Traditional culture-based methods have incompletely defined the etiology of common recalcitrant human fungal skin diseases including athlete’s foot and toenail infections. Skin protects humans from invasion by pathogenic microorganisms, while providing a home for diverse commensal microbiota1. Bacterial genomic sequence data have generated novel hypotheses about species and community structures underlying human disorders2,3,4. However, microbial diversity is not limited to bacteria; microorganisms such as fungi also play major roles in microbial community stability, human health and disease5. Genomic methodologies to identify fungal species and communities have been limited compared with tools available for bacteria6. Fungal evolution can be reconstructed with phylogenetic markers, including ribosomal RNA gene regions and other highly conserved genes7. Here, we sequenced and analyzed fungal communities of 14 skin sites in 10 healthy adults. Eleven core body and arm sites were dominated by Malassezia fungi, with species-level classifications revealing greater topographical resolution between sites. By contrast, three foot sites, plantar heel, toenail, and toeweb, exhibited tremendous fungal diversity. Concurrent analysis of bacterial and fungal communities demonstrated that skin physiologic attributes and topography differentially shape these two microbial communities. These results provide a framework for future investigation of interactions between pathogenic and commensal fungal and bacterial communities in maintaining human health and contributing to disease pathogenesis.
fungi; genome; skin; dermatology; microbiome; Malassezia; ITS; 18S rRNA
Allostery is an intrinsic property of many globular proteins and enzymes that is indispensable for cellular regulatory and feedback mechanisms. Recent theoretical1 and empirical2 observations indicate that allostery is also manifest in intrinsically disordered proteins (IDPs), which account for a significant proportion of the proteome3,4. Many IDPs are promiscuous binders that interact with multiple partners and frequently function as molecular hubs in protein interaction networks. The adenovirus early region 1A (E1A) oncoprotein is a prime example of a molecular hub IDP5. E1A can induce drastic epigenetic reprogramming of the cell within hours after infection, through interactions with a diverse set of partners that include key host regulators like the general transcriptional coactivator CREB binding protein (CBP), its paralog p300, and the retinoblastoma protein (pRb)6,7. Little is known about the allosteric effects at play in E1A-CBP-pRb interactions, or more generally in hub IDP interaction networks. Here, we utilized single-molecule Förster/fluorescence resonance energy transfer (smFRET) to study coupled binding and folding processes in the ternary E1A system. The low concentrations used in these high-sensitivity experiments proved essential for these studies, which are challenging due to a combination of E1A aggregation propensity and high-affinity binding interactions. Our data revealed that E1A-CBP-pRb interactions display either positive or negative cooperativity, depending on the available E1A interaction sites. This striking cooperativity switch enables fine-tuning of the thermodynamic accessibility of the ternary vs. binary E1A complexes, and may permit a context-specific tuning of associated downstream signaling outputs. Such a modulation of allosteric interactions is likely a common mechanism in molecular hub IDP function.
adenovirus E1A; p300/CBP; retinoblastoma protein; intrinsically disordered protein; allostery; single-molecule fluorescence
Cytosolic DNA arising from intracellular bacteria or viral infections is a powerful pathogen-associated molecular pattern (PAMP) that leads to innate immune host defense by the production of type I interferon and inflammatory cytokines. Recognition of cytosolic DNA by the recently discovered cyclic-GMP-AMP (cGA) synthase (cGAS) induces the production of cGA to activate the stimulator of interferon genes (STING). Here we report the crystal structure of cGAS alone and in complex with DNA, ATP and GTP along with functional studies. Our results explain cGAS’ broad specificity DNA sensing, show how cGAS catalyzes di-nucleotide formation and indicate activation by a DNA-induced structural switch. cGAS possesses a remarkable structural similarity to the antiviral cytosolic dsRNA sensor 2’-5’oligoadenylate synthase (OAS1), but contains a unique zinc-thumb that recognizes B-form dsDNA. Our results mechanistically unify dsRNA and dsDNA innate immune sensing by OAS1 and cGAS nucleotidyl transferases.
Regulatory T cells (Tregs) play a crucial role in the immune system by preventing autoimmunity, limiting immunopathology, and maintaining immune homeostasis1. However, they also represent a major barrier to effective anti-tumor immunity and sterilizing immunity to chronic viral infections1. The transcription factor Foxp3 plays a major role in the development and programming of Treg cells2,3. The relative stability of Tregs at inflammatory disease sites has been highly contentious4-6. There is considerable interest in identifying pathways that control Treg stability as many immune-mediated diseases are characterized by either exacerbated or limited Treg function. Here we show that the immune cell-expressed ligand semaphorin-4a (Sema4a) and the Treg-expressed receptor neuropilin-1 (Nrp1) interact to potentiate Treg function and survival in vitro and in inflammatory sites in vivo. Nrp1 is dispensable for suppression of autoimmunity and maintenance of immune homeostasis, but is required by Tregs to limit anti-tumor immune responses and to cure established inflammatory colitis. Sema4a ligation of Nrp1 restrained Akt phosphorylation cellularly and at the immunologic synapse (IS) via phosphatase and tensin homolog (PTEN), which increased nuclear localization of the transcription factor Foxo3a. The Nrp1-induced transcriptome promoted Treg stability by enhancing quiescence/survival factors while inhibiting programs that promote differentiation. Importantly, this Nrp1-dependent molecular program is evident in intratumoral Tregs. Our data support a model in which Treg stability can be subverted in certain inflammatory sites, but is maintained by a Sema4a:Nrp1 axis, highlighting this pathway as a potential therapeutic target that could limit Treg-mediated tumor-induced tolerance without inducing autoimmunity.
Most eukaryotic messenger RNA precursors (pre-mRNAs) undergo extensive maturational processing, including 3’-end cleavage and polyadenylation1–8. Despite the characterization of a large number of proteins that are required for the cleavage reaction, the identity of the endoribonuclease is not known4,9,10. Recent analyses suggested that the 73 kD subunit of cleavage and polyadenylation specificity factor (CPSF-73) may be the endonuclease for this and related reactions10–15, although no direct data confirmed this. Here we report the crystal structures of human CPSF-73 at 2.1 Å resolution, complexed with zinc ions and a sulfate that may mimic the phosphate group of the substrate, and the related yeast protein CPSF-100 (Ydh1p) at 2.5 Å resolution. Both CPSF-73 and CPSF-100 contain two domains, a metallo-β-lactamase domain and a novel β-CASP domain. The active site of CPSF-73, with two zinc ions, is located at the interface of the two domains. Purified recombinant CPSF-73 possesses endoribonuclease activity, and mutations that disrupt zinc binding in the active site abolish this activity. Our studies provide the first direct experimental evidence that CPSF-73 is the pre-mRNA 3’-end processing endonuclease.
polyadenylation; metallo-β-lactamase; pre-mRNA processing; Artemis; V(D)J recombination; double-strand break repair
Primary triple negative breast cancers (TNBC) represent approximately 16% of all breast cancers1 and are a tumour type defined by exclusion, for which comprehensive landscapes of somatic mutation have not been determined. Here we show in 104 early TNBC cases, that at the time of diagnosis these cancers exhibit a wide and continuous spectrum of genomic evolution, with some exhibiting only a handful of somatic aberrations in a few pathways, whereas others contain hundreds of somatic events and multiple pathways implicated. Integration with matched whole transcriptome sequence data revealed that only ~36% of mutations are expressed. By examining single nucleotide variant (SNV) allelic abundance derived from deep re-sequencing (median >20,000 fold) measurements in 2414 somatic mutations, we determine for the first time in an epithelial tumour, the relative abundance of clonal genotypes among cases in the population. We show that TNBC vary widely and continuously in their clonal frequencies at the time of diagnosis, with basal subtype TNBC2,3 exhibiting more variation than non-basal TNBC. Although p53 and PIK3CA/PTEN somatic mutations appear clonally dominant compared with other pathways, in some tumours their clonal frequencies are incompatible with founder status. Mutations in cytoskeletal and cell shape/motility proteins occurred at lower clonal frequencies, suggesting they occurred later during tumour progression. Taken together our results show that future attempts to dissect the biology and therapeutic responses of TNBC will require the determination of individual tumour clonal genotypes.
The pluripotent state, which is first established in the primitive ectoderm cells (PE) of blastocysts, is lost progressively and irreversibly during subsequent development1. For example, development of postimplantation epiblast from PE involves significant transcriptional and epigenetic changes, including DNA methylation and X inactivation2, which creates a robust epigenetic barrier and prevents their reversion to a PE-like state. Epiblast cells are refractory to leukaemia inhibitory factor (LIF)-STAT3 signaling, but they respond to Activin/bFGF to form self-renewing epiblast stem cells (EpiSC), which exhibit essential properties of epiblast cells3,4, that differ from embryonic stem cells (ESC) derived from PE5. Here we show reprogramming of advanced epiblast cells from E5.5 - E7.5 embryos with uniform expression of N-cadherin and inactive X chromosome, to ES-like cells (rESC) in response to LIF-STAT3 signaling. Cultured epiblast cells (cEpi) overcome the epigenetic barrier progressively as they proceed with the erasure of key properties of epiblast cells, involving DNA demethylation, X reactivation and expression of E-cadherin. The accompanying changes in the transcriptome result in a loss of phenotypic and epigenetic memory of epiblast cells. Notably, using this new approach, we report reversion of established EpiSC to rESC. Furthermore, unlike epiblast and EpiSC, rESC contribute to somatic tissues and germ cells in chimeras. This is a tractable model to investigate signaling molecule induced epigenetic reprogramming that can promote reacquisition of the fundamental pluripotent state.
Recent molecular studies have revealed that, even when derived from a seemingly
homogenous population, individual cells can exhibit substantial differences in gene expression,
protein levels, and phenotypic output1–5, with important functional consequences4,5. Existing studies of cellular
heterogeneity, however, have typically measured only a few pre-selected RNAs1,2 or proteins5,6 simultaneously
because genomic profiling methods3 could not be
applied to single cells until very recently7–10. Here, we use single-cell RNA-Seq
to investigate heterogeneity in the response of bone marrow derived dendritic cells (BMDCs) to
lipopolysaccharide (LPS). We find extensive, and previously unobserved, bimodal variation in mRNA
abundance and splicing patterns, which we validate by RNA-fluorescence in situ
hybridization (RNA-FISH) for select transcripts. In particular, hundreds of key immune genes are
bimodally expressed across cells, surprisingly even for genes that are very highly expressed at the
population average. Moreover, splicing patterns demonstrate previously unobserved levels of
heterogeneity between cells. Some of the observed bimodality can be attributed to closely related,
yet distinct, known maturity states of BMDCs; other portions reflect differences in the usage of key
regulatory circuits. For example, we identify a module of 137 highly variable, yet co-regulated,
antiviral response genes. Using cells from knockout mice, we show that variability in this module
may be propagated through an interferon feedback circuit involving the transcriptional regulators
Stat2 and Irf7. Our study demonstrates the power and promise of single-cell genomics in uncovering
functional diversity between cells and in deciphering cell states and circuits.
The protein-tyrosine phosphatase SHP-1 plays critical roles in immune signaling, but how mutations in SHP-1 cause inflammatory disease in humans remains poorly defined1. Mice homozygous for the Y208N amino acid substitution in the carboxy-terminus of SHP-1 (referred to as Ptpn6spin mice) spontaneously develop a severe inflammatory syndrome that resembles neutrophilic dermatosis in humans and is characterized by persistent footpad swelling and suppurative inflammation2,3. Here, we report that RIP1-regulated IL-1α production by hematopoietic cells critically mediates chronic inflammatory disease in Ptpn6spin mice, whereas inflammasome signaling and IL-1β-mediated events were dispensable. IL-1α was also critical for exacerbated inflammatory responses and unremitting tissue damage upon footpad microabrasion of Ptpn6spin mice. Intriguingly, pharmacological and genetic blockade of the kinase RIP1 protected against wound-induced inflammation and tissue damage in Ptpn6spin mice, whereas RIP3 deletion failed to do so. Moreover, RIP1-mediated inflammatory cytokine production was attenuated by NF-κB and ERK inhibition. Together, our results suggest that wound-induced tissue damage and chronic inflammation in Ptpn6spin mice are critically dependent on RIP1-mediated IL-1α production, whereas inflammasome signaling and RIP3-mediated necroptosis were dispensable. Thus, we have unravelled a novel inflammatory circuit in which RIP1-mediated IL-1α secretion in response to deregulated SHP-1 activity triggers an inflammatory destructive disease that proceeds independently of inflammasomes and programmed necrosis.
SHP-1; Ptpn6; inflammasome; NOD-like receptor; caspase; interleukin; RIP1; RIP3; cell death; inflammation
Congenital heart disease (CHD) is the most frequent birth defect, affecting 0.8% of live births1. Many cases occur sporadically and impair reproductive fitness, suggesting a role for de novo mutations. By analysis of exome sequencing of parent-offspring trios, we compared the incidence of de novo mutations in 362 severe CHD cases and 264 controls. CHD cases showed a significant excess of protein-altering de novo mutations in genes expressed in the developing heart, with an odds ratio of 7.5 for damaging mutations. Similar odds ratios were seen across major classes of severe CHD. We found a marked excess of de novo mutations in genes involved in production, removal or reading of H3K4 methylation (H3K4me), or ubiquitination of H2BK120, which is required for H3K4 methylation2–4. There were also two de novo mutations in SMAD2; SMAD2 signaling in the embryonic left-right organizer induces demethylation of H3K27me5. H3K4me and H3K27me mark `poised' promoters and enhancers that regulate expression of key developmental genes6. These findings implicate de novo point mutations in several hundred genes that collectively contribute to ~10% of severe CHD.
Adult stem cells undergo asymmetric cell division to self-renew and give rise to differentiated cells that comprise mature tissue1. Sister chromatids may be distinguished and segregated non-randomly in asymmetrically dividing stem cells2, although the underlying mechanism and the purpose it may serve remain elusive. We developed the CO-FISH (chromosome orientation fluorescence in situ hybridization) technique3 with single-chromosome resolution and show that sister chromatids of X and Y chromosomes, but not autosomes, are segregated non-randomly during asymmetric divisions of Drosophila male germline stem cells (GSCs). This provides the first direct evidence that two sister chromatids containing identical genetic information can be distinguished and segregated non-randomly during asymmetric stem cell divisions. We further show that the centrosome, SUN-KASH nuclear envelope proteins, and Dnmt2 are required for non-random sister chromatid segregation. Our data suggest that the information on X and Y chromosomes that enables non-random segregation is primed during gametogenesis in the parents. Moreover, we show that sister chromatid segregation is randomized in GSC overproliferation and dedifferentiated GSCs. We propose that non-random sister chromatid segregation may serve to transmit distinct information carried on two sister chromatids to the daughters of asymmetrically dividing stem cells.
Early life dietary transitions reflect fundamental aspects of primate evolution and are important determinants of health in contemporary human populations1,2. Weaning is critical to developmental and reproductive rates; early weaning can have detrimental health effects but enables shorter inter-birth intervals, which influences population growth3. Uncovering early life dietary history in fossils is hampered by the absence of prospectively-validated biomarkers that are not modified during fossilisation4. Here we show that major dietary shifts in early life manifest as compositional variations in dental tissues. Teeth from human children and captive macaques, with prospectively-recorded diet histories, demonstrate that barium (Ba) distributions accurately reflect dietary transitions from the introduction of mother’s milk and through the weaning process. We also document transitions in a Middle Palaeolithic juvenile Neanderthal, which shows a pattern of exclusive breastfeeding for seven months, followed by seven months of supplementation. After this point, Ba levels in enamel returned to baseline prenatal levels, suggesting an abrupt cessation of breastfeeding at 1.2 years of age. Integration of Ba spatial distributions and histological mapping of tooth formation enables novel studies of the evolution of human life history, dietary ontogeny in wild primates, and human health investigations through accurate reconstructions of breastfeeding history.
Defining mechanisms by which Plasmodium virulence is regulated is central to understanding the pathogenesis of human malaria. Serial blood passage of Plasmodium through rodents1-3, primates4 or humans5 increases parasite virulence, suggesting that vector transmission regulates Plasmodium virulence within the mammalian host. In agreement, disease severity can be modified by vector transmission6-8, which is assumed to ‘reset’ Plasmodium to its original character3. However, direct evidence that vector transmission regulates Plasmodium virulence is lacking. Here we utilise mosquito transmission of serially blood passaged (SBP) Plasmodium chabaudi chabaudi9 to interrogate regulation of parasite virulence. Analysis of SBP P.c. chabaudi before and after mosquito transmission demonstrates that vector transmission intrinsically modifies the asexual blood-stage parasite, which in turn, modifies the elicited mammalian immune response, which in turn, attenuates parasite growth and associated pathology. Attenuated parasite virulence associates with modified expression of the pir multi-gene family. Vector transmission of Plasmodium therefore regulates gene expression of probable variant antigens in the erythrocytic cycle, modifies the elicited mammalian immune response, and thus regulates parasite virulence. These results place the mosquito at the centre of our efforts to dissect mechanisms of protective immunity to malaria for the development of an effective vaccine.
Detecting genomic changes represents a critical step in cellular responses to DNA damage. Here, we show that tyrosine phosphorylation of the protein acetyltransferase KAT5 (Tip60) increases in response to DNA damage in a manner that promotes KAT5 binding to the histone mark H3K9me3. This in turn triggers KAT5-mediated acetylation of the ATM kinase, promoting DNA-damage checkpoint activation and cell survival. We also establish that chromatin alterations per se can enhance KAT5 tyrosine phosphorylation and ATM-dependent signaling, and identify the proto-oncogene c-Abl as mediating this modification. These findings define KAT5 as a key sensor for genomic and chromatin perturbations, and highlight a prime role for c-Abl in such events.
Heart attacks occur when lipoprotein-driven inflammation called
atherosclerosis triggers blood clotting in the arteries. It seems that the
attacks can, in turn, accelerate atherosclerosis by fanning the
Cyclic-nucleotide-gated (CNG) channels in outer segments of vertebrate photoreceptors generate electrical signals in response to changes in cyclic GMP concentration during phototransduction1. CNG channels also allow the influx of Ca2+, which is essential for photoreceptor adaptation2. In cone photoreceptors, cGMP triggers an increase in membrane capacitance indicative of exocytosis, suggesting that CNG channels are also involved in synaptic function3. Here we examine whether CNG channels reside in cone terminals and whether they regulate neurotransmitter release, specifically in response to nitric oxide (NO), a retrograde transmitter that increases cGMP synthesis and potentiates synaptic transmission in the brain4–6. Using intact retina, we show that endogenous NO modulates synapses between cones and horizontal cells. In experiments on isolated cones, we show directly that CNG channels occur in clusters and are indirectly activated by S-nitrosocysteine (SNC), an NO donor. Furthermore, both SNC and pCPT–cGMP, a membrane-permeant analogue of cGMP, trigger the release of transmitter from the cone terminals. The NO-induced transmitter release is suppressed by guanylate cyclase inhibitors and prevented by direct activation of CNG channels, indicating that their activation is required for NO to elicit release. These results expand our view of CNG channel function to include the regulation of synaptic transmission and mediation of the presynaptic effects of NO.
Human language, as well as birdsong, relies on the ability to arrange vocal elements in novel sequences. However, little is known about the ontogenetic origin of this capacity. We tracked the development of vocal combinatorial capacity in three species of vocal learners, combining an experimental approach in zebra finches with an analysis of natural development of vocal transitions in Bengalese finches and pre-lingual human infants and found a common, stepwise pattern of acquiring vocal transitions across species. In our first study, juvenile zebra finches were trained to perform one song and then the training target was altered, prompting the birds to swap syllable order, or insert a new syllable into a string. All birds solved these permutation tasks in a series of steps, gradually approximating the target sequence by acquiring novel pair-wise syllable transitions, sometimes too slowly to fully accomplish the task. Similarly, in the more complex songs of Bengalese finches, branching points and bidirectional transitions in song-syntax were acquired in a stepwise manner, starting from a more restrictive set of vocal transitions. The babbling of pre-lingual human infants revealed a similar developmental pattern: instead of a single developmental shift from reduplicated to variegated babbling (i.e., from repetitive to diverse sequences), we observed multiple shifts, where each novel syllable type slowly acquired a diversity of pair-wise transitions, asynchronously over development. Collectively, these results point to a common generative process that is conserved across species, suggesting that the long-noted gap between perceptual versus motor combinatorial capabilities in human infants1 may arise from the challenges in constructing new pair-wise transitions.
Cholesterol is a structural component of the cell, indispensable for normal cellular function, but its excess often leads to abnormal proliferation, migration, inflammatory responses and/or cell death. To prevent cholesterol overload, ATP-binding cassette (ABC) transporters mediate cholesterol efflux from the cells to apolipoprotein A-I (ApoA-I) and to the ApoA-I-containing high-density lipoprotein (HDL)1-3. Maintaining efficient cholesterol efflux is essential for normal cellular function4-6. However, the role of cholesterol efflux in angiogenesis and the identity of its local regulators are poorly understood. Here we show that ApoA-I binding protein (AIBP) accelerates cholesterol efflux from endothelial cells (EC) to HDL and thereby regulates angiogenesis. AIBP/HDL-mediated cholesterol depletion reduces lipid rafts, interferes with VEGFR2 dimerization and signaling, and inhibits VEGF-induced angiogenesis in vitro and mouse aortic neovascularization ex vivo. Remarkably, Aibp regulates the membrane lipid order in embryonic zebrafish vasculature and functions as a non-cell autonomous regulator of zebrafish angiogenesis. Aibp knockdown results in dysregulated sprouting/branching angiogenesis, while forced Aibp expression inhibits angiogenesis. Dysregulated angiogenesis is phenocopied in Abca1/Abcg1-deficient embryos, and cholesterol levels are increased in Aibp-deficient and Abca1/Abcg1-deficient embryos. Our findings demonstrate that secreted AIBP positively regulates cholesterol efflux from EC and that effective cholesterol efflux is critical for proper angiogenesis.
The RNA-induced silencing complex, comprising Argonaute and guide RNA, mediates RNA interference. Here we report the 3.2 Å crystal structure of Kluyveromyces Argonaute (KpAGO) fortuitously complexed with guide RNA originating from small-RNA duplexes autonomously loaded and processed by recombinant KpAGO. Despite their diverse sequences, guide-RNA nucleotides 1–8 are positioned similarly, with sequence-independent contacts to bases, phosphates and 2′-hydroxyl groups pre-organizing the backbone of nucleotides 2–8 in a near–A-form conformation. Compared with prokaryotic Argonautes, KpAGO has numerous surface-exposed insertion segments, with a cluster of conserved insertions repositioning the N domain to enable full propagation of guide–target pairing. Compared with Argonautes in inactive conformations, KpAGO has a hydrogen-bond network that stabilizes an expanded and repositioned loop, which inserts an invariant glutamate into the catalytic pocket. Mutation analyses and analogies to Ribonuclease H indicate that insertion of this glutamate finger completes a universally conserved catalytic tetrad, thereby activating Argonaute for RNA cleavage.
Anti-diabetic drugs that activate the protein PPARγ had a bright start but soon lost appeal due to undesirable side effects. Subtle modifications may once again make them suitable for treating diabetes.
The germ cell lineage exhibits unique characteristics, which are essential towards generating totipotency. Among the distinctive events in this lineage is DNA demethylation and the erasure of parental imprints, which occur on embryonic day 11.5 (E11.5) after the primordial germ cells (PGCs) have entered into the developing gonads 12. Little is yet known about the mechanism involved, except that this appears to be an active process. Here we have examined the associated changes in the chromatin to gain further insights into this reprogramming event. We show that chromatin changes during this process occur in two-steps. The first changes observed in nascent PGCs at E8.5 establish a distinctive chromatin signature with some characteristics associated with pluripotency. Subsequently, at E11.5 when these PGCs are residing in the gonads, major changes occur in nuclear architecture with an extensive erasure of several histone modification marks along with exchange of histone variants. Furthermore, at this time, the histone chaperones, HIRA and NAP1, which are implicated in histone exchange, show accumulation in PGC nuclei undergoing reprogramming. We thus suggest that the mechanism of histone replacement is critical for these chromatin rearrangements to occur. The striking chromatin changes we show here are intimately linked with the process of genome-wide DNA demethylation. Based on the timing of the observed events, we propose, that if DNA demethylation entails DNA repair based mechanism, the evident histone replacement would rather than being a prerequisite, represent a repair-induced response event.