We measured half-lives of 21,248 mRNA 3′ isoforms in yeast by rapidly depleting RNA polymerase II from the nucleus and performing direct RNA sequencing throughout the decay process. Interestingly, half-lives of mRNA isoforms from the same gene, including nearly identical isoforms, often vary widely. Based on clusters of isoforms with different half-lives, we identify hundreds of sequences conferring stabilization or destabilization upon mRNAs terminating downstream. One class of stabilizing element is a polyU sequence that can interact with poly(A) tails, inhibit the association of poly(A)-binding protein, and confer increased stability upon introduction into ectopic transcripts. More generally, destabilizing and stabilizing elements are linked to the propensity of the poly(A) tail to engage in double-stranded structures. Isoforms engineered to fold into 3′ stem-loop structures not involving the poly(A) tail exhibit even longer half-lives. We suggest that double-stranded structures at 3′ ends are a major determinant of mRNA stability.
Cognitive function is tightly related to metabolic state but the locus of this control is not well understood. Synapses are thought to present large ATP demands however it is unclear how fuel availability and electrical activity impact synaptic ATP levels, and how ATP availability controls synaptic function. We developed a quantitative genetically-encoded optical reporter of presynaptic ATP, Syn-ATP, and find that electrical activity imposes large metabolic demands that are met via activity-driven control of both glycolysis and mitochondrial function. We discovered that the primary source of activity-driven metabolic demand is the synaptic vesicle cycle. In metabolically intact synapses, activity-driven ATP synthesis is well matched to the energetic needs of synaptic function which at steady state results in ~ 106 free ATPs per nerve terminal. Despite this large reservoir of ATP we find that several key aspects of presynaptic function are severely impaired following even brief interruptions in activity-stimulated ATP synthesis.
Bacterial and viral mRNAs are often polycistronic. Akin to alternative splicing, alternative translation of polycistronic messages is a mechanism to generate protein diversity and regulate gene function. Although a few examples exist, the use of polycistronic messages in mammalian cells is not widely appreciated. Here we report an example of alternative translation as a means of regulating innate immune signaling. MAVS, a regulator of antiviral innate immunity, is expressed from a bicistronic mRNA encoding a second protein, miniMAVS. This truncated variant interferes with interferon production induced by full length MAVS, whereas both proteins positively regulate cell death. To identify other polycistronic messages, we carried out genome-wide ribosomal profiling and identified a class of antiviral truncated variants. This study therefore reveals the existence of a functionally important bicistronic antiviral mRNA, and suggests a widespread role for polycistronic mRNAs in the innate immune system.
mTORC1 promotes cell growth in response to nutrients and growth factors. Insulin activates mTORC1 through the PI3K-Akt pathway, which inhibits the TSC1-TSC2-TBC1D7 complex (the TSC complex) to turn on Rheb, an essential activator of mTORC1. However, the mechanistic basis of how this pathway integrates with nutrient-sensing pathways is unknown. We demonstrate that insulin stimulates acute dissociation of the TSC complex from the lysosomal surface, where subpopulations of Rheb and mTORC1 reside. The TSC complex associates with the lysosome in a Rheb-dependent manner, and its dissociation in response to insulin requires Akt-mediated TSC2 phosphorylation. Loss of the PTEN tumor suppressor results in constitutive activation of mTORC1 through the Akt-dependent dissociation of the TSC complex from the lysosome. These findings provide a unifying mechanism by which independent pathways affecting the spatial recruitment of mTORC1 and the TSC complex to Rheb at the lysosomal surface serve to integrate diverse growth signals.
Despite 30 years of effort, there is no effective vaccine for HIV-1. However, antibodies can prevent HIV-1 infection in humanized mice and macaques when passively transferred. New single-cell-based methods have uncovered many broad and potent donor-derived antibodies, and structural studies have revealed the molecular bases for their activities. The new data suggest why such antibodies are difficult to elicit and inform HIV-1 vaccine development efforts. In addition to protecting against infection, the newly identified antibodies can suppress active infections in mice and macaques, suggesting they could be valuable additions to anti-HIV-1 therapies and to strategies to eradicate HIV-1 infection.
Reprogramming somatic cells to induced pluripotency by Yamanaka factors is usually slow and inefficient, and is thought to be a stochastic process. We identified a privileged somatic cell state, from which acquisition of pluripotency could occur in a non-stochastic manner. Subsets of murine hematopoietic progenitors are privileged, whose progeny cells predominantly adopt the pluripotent fate with activation of endogenous Oct4 locus after 4–5 divisions in reprogramming conditions. Privileged cells display an ultrafast cell cycle of ~8 hours. In fibroblasts, a subpopulation cycling at a similar ultrafast speed is observed after 6 days of factor expression, and is increased by p53-knockdown. This ultrafast-cycling population accounts for >99% of the bulk reprogramming activity in wildtype or p53-knockdown fibroblasts. Our data demonstrate that the stochastic nature of reprogramming can be overcome in a privileged somatic cell state, and suggest that cell cycle acceleration toward a critical threshold is an important bottleneck for reprogramming.
Differentiated cells possess a remarkable genomic plasticity that can be manipulated to reverse or change developmental commitments. Here, we show that the leprosy bacterium hijacks this property to reprogram adult Schwann cells, its preferred host niche, to a stage of progenitor/stem-like cells (pSLC) of mesenchymal traits by downregulating Schwann cell lineage/differentiation-associated genes and upregulating genes mostly of mesoderm development. Reprogramming accompanies epigenetic changes and rendered infected cells highly plastic, migratory and immunomodulatory. We provide evidence that acquisition of these properties by pSLC promotes bacterial spread by two distinct mechanisms: direct differentiation to mesenchymal tissues, including skeletal and smooth muscles, and by forming granuloma-like structures and subsequently release bacteria-laden macrophages. These findings support a model of host cell reprogramming in which a bacterial pathogen uses the plasticity of its cellular niche for promoting dissemination of infection, and provide an unexpected link between cellular reprogramming and host-pathogen interaction.
Glioblastomas (GBMs) are the most common and malignant primary brain tumors and are aggressively treated with surgery, chemotherapy, and radiotherapy. Despite this treatment, recurrence is inevitable and survival has improved minimally over the last 50 years. Recent studies have suggested that GBMs exhibit both heterogeneity and instability of differentiation states and varying sensitivities of these states to radiation. Here, we employed an iterative combined theoretical and experimental strategy that takes into account tumor cellular heterogeneity and dynamically acquired radioresistance to predict the effectiveness of different radiation schedules. Using this model, we identified two delivery schedules predicted to significantly improve efficacy by taking advantage of the dynamic instability of radioresistance. These schedules led to superior survival in mice. Our interdisciplinary approach may also be applicable to other human cancer types treated with radiotherapy and, hence, may lay the foundation for significantly increasing the effectiveness of a mainstay of oncologic therapy.
Lung stem cells are instructed to produce lineage-specific progeny through unknown factors in their microenvironment. We used clonal three-dimensional (3D) co-cultures of endothelial cells and distal lung stem cells, bronchioalveolar stem cells (BASCs), to probe the instructive mechanisms. Single BASCs had bronchiolar and alveolar differentiation potential in lung endothelial cell co-cultures. Gain and loss of function experiments showed BMP4-Bmpr1a signaling triggers calcineurin/NFATc1-dependent expression of Thrombospondin-1 (Tsp1) in lung endothelial cells to drive alveolar lineage-specific BASC differentiation. Tsp1-null mice exhibited defective alveolar injury repair, confirming a crucial role for the BMP4-NFATc1-TSP1 axis in lung epithelial differentiation and regeneration in vivo. Discovery of this pathway points to methods to direct the derivation of specific lung epithelial lineages from multipotent cells. These findings elucidate a pathway that may be a critical target in lung diseases and provide new tools to understand the mechanisms of respiratory diseases at the single cell level.
Lung stem cells; Lineage; Differentiation; Endothelial cells; Tsp1; Bmp4
The critical impact microbiota have on health and disease make the interaction between host and microbiome increasingly important as we evaluate therapeutics. Here we highlight growing evidence that beyond disease, microbes also affect the most fundamental of host physiological phenotypes, the rate of aging itself.
Therapy-resistant microenvironments represent a major barrier towards effective elimination of disseminated malignancies. Here, we show that select microenvironments can underlie resistance to antibody-based therapy. Using a humanized model of treatment-refractory B-cell leukemia, we find that infiltration of leukemia cells into the bone marrow rewires the tumor microenvironment to inhibit engulfment of antibody-targeted tumor cells. Resistance to macrophage-mediated killing can be overcome by combination regimens involving therapeutic antibodies and chemotherapy. Specifically, the nitrogen mustard cyclophosphamide induces an acute secretory activating phenotype (ASAP), releasing CCL4, IL8, VEGF and TNFα from treated tumor cells. These factors induce macrophage infiltration and phagocytic activity in the bone marrow. Thus, the acute induction of stress-related cytokines can effectively target cancer cells for removal by the innate immune system. This synergistic chemo-immunotherapeutic regimen represents a potent strategy for using conventional anti-cancer agents to alter the tumor microenvironment and promote the efficacy of targeted therapeutics.
The extended amygdala has dominated research on the neural circuitry of
fear and anxiety, but the septo-hippocampal axis plays an important role as
well. The lateral septum (LS) is thought to suppress fear and anxiety, through
its outputs to the hypothalamus. However, this structure has not yet been
dissected using modern tools. The type 2 CRF receptor (Crfr2)
marks a subset of LS neurons, whose functional connectivity we have investigated
using optogenetics. Crfr2+ cells include
GABAergic projection neurons that connect with the anterior hypothalamus.
Surprisingly, we find that these LS outputs enhance stress-induced behavioral
measures of anxiety. Furthermore, transient activation of
Crfr2+ neurons promotes, while
inhibition suppresses, persistent anxious behaviors. LS
Crfr2+ outputs also positively regulate
circulating corticosteroid levels. These data identify a subset of LS projection
neurons that promote, rather than suppress, stress-induced behavioral and
endocrinological dimensions of persistent anxiety states, and provide a cellular
point-of-entry to LS circuitry.
Vascular permeability is frequently associated with inflammation and triggered by a cohort of secreted permeability factors such as VEGF. Here we show that the physiological vascular permeability that precedes implantation is directly controlled by progesterone receptor (PR) and is independent of VEGF. Both global and endothelial-specific deletion of PR block physiological vascular permeability in the uterus whereas misexpression of PR in the endothelium of other organs results in ectopic vascular leakage. Integration of an endothelial genome-wide transcriptional profile with ChIP-sequencing revealed that PR induces a NR4A1 (Nur77/TR3)-dependent transcriptional program that broadly regulates vascular permeability in response to progesterone. Silencing of NR4A1 blocks PR-mediated permeability responses indicating a direct link between PR and NR4A1. This program triggers concurrent suppression of several junctional proteins and leads to an effective, timely and venous-specific regulation of vascular barrier function that is critical to embryo implantation.
Two recent studies identify how sex-specific pheromonal factors in flies
and worms alter lifespan through metabolic pathways that are shared with mammals
Sex differences in human lifespans imply non-autonomous effects modulated by
sex-specific gene-environment interactions which could still include pheromonal
The response to DNA damage, which regulates nuclear processes such as DNA repair, transcription, and cell cycle, has been studied thoroughly. However, the cytoplasmic response to DNA damage is poorly understood. Here, we demonstrate that DNA damage triggers dramatic reorganization of the Golgi, resulting in its dispersal throughout the cytoplasm. We further show that DNA-damage-induced Golgi dispersal requires GOLPH3/MYO18A/F-actin and the DNA damage protein kinase, DNA-PK. In response to DNA damage, DNA-PK phosphorylates GOLPH3, resulting in increased interaction with MYO18A, which applies a tensile force to the Golgi. Interference with the Golgi DNA damage response by depletion of DNA-PK, GOLPH3, or MYO18A reduces survival after DNA damage, whereas over-expression of GOLPH3, as is observed frequently in human cancers, confers resistance to killing by DNA-damaging agents. Identification of the DNA-damage-induced Golgi response reveals an unexpected pathway through DNA-PK, GOLPH3, and MYO18A that regulates cell survival following DNA damage.
Specification of primordial germ cells (PGCs) marks the beginning of the totipotent state. However, without a tractable experimental model, the mechanism of human PGC (hPGC) specification remains unclear. Here we demonstrate specification of hPGC-like cells (hPGCLCs) from germline competent pluripotent stem cells. The characteristics of hPGCLCs are consistent with the embryonic hPGCs and a germline seminoma that share a CD38 cell surface marker, which collectively defines likely progression of the early human germline. Remarkably, SOX17 is the key regulator of hPGC-like fate, while BLIMP1 represses endodermal and other somatic genes during specification of hPGCLCs. Notable mechanistic differences between mouse and human PGC specification could be attributed to their divergent embryonic development and pluripotent states, which might affect other early cell fate decisions. We have established a foundation for future studies on resetting of the epigenome in hPGCLCs and hPGCs, for totipotency and the transmission of genetic and epigenetic information.
The mammalian radiation has corresponded with rapid changes in noncoding regions of the genome, but we lack a comprehensive understanding of regulatory evolution in mammals. Here, we track the evolution of promoters and enhancers active in liver across 20 mammalian species from six diverse orders by profiling genomic enrichment of H3K27 acetylation and H3K4 trimethylation. We report that rapid evolution of enhancers is a universal feature of mammalian genomes. Most of the recently evolved enhancers arise from ancestral DNA exaptation, rather than lineage-specific expansions of repeat elements. In contrast, almost all liver promoters are partially or fully conserved across these species. Our data further reveal that recently evolved enhancers can be associated with genes under positive selection, demonstrating the power of this approach for annotating regulatory adaptations in genomic sequences. These results provide important insight into the functional genetics underpinning mammalian regulatory evolution.
•Rapid enhancer and slow promoter evolution across genomes of 20 mammalian species•Enhancers are rarely conserved across these mammals•Recently evolved enhancers dominate mammalian regulatory landscapes•Unbiased mapping links candidate enhancers with lineage-specific positive selection
Comparative functional genomic analysis in 20 mammalian species reveals distinct features for the evolution of enhancers, in comparison to those of promoters, across 180 million years.
Viable yet damaged cells can accumulate during development and aging. Although eliminating those cells may benefit organ function, identification of this less fit cell population remains challenging. Previously, we identified a molecular mechanism, based on “fitness fingerprints” displayed on cell membranes, which allows direct fitness comparison among cells in Drosophila. Here, we study the physiological consequences of efficient cell selection for the whole organism. We find that fitness-based cell culling is naturally used to maintain tissue health, delay aging, and extend lifespan in Drosophila. We identify a gene, azot, which ensures the elimination of less fit cells. Lack of azot increases morphological malformations and susceptibility to random mutations and accelerates tissue degeneration. On the contrary, improving the efficiency of cell selection is beneficial for tissue health and extends lifespan.
•Fitness-based cell culling maintains tissue health•Azot ensures the elimination of less fit cells•Lack of azot accelerates tissue degeneration•Improving the efficiency of cell selection extends lifespan
Elimination of less fit cells ensured by the expression of a cell-fitness checkpoint, Azot, is important for maintaining tissue health and prolonging lifespan in Drosophila.
Proper regulation of chromatin structure is necessary for the maintenance of cell type-specific gene expression patterns. The embryonic stem cell (ESC) expression pattern governs self-renewal and pluripotency. Here, we present an RNAi screen in mouse ESCs of 1008 loci encoding chromatin proteins. We identified 68 proteins that exhibit diverse phenotypes upon knockdown (KD), including seven subunits of the Tip60-p400 complex. Phenotypic analyses revealed that Tip60-p400 is necessary to maintain characteristic features of ESCs. We show that p400 localization to the promoters of both silent and active genes is dependent upon histone H3 lysine 4 trimethylation (H3K4me3). Furthermore, the Tip60-p400 KD gene expression profile is enriched for developmental regulators and significantly overlaps with that of the transcription factor Nanog. Depletion of Nanog reduces p400 binding to target promoters without affecting H3K4me3 levels. Together, these data indicate that Tip60-p400 integrates signals from Nanog and H3K4me3 to regulate gene expression in ESCs.
A clear relationship exists between visceral obesity and type 2 diabetes, whereas subcutaneous obesity is comparatively benign. Here we show that adipocyte-specific deletion of the coregulatory protein PRDM16 caused minimal effects on classical brown fat but markedly inhibited beige adipocyte function in subcutaneous fat following cold exposure or β3-agonist treatment. These animals developed obesity on a high fat diet, with severe insulin resistance and hepatic steatosis. They also showed altered fat distribution with markedly increased subcutaneous adiposity. Subcutaneous adipose tissue in mutant mice acquired many key properties of visceral fat, including decreased thermogenic and increased inflammatory gene expression and increased macrophage accumulation. Transplantation of subcutaneous fat into mice with diet-induced obesity showed a loss of metabolic benefit when tissues were derived from PRDM16 mutant animals. These findings indicate that PRDM16 and beige adipocytes are required for the “browning” of white fat and the healthful effects of subcutaneous adipose tissue.
Successful infection depends on the ability of the pathogen to gain nutrients from the host. The extracellular pathogenic bacterium group A Streptococcus (GAS) causes a vast array of human diseases. By using the quorum sensing sil system as a reporter, we found that during adherence to host cells GAS delivers streptolysin toxins creating endoplasmic reticulum stress. This in turn, increases asparagine (ASN) synthetase expression and the production of ASN. The released ASN is sensed by the bacteria altering the expression of ~17% of GAS genes of which about 1/3 are dependent on the two-component system TrxSR. The expression of the streptolysin toxins is strongly upregulated whereas genes linked to proliferation are downregulated in ASN absence. Asparaginase a widely used chemotherapeutic agent, arrests GAS growth in human blood and blocks GAS proliferation in a mouse model of human bacteremia. These results delineate a pathogenic pathway and propose a new therapeutic strategy against GAS infections.
group A streptococcus; streptolysin S; streptolysin O; quorum sensing; virulence; endoplasmic reticulum-stress; asparagine; transcriptome profiling; RNA-seq analyses; asparagine synthetase; asparaginase