops through release of paused polymerase.
Rheumatoid arthritis is a chronic autoinflammatory disease that affects 1–2% of the world’s population and is characterized by widespread joint inflammation. Interleukin-1 is an important mediator of cartilage destruction in rheumatic diseases, but our understanding of the upstream mechanisms leading to production of interleukin-1β in rheumatoid arthritis is limited by the absence of suitable mouse models of the disease in which inflammasomes contribute to pathology. Myeloid-cell-specific deletion of the rheumatoid arthritis susceptibility gene A20/Tnfaip3 in mice (A20myel-KO mice) triggers a spontaneous erosive polyarthritis that resembles rheumatoid arthritis in patients. Rheumatoid arthritis in A20myel-KO mice is not rescued by deletion of tumour necrosis factor receptor 1 (ref. 2). Here we show, however, that it crucially relies on the Nlrp3 inflammasome and interleukin-1 receptor signalling. Macrophages lacking A20 have increased basal and lipopolysaccharide-induced expression levels of the inflammasome adaptor Nlrp3 and proIL-1β. As a result, A20-deficiency in macrophages significantly enhances Nlrp3 inflammasome-mediated caspase-1 activation, pyroptosis and interleukin-1β secretion by soluble and crystalline Nlrp3 stimuli. In contrast, activation of the Nlrc4 and AIM2 inflammasomes is not altered. Importantly, increased Nlrp3 inflammasome activation contributes to the pathology of rheumatoid arthritis in vivo, because deletion of Nlrp3, caspase-1 and the interleukin-1 receptor markedly protects against rheumatoid-arthritis-associated inflammation and cartilage destruction in A20myel-KO mice. These results reveal A20 as a novel negative regulator of Nlrp3 inflammasome activation, and describe A20myel-KO mice as the first experimental model to study the role of inflammasomes in the pathology of rheumatoid arthritis.
Myeloproliferative neoplasms (MPNs) are diseases caused by mutations in the haematopoietic stem cell (HSC) compartment. Most MPN patients have a common acquired mutation of Janus kinase 2 (JAK2) gene in HSCs that renders this kinase constitutively active, leading to uncontrolled cell expansion. The bone marrow microenvironment might contribute to the clinical outcomes of this common event. We previously showed that bone marrow nestin+ mesenchymal stem cells (MSCs) innervated by sympathetic nerve fibres regulate normal HSCs. Here we demonstrate that abrogation of this regulatory circuit is essential for MPN pathogenesis. Sympathetic nerve fibres, supporting Schwann cells and nestin+ MSCs are consistently reduced in the bone marrow of MPN patients and mice expressing the human JAK2(V617F) mutation in HSCs. Unexpectedly, MSC reduction is not due to differentiation but is caused by bone marrow neural damage and Schwann cell death triggered by interleukin-1β produced by mutant HSCs. In turn, in vivo depletion of nestin+ cells or their production of CXCL12 expanded mutant HSC number and accelerated MPN progression. In contrast, administration of neuroprotective or sympathomimetic drugs prevented mutant HSC expansion. Treatment with β3-adrenergic agonists that restored the sympathetic regulation of nestin+ MSCs prevented the loss of these cells and blocked MPN progression by indirectly reducing the number of leukaemic stem cells. Our results demonstrate that mutant-HSC-driven niche damage critically contributes to disease manifestation in MPN and identify niche-forming MSCs and their neural regulation as promising therapeutic targets.
The proton gradient is a principal energy source for respiration-dependent active transport, but the structural mechanisms of proton-coupled transport processes are poorly understood. YiiP is a proton-coupled zinc transporter found in the cytoplasmic membrane of Escherichia coli. Its transport site receives protons from water molecules that gain access to its hydrophobic environment and transduces the energy of an inward proton gradient to drive Zn(ii) efflux. This membrane protein is a well-characterized member of the family of cation diffusion facilitators that occurs at all phylogenetic levels. Here we show, using X-ray-mediated hydroxyl radical labelling of YiiP and mass spectrometry, that Zn(ii) binding triggers a highly localized, all-or-nothing change of water accessibility to the transport site and an adjacent hydrophobic gate. Millisecond time-resolved dynamics reveal a concerted and reciprocal pattern of accessibility changes along a transmembrane helix, suggesting a rigid-body helical re-orientation linked to Zn(ii) binding that triggers the closing of the hydrophobic gate. The gated water access to the transport site enables a stationary proton gradient to facilitate the conversion of zinc-binding energy to the kinetic power stroke of a vectorial zinc transport. The kinetic details provide energetic insights into a proton-coupled active-transport reaction.
‘Gain’ of supernumerary copies of the 8q24.21 chromosomal region has been shown to be common in many human cancers and is associated with poor prognosis. The well-characterized myelocytomatosis (MYC) oncogene resides in the 8q24.21 region and is consistently co-gained with an adjacent ‘gene desert’ of approximately 2 megabases that contains the long non-coding RNA gene PVT1, the CCDC26 gene candidate and the GSDMC gene. Whether low copy-number gain of one or more of these genes drives neoplasia is not known. Here we use chromosome engineering in mice to show that a single extra copyof either the Myc gene or the region encompassing Pvt1, Ccdc26 and Gsdmc fails to advance cancer measurably, whereas a single supernumerary segment encompassing all four genes successfully promotes cancer. Gain of PVT1 long non-coding RNA expression was required for high MYC protein levels in 8q24-amplified human cancer cells. PVT1 RNA and MYC protein expression correlated in primary human tumours, and copy number of PVT1 was co-increased in more than 98% of MYC-copy-increase cancers. Ablation of PVT1 from MYC-driven colon cancer line HCT116 diminished its tumorigenic potency. As MYC protein has been refractory to small-molecule inhibition, the dependence of high MYC protein levels on PVT1 long non-coding RNA provides a much needed therapeutic target.
What gives quantum computers that extra oomph over their classical digital counterparts? An intrinsic, measurable aspect of quantum mechanics called contextuality, it now emerges.
The finding that phosphoinositide-3-OH kinaseδ restrains the antitumour immune response by promoting the action of suppressive immune cells may broaden the applicability of drugs targeting this enzyme to multiple cancers.
Quantum computing promises advantages over classical computing for certain problems; now‘quantum contextuality’ — a generalization of the concept of quantum non-locality — is shown to be a critical resource that gives the most promising class of quantum computers their power.
The Eucalyptus grandis genome has been sequenced, revealing the greatest number of tandem duplications of any plant genome sequenced so far, and the highest diversity of genes for specialized metabolites that act as chemical defence and provide unique pharmaceutical oils; genome sequencing of the sister species E. globulus and a set of inbred E. grandis tree genomes reveals dynamic genome evolution and hotspots of inbreeding depression.
Large-scale single-cell RNA-seq of stimulated primary mouse bone-marrow-derived dendritic cells highlights positive and negative intercellular signalling pathways that promote and restrain cellular variation.
Inhibitors against the p110δ isoform of phosphoinositide-3-OH kinase (PI(3)K) have shown remarkable therapeutic efficacy in some human leukaemias. As p110δ is primarily expressed in leukocytes, drugs against p110δ have not been considered for the treatment of solid tumours. Here we report that p110δ inactivation in mice protects against a broad range of cancers, including non-haematological solid tumours. We demonstrate that p110δ inactivation in regulatory T cells unleashes CD8+ cytotoxic T cells and induces tumour regression. Thus, p110δ inhibitors can break tumour-induced immune tolerance and should be considered for wider use in oncology.
Undernourished children fall behind not only on growth, but also on maturation of their intestinal bacterial communities, according to a study comparing acutely malnourished and healthy Bangladeshi children.
The enzyme parkin is known to promote disposal of organelles called mitochondria that have suffered damage. The identification of an enzyme that opposes parkin demonstrates how a delicate balance is maintained in the cell.
Damaged mitochondria are removed by mitophagy, and defects in mitophagy are linked to Parkinson’s disease; here it is shown that USP30, a deubiquitinase localized to mitochondria, antagonizes mitophagy by removing the ubiquitin tags put in place by Parkin, USP30 inhibition is therefore potentially beneficial for Parkinson’s disease by promoting mitochondrial clearance and quality control.
Therapeutic food interventions have reduced mortality in children with severe acute malnutrition (SAM), but incomplete restoration of healthy growth remains a major problem. The relationships between the type of nutritional intervention, the gut microbiota, and therapeutic responses are unclear. In the current study, bacterial species whose proportional representation define a healthy gut microbiota as it assembles during the first two postnatal years were identified by applying a machine-learning-based approach to 16S ribosomal RNA data sets generated from monthly faecal samples obtained from birth onwards in a cohort of children living in an urban slum of Dhaka, Bangladesh, who exhibited consistently healthy growth. These age-discriminatory bacterial species were incorporated into a model that computes a‘relative microbiota maturity index’ and ‘microbiota-for-age Z-score’ that compare postnatal assembly (defined here as maturation) of a child’s faecal microbiota relative to healthy children of similar chronologic age. The model was applied to twins and triplets (to test for associations of these indices with genetic and environmental factors, including diarrhoea), children with SAM enrolled in a randomized trial of two food interventions, and children with moderate acute malnutrition. Our results indicate that SAM is associated with significant relative microbiota immaturity that is only partially ameliorated following two widely used nutritional interventions. Immaturity is also evident in less severe forms of malnutrition and correlates with anthropometric measurements. Microbiota maturity indices provide a microbial measure of human postnatal development, a way of classifying malnourished states, and a parameter for judging therapeutic efficacy. More prolonged interventions with existing or new therapeutic foods and/or addition of gut microbes may be needed to achieve enduring repair of gut microbiota immaturity in childhood malnutrition and improve clinical outcomes.
Transcriptional enhancers are crucial regulators of gene expression and animal development and the characterization of their genomic organization, spatiotemporal activities and sequence properties is a key goal in modern biology. Here we characterize the in vivo activity of 7,705 Drosophila melanogaster enhancer candidates covering 13.5% of the non-coding non-repetitive genome throughout embryogenesis. 3,557 (46%) candidates are active, suggesting a high density with 50,000 to 100,000 developmental enhancers genome-wide. The vast majority of enhancers display specific spatial patterns that are highly dynamic during development. Most appear to regulate their neighbouring genes, suggesting that the cis-regulatory genome is organized locally into domains, which are supported by chromosomal domains, insulator binding and genome evolution. However, 12 to 21 per cent of enhancers appear to skip non-expressed neighbours and regulate a more distal gene. Finally, we computationally identify cis-regulatory motifs that are predictive and required for enhancer activity, as we validate experimentally. This work provides global insights into the organization of an animal regulatory genome and the make-up of enhancer sequences and confirms and generalizes principles from previous studies. All enhancer patterns are annotated manually with a controlled vocabulary and all results are available through a web interface (http://enhancers.starklab.org), including the raw images of all microscopy slides for manual inspection at arbitrary zoom levels.
A unique property of many adult stem cells is their ability to exist in a non-cycling, quiescent state. Although quiescence serves an essential role in preserving stem cell function until the stem cell is needed in tissue homeostasis or repair, defects in quiescence can lead to an impairment in tissue function. The extent to which stem cells can regulate quiescence is unknown. Here we show that the stem cell quiescent state is composed of two distinct functional phases, G0 and an‘alert’ phase we term GAlert. Stem cells actively and reversibly transition between these phases in response to injury-induced systemic signals. Using genetic mouse models specific to muscle stem cells (or satellite cells), we show that mTORC1 activity is necessary and sufficient for the transition of satellite cells from G0 into GAlert and that signalling through the HGF receptor cMet is also necessary. We also identify G0-to-GAlert transitions in several populations of quiescent stem cells. Quiescent stem cells that transition into GAlert possess enhanced tissue regenerative function. We propose that the transition of quiescent stem cells into GAlert functions as an ‘alerting’ mechanism, an adaptive response that positions stem cells to respond rapidly under conditions of injury and stress, priming them for cell cycle entry.
Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits. Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. Here we show thatα-ketoglutarate (α-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans. ATP synthase subunit β is identified as a novel binding protein of α-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution. Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan. We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit β and is dependent on target of rapamycin (TOR) downstream. Endogenous α-KG levels are increased on starvation and α-KG does not extend the lifespan of dietary-restricted animals, indicating that α-KG is a key metabolite that mediates longevity by dietary restriction. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases.
2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components and in the hydroxylation of transcription factors and splicing factor proteins. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA and ribosomal proteins have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans raises questions as to their structural and evolutionary relationships. In Escherichia coli, YcfD catalyses arginine hydroxylation in the ribosomal protein L16; in humans, MYC-induced nuclear antigen (MINA53; also known as MINA) and nucleolar protein 66 (NO66) catalyse histidine hydroxylation in the ribosomal proteins RPL27A and RPL8, respectively. The functional assignments of ROXs open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in the residue and protein selectivities of prokaryotic and eukaryotic ROXs, comparison of the crystal structures of E. coli YcfD and Rhodothermus marinus YcfD with those of human MINA53 and NO66 reveals highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-dependent oxygenases. ROX structures with and without their substrates support their functional assignments as hydroxylases but not demethylases, and reveal how the subfamily has evolved to catalyse the hydroxylation of different residue side chains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone Nε-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases catalysing modifications of translational and transcriptional machinery. The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate-oxidizing species reacts. This coordination flexibility has probably contributed to the evolution of the wide range of reactions catalysed by oxygenases.
The global shortening of messenger RNAs through alternative polyadenylation (APA) that occurs during enhanced cellular proliferation represents an important, yet poorly understood mechanism of regulated gene expression. The 3′ untranslated region (UTR) truncation of growth-promoting mRNA transcripts that relieves intrinsic microRNA- and AU-rich-element-mediated repression has been observed to correlate with cellular transformation; however, the importance to tumorigenicity of RNA 3′-end-processing factors that potentially govern APA is unknown. Here we identify CFIm25 as a broad repressor of proximal poly(A) site usage that, when depleted, increases cell proliferation. Applying a regression model on standard RNA-sequencing data for novel APA events, we identified at least 1,450 genes with shortened 3′ UTRs after CFIm25 knockdown, representing 11% of significantly expressed mRNAs in human cells. Marked increases in the expression of several known oncogenes, including cyclin D1, are observed as a consequence of CFIm25 depletion. Importantly, we identified a subset of CFIm25-regulated APA genes with shortened 3′ UTRs in glioblastoma tumours that have reduced CFIm25 expression. Downregulation of CFIm25 expression in glioblastoma cells enhances their tumorigenic properties and increases tumour size, whereas CFIm25 overexpression reduces these properties and inhibits tumour growth. These findings identify a pivotal role of CFIm25 in governing APA and reveal a previously unknown connection between CFIm25 and glioblastoma tumorigenicity.
Sulphur is an essential element for life and is ubiquitous in living systems. Yet how the sulphur atom is incorporated into many sulphur-containing secondary metabolites is poorly understood. For bond formation between carbon and sulphur in primary metabolites, the major ionic sulphur sources are the persulphide and thiocarboxylate groups on sulphur-carrier (donor) proteins. Each group is post-translationally generated through the action of a specific activating enzyme. In all reported bacterial cases, the gene encoding the enzyme that catalyses the carbon–sulphur bond formation reaction and that encoding the cognate sulphur-carrier protein exist in the same gene cluster. To study the production of the 2-thiosugar moiety in BE-7585A, an antibiotic from Amycolatopsis orientalis, we identified a putative 2-thioglucose synthase, BexX, whose protein sequence and mode of action seem similar to those of ThiG, the enzyme that catalyses thiazole formation in thiamine biosynthesis. However, no gene encoding a sulphur-carrier protein could be located in the BE-7585A cluster. Subsequent genome sequencing uncovered a few genes encoding sulphur-carrier proteins that are probably involved in the biosynthesis of primary metabolites but only one activating enzyme gene in the A. orientalis genome. Further experiments showed that this activating enzyme can adenylate each of these sulphur-carrier proteins and probably also catalyses the subsequent thiolation, through its rhodanese domain. A proper combination of these sulphur-delivery systems is effective for BexX-catalysed 2-thioglucose production. The ability of BexX to selectively distinguish sulphur-carrier proteins is given a structural basis using X-ray crystallography. This study is, to our knowledge, the first complete characterization of thiosugar formation in nature and also demonstrates the receptor promiscuity of the A. orientalis sulphur-delivery system. Our results also show that co-opting the sulphur-delivery machinery of primary metabolism for the biosynthesis of sulphur-containing natural products is probably a general strategy found in nature.
PTEN encodes a lipid phosphatase that is underexpressed in many cancers owing to deletions, mutations or gene silencing. PTEN dephosphorylates phosphatidylinositol (3,4,5)-triphosphate, thereby opposing the activity of class I phosphatidylinositol 3-kinases that mediate growth- and survival-factor signalling through phosphatidylinositol 3-kinase effectors such as AKT and mTOR. To determine whether continued PTEN inactivation is required to maintain malignancy, here we generate an RNA interference-based transgenic mouse model that allows tetracycline-dependent regulation of PTEN in a time- and tissue-specific manner. Postnatal Pten knockdown in the haematopoietic compartment produced highly disseminated T-cell acute lymphoblastic leukaemia. Notably, reactivation of PTEN mainly reduced T-cell leukaemia dissemination but had little effect on tumour load in haematopoietic organs. Leukaemia infiltration into the intestine was dependent on CCR9 G-protein-coupled receptor signalling, which was amplified by PTEN loss. Our results suggest that in the absence of PTEN, G-protein-coupled receptors may have an unanticipated role in driving tumour growth and invasion in an unsupportive environment. They further reveal that the role of PTEN loss in tumour maintenance is not invariant and can be influenced by the tissue microenvironment, thereby producing a form of intratumoral heterogeneity that is independent of cancer genotype.