Loss of epigenetic information as a cause of mammalian aging.

All living things experience an increase in entropy, manifested as a loss of genetic and epigenetic information. In yeast, epigenetic information is lost over time due to the relocalization of chromatin-modifying proteins to DNA breaks, causing cells to lose their identity, a hallmark of yeast aging. Using a system called "ICE" (inducible changes to the epigenome), we find that the act of faithful DNA repair advances aging at physiological, cognitive, and molecular levels, including erosion of the epigenetic landscape, cellular exdifferentiation, senescence, and advancement of the DNA methylation clock, which can be reversed by OSK-mediated rejuvenation. These data are consistent with the information theory of aging, which states that a loss of epigenetic information is a reversible cause of aging.

Metabolic Dynamics and Prediction of Gestational Age and Time to Delivery in Pregnant Women.

Metabolism during pregnancy is a dynamic and precisely programmed process, the failure of which can bring devastating consequences to the mother and fetus. To define a high-resolution temporal profile of metabolites during healthy pregnancy, we analyzed the untargeted metabolome of 784 weekly blood samples from 30 pregnant women. Broad changes and a highly choreographed profile were revealed: 4,995 metabolic features (of 9,651 total), 460 annotated compounds (of 687 total), and 34 human metabolic pathways (of 48 total) were significantly changed during pregnancy. Using linear models, we built a metabolic clock with five metabolites that time gestational age in high accordance with ultrasound (R = 0.92). Furthermore, two to three metabolites can identify when labor occurs (time to delivery within two, four, and eight weeks, AUROC ≥ 0.85). Our study represents a weekly characterization of the human pregnancy metabolome, providing a high-resolution landscape for understanding pregnancy with potential clinical utilities.

A Compendium of Genetic Modifiers of Mitochondrial Dysfunction Reveals Intra-organelle Buffering.

Mitochondrial dysfunction is associated with a spectrum of human conditions, ranging from rare, inborn errors of metabolism to the aging process. To identify pathways that modify mitochondrial dysfunction, we performed genome-wide CRISPR screens in the presence of small-molecule mitochondrial inhibitors. We report a compendium of chemical-genetic interactions involving 191 distinct genetic modifiers, including 38 that are synthetic sick/lethal and 63 that are suppressors. Genes involved in glycolysis (PFKP), pentose phosphate pathway (G6PD), and defense against lipid peroxidation (GPX4) scored high as synthetic sick/lethal. A surprisingly large fraction of suppressors are pathway intrinsic and encode mitochondrial proteins. A striking example of such "intra-organelle" buffering is the alleviation of a chemical defect in complex V by simultaneous inhibition of complex I, which benefits cells by rebalancing redox cofactors, increasing reductive carboxylation, and promoting glycolysis. Perhaps paradoxically, certain forms of mitochondrial dysfunction may best be buffered with "second site" inhibitors to the organelle.

Phosphoproteome Integration Reveals Patient-Specific Networks in Prostate Cancer.

We used clinical tissue from lethal metastatic castration-resistant prostate cancer (CRPC) patients obtained at rapid autopsy to evaluate diverse genomic, transcriptomic, and phosphoproteomic datasets for pathway analysis. Using Tied Diffusion through Interacting Events (TieDIE), we integrated differentially expressed master transcriptional regulators, functionally mutated genes, and differentially activated kinases in CRPC tissues to synthesize a robust signaling network consisting of druggable kinase pathways. Using MSigDB hallmark gene sets, six major signaling pathways with phosphorylation of several key residues were significantly enriched in CRPC tumors after incorporation of phosphoproteomic data. Individual autopsy profiles developed using these hallmarks revealed clinically relevant pathway information potentially suitable for patient stratification and targeted therapies in late stage prostate cancer. Here, we describe phosphorylation-based cancer hallmarks using integrated personalized signatures (pCHIPS) that shed light on the diversity of activated signaling pathways in metastatic CRPC while providing an integrative, pathway-based reference for drug prioritization in individual patients.

Interleukin-10 receptor signaling promotes the maintenance of a PD-1int TCF-1+ CD8+ T cell population that sustains anti-tumor immunity.

T cell exhaustion limits anti-tumor immunity and responses to immunotherapy. Here, we explored the microenvironmental signals regulating T cell exhaustion using a model of chronic lymphocytic leukemia (CLL). Single-cell analyses identified a subset of PD-1hi, functionally impaired CD8+ T cells that accumulated in secondary lymphoid organs during disease progression and a functionally competent PD-1int subset. Frequencies of PD-1int TCF-1+ CD8+ T cells decreased upon Il10rb or Stat3 deletion, leading to accumulation of PD-1hi cells and accelerated tumor progression. Mechanistically, inhibition of IL-10R signaling altered chromatin accessibility and disrupted cooperativity between the transcription factors NFAT and AP-1, promoting a distinct NFAT-associated program. Low IL10 expression or loss of IL-10R-STAT3 signaling correlated with increased frequencies of exhausted CD8+ T cells and poor survival in CLL and in breast cancer patients. Thus, balance between PD-1hi, exhausted CD8+ T cells and functional PD-1int TCF-1+ CD8+ T cells is regulated by cell-intrinsic IL-10R signaling, with implications for immunotherapy.

A molecular mechanism for environmental sex determination.

Daphnia produce genetically identical males and females; their sex is determined by environmental conditions. Recently, Kato et al. identified isoform switching events in Daphnia as a gene regulatory mechanism for sex-specific development. This finding uncovers the impact of alternative usage of gene isoforms on this extreme phenotypic plasticity trait.

HCR spectral imaging: 10-plex, quantitative, high-resolution RNA and protein imaging in highly autofluorescent samples.

Signal amplification based on the mechanism of hybridization chain reaction (HCR) provides a unified framework for multiplex, quantitative, high-resolution imaging of RNA and protein targets in highly autofluorescent samples. With conventional bandpass imaging, multiplexing is typically limited to four or five targets owing to the difficulty in separating signals generated by fluorophores with overlapping spectra. Spectral imaging has offered the conceptual promise of higher levels of multiplexing, but it has been challenging to realize this potential in highly autofluorescent samples, including whole-mount vertebrate embryos. Here, we demonstrate robust HCR spectral imaging with linear unmixing, enabling simultaneous imaging of ten RNA and/or protein targets in whole-mount zebrafish embryos and mouse brain sections. Further, we demonstrate that the amplified and unmixed signal in each of the ten channels is quantitative, enabling accurate and precise relative quantitation of RNA and/or protein targets with subcellular resolution, and RNA absolute quantitation with single-molecule resolution, in the anatomical context of highly autofluorescent samples.

Multigenerational Regulation of the Caenorhabditis elegans Chromatin Landscape by Germline Small RNAs.

In animals, small noncoding RNAs that are expressed in the germline and transmitted to progeny control gene expression to promote fertility. Germline-expressed small RNAs, including endogenous small interfering RNAs (endo-siRNAs) and Piwi-interacting RNAs (piRNAs), drive the repression of deleterious transcripts such as transposons, repetitive elements, and pseudogenes. Recent studies have highlighted an important role for small RNAs in transgenerational epigenetic inheritance via regulation of heritable chromatin marks; therefore, small RNAs are thought to convey an epigenetic memory of genomic self and nonself elements. Small RNA pathways are highly conserved in metazoans and have been best described for the model organism Caenorhabditis elegans. In this review, we describe the biogenesis, regulation, and function of C. elegans endo-siRNAs and piRNAs, along with recent insights into how these distinct pathways are integrated to collectively regulate germline gene expression, transgenerational epigenetic inheritance, and ultimately, animal fertility.

Half a century of global decline in oceanic sharks and rays.

Overfishing is the primary cause of marine defaunation, yet declines in and increasing extinction risks of individual species are difficult to measure, particularly for the largest predators found in the high seas1-3. Here we calculate two well-established indicators to track progress towards Aichi Biodiversity Targets and Sustainable Development Goals4,5: the Living Planet Index (a measure of changes in abundance aggregated from 57 abundance time-series datasets for 18 oceanic shark and ray species) and the Red List Index (a measure of change in extinction risk calculated for all 31 oceanic species of sharks and rays). We find that, since 1970, the global abundance of oceanic sharks and rays has declined by 71% owing to an 18-fold increase in relative fishing pressure. This depletion has increased the global extinction risk to the point at which three-quarters of the species comprising this functionally important assemblage are threatened with extinction. Strict prohibitions and precautionary science-based catch limits are urgently needed to avert population collapse6,7, avoid the disruption of ecological functions and promote species recovery8,9.

The Gene-Silencing Protein MORC-1 Topologically Entraps DNA and Forms Multimeric Assemblies to Cause DNA Compaction.

Microrchidia (MORC) ATPases are critical for gene silencing and chromatin compaction in multiple eukaryotic systems, but the mechanisms by which MORC proteins act are poorly understood. Here, we apply a series of biochemical, single-molecule, and cell-based imaging approaches to better understand the function of the Caenorhabditis elegans MORC-1 protein. We find that MORC-1 binds to DNA in a length-dependent but sequence non-specific manner and compacts DNA by forming DNA loops. MORC-1 molecules diffuse along DNA but become static as they grow into foci that are topologically entrapped on DNA. Consistent with the observed MORC-1 multimeric assemblies, MORC-1 forms nuclear puncta in cells and can also form phase-separated droplets in vitro. We also demonstrate that MORC-1 compacts nucleosome templates. These results suggest that MORCs affect genome structure and gene silencing by forming multimeric assemblages to topologically entrap and progressively loop and compact chromatin.

The quantitative genetics of gene expression in Mimulus guttatus.

Gene expression can be influenced by genetic variants that are closely linked to the expressed gene (cis eQTLs) and variants in other parts of the genome (trans eQTLs). We created a multiparental mapping population by sampling genotypes from a single natural population of Mimulus guttatus and scored gene expression in the leaves of 1,588 plants. We find that nearly every measured gene exhibits cis regulatory variation (91% have FDR < 0.05). cis eQTLs are usually allelic series with three or more functionally distinct alleles. The cis locus explains about two thirds of the standing genetic variance (on average) but varies among genes and tends to be greatest when there is high indel variation in the upstream regulatory region and high nucleotide diversity in the coding sequence. Despite mapping over 10,000 trans eQTL / affected gene pairs, most of the genetic variance generated by trans acting loci remains unexplained. This implies a large reservoir of trans acting genes with subtle or diffuse effects. Mapped trans eQTLs show lower allelic diversity but much higher genetic dominance than cis eQTLs. Several analyses also indicate that trans eQTLs make a substantial contribution to the genetic correlations in expression among different genes. They may thus be essential determinants of "gene expression modules," which has important implications for the evolution of gene expression and how it is studied by geneticists.

A large-effect fitness trade-off across environments is explained by a single mutation affecting cold acclimation.

Identifying the genetic basis of local adaptation and fitness trade-offs across environments is a central goal of evolutionary biology. Cold acclimation is an adaptive plastic response for surviving seasonal freezing, and costs of acclimation may be a general mechanism for fitness trade-offs across environments in temperate zone species. Starting with locally adapted ecotypes of Arabidopsis thaliana from Italy and Sweden, we examined the fitness consequences of a naturally occurring functional polymorphism in CBF2. This gene encodes a transcription factor that is a major regulator of cold-acclimated freezing tolerance and resides within a locus responsible for a genetic trade-off for long-term mean fitness. We estimated the consequences of alternate genotypes of CBF2 on 5-y mean fitness and fitness components at the native field sites by comparing near-isogenic lines with alternate genotypes of CBF2 to their genetic background ecotypes. The effects of CBF2 were validated at the nucleotide level using gene-edited lines in the native genetic backgrounds grown in simulated parental environments. The foreign CBF2 genotype in the local genetic background reduced long-term mean fitness in Sweden by more than 10%, primarily via effects on survival. In Italy, fitness was reduced by more than 20%, primarily via effects on fecundity. At both sites, the effects were temporally variable and much stronger in some years. The gene-edited lines confirmed that CBF2 encodes the causal variant underlying this genetic trade-off. Additionally, we demonstrated a substantial fitness cost of cold acclimation, which has broad implications for potential maladaptive responses to climate change.

Streptolysin S is required for Streptococcus pyogenes nasopharyngeal and skin infection in HLA-transgenic mice.

Streptococcus pyogenes is a human-specific pathogen that commonly colonizes the upper respiratory tract and skin, causing a wide variety of diseases ranging from pharyngitis to necrotizing fasciitis and toxic shock syndrome. S. pyogenes has a repertoire of secreted virulence factors that promote infection and evasion of the host immune system including the cytolysins streptolysin O (SLO) and streptolysin S (SLS). S. pyogenes does not naturally infect the upper respiratory tract of mice although mice transgenic for MHC class II human leukocyte antigens (HLA) become highly susceptible. Here we used HLA-transgenic mice to assess the role of both SLO and SLS during both nasopharyngeal and skin infection. Using S. pyogenes MGAS8232 as a model strain, we found that an SLS-deficient strain exhibited a 100-fold reduction in bacterial recovery from the nasopharynx and a 10-fold reduction in bacterial burden in the skin, whereas an SLO-deficient strain did not exhibit any infection defects in these models. Furthermore, depletion of neutrophils significantly restored the bacterial burden of the SLS-deficient bacteria in skin, but not in the nasopharynx. In mice nasally infected with the wildtype S. pyogenes, there was a marked change in localization of the tight junction protein ZO-1 at the site of infection, demonstrating damage to the nasal epithelia that was absent in mice infected with the SLS-deficient strain. Overall, we conclude that SLS is required for the establishment of nasopharyngeal infection and skin infection in HLA-transgenic mice by S. pyogenes MGAS8232 and provide evidence that SLS contributes to nasopharyngeal infection through the localized destruction of nasal epithelia.

Use of HSC Targeted LNP to Generate a Mouse Model of Lethal α-Thalassemia and Treatment via Lentiviral Gene Therapy.

-Thalassemia (AT) is one of the most commonly occurring inherited hematological diseases. However, few treatments are available, and allogeneic bone marrow transplantation (BMT) is the only available therapeutic option for patients with severe AT. Research into AT has remained limited due to a lack of adult mouse models, with severe AT typically resulting in in utero lethality. By using a lipid nanoparticle (LNP) targeting the receptor CD117 and delivering a Cre mRNA (mRNACreLNPCD117), we were able to delete floxed -globin genes at high efficiency in hematopoietic stem cells (HSC) ex vivo. These cells were then engrafted in the absence or presence of a novel α-globin expressing lentiviral vector (ALS20I). Myeloablated mice transplanted with mRNACreLNPCD117-treated HSC showed a complete knockout of -globin genes. They demonstrated a phenotype characterized by the synthesis of hemoglobin H (-tetramers,  or HbH), aberrant erythropoiesis, and abnormal organ morphology, culminating in lethality approximately eight weeks following engraftment. Mice receiving mRNACreLNPCD117-treated HSC with at least one copy of ALS20I survived long-term with normalization of erythropoiesis, decreased the production of HbH, and ameliorated the abnormal organ morphology. Furthermore, we tested ALS20I in erythroid progenitors derived from -globin-KO CD34+ and cells isolated from patients with both deletional and non-deletional HbH disease, demonstrating improvement in -globin/-globin mRNA ratio and reduction in the formation of HbH by HPLC. Our results demonstrate the broad applicability of LNP for disease modeling, characterization of a novel severe mouse model of AT, and the efficacy of ALS20I for treating AT.

Climate change challenges, plant science solutions.

Climate change is a defining challenge of the 21st century, and this decade is a critical time for action to mitigate the worst effects on human populations and ecosystems. Plant science can play an important role in developing crops with enhanced resilience to harsh conditions (e.g. heat, drought, salt stress, flooding, disease outbreaks) and engineering efficient carbon-capturing and carbon-sequestering plants. Here, we present examples of research being conducted in these areas and discuss challenges and open questions as a call to action for the plant science community.

A few essential genetic loci distinguish Penstemon species with flowers adapted to pollination by bees or hummingbirds.

In the formation of species, adaptation by natural selection generates distinct combinations of traits that function well together. The maintenance of adaptive trait combinations in the face of gene flow depends on the strength and nature of selection acting on the underlying genetic loci. Floral pollination syndromes exemplify the evolution of trait combinations adaptive for particular pollinators. The North American wildflower genus Penstemon displays remarkable floral syndrome convergence, with at least 20 separate lineages that have evolved from ancestral bee pollination syndrome (wide blue-purple flowers that present a landing platform for bees and small amounts of nectar) to hummingbird pollination syndrome (bright red narrowly tubular flowers offering copious nectar). Related taxa that differ in floral syndrome offer an attractive opportunity to examine the genomic basis of complex trait divergence. In this study, we characterized genomic divergence among 229 individuals from a Penstemon species complex that includes both bee and hummingbird floral syndromes. Field plants are easily classified into species based on phenotypic differences and hybrids displaying intermediate floral syndromes are rare. Despite unambiguous phenotypic differences, genome-wide differentiation between species is minimal. Hummingbird-adapted populations are more genetically similar to nearby bee-adapted populations than to geographically distant hummingbird-adapted populations, in terms of genome-wide dXY. However, a small number of genetic loci are strongly differentiated between species. These approximately 20 "species-diagnostic loci," which appear to have nearly fixed differences between pollination syndromes, are sprinkled throughout the genome in high recombination regions. Several map closely to previously established floral trait quantitative trait loci (QTLs). The striking difference between the diagnostic loci and the genome as whole suggests strong selection to maintain distinct combinations of traits, but with sufficient gene flow to homogenize the genomic background. A surprisingly small number of alleles confer phenotypic differences that form the basis of species identity in this species complex.

Inhibition of the C1s Protease and the Classical Complement Pathway by 6-(4-Phenylpiperazin-1-yl)Pyridine-3-Carboximidamide and Chemical Analogs.

The classical pathway (CP) is a potent mechanism for initiating complement activity and is a driver of pathology in many complement-mediated diseases. The CP is initiated via activation of complement component C1, which consists of the pattern recognition molecule C1q bound to a tetrameric assembly of proteases C1r and C1s. Enzymatically active C1s provides the catalytic basis for cleavage of the downstream CP components, C4 and C2, and is therefore an attractive target for therapeutic intervention in CP-driven diseases. Although an anti-C1s mAb has been Food and Drug Administration approved, identifying small-molecule C1s inhibitors remains a priority. In this study, we describe 6-(4-phenylpiperazin-1-yl)pyridine-3-carboximidamide (A1) as a selective, competitive inhibitor of C1s. A1 was identified through a virtual screen for small molecules that interact with the C1s substrate recognition site. Subsequent functional studies revealed that A1 dose-dependently inhibits CP activation by heparin-induced immune complexes, CP-driven lysis of Ab-sensitized sheep erythrocytes, CP activation in a pathway-specific ELISA, and cleavage of C2 by C1s. Biochemical experiments demonstrated that A1 binds directly to C1s with a Kd of ∼9.8 µM and competitively inhibits its activity with an inhibition constant (Ki) of ∼5.8 µM. A 1.8-Å-resolution crystal structure revealed the physical basis for C1s inhibition by A1 and provided information on the structure-activity relationship of the A1 scaffold, which was supported by evaluating a panel of A1 analogs. Taken together, our work identifies A1 as a new class of small-molecule C1s inhibitor and lays the foundation for development of increasingly potent and selective A1 analogs for both research and therapeutic purposes.

Plasticity of ether lipids promotes ferroptosis susceptibility and evasion.

Ferroptosis-an iron-dependent, non-apoptotic cell death process-is involved in various degenerative diseases and represents a targetable susceptibility in certain cancers1. The ferroptosis-susceptible cell state can either pre-exist in cells that arise from certain lineages or be acquired during cell-state transitions2-5. However, precisely how susceptibility to ferroptosis is dynamically regulated remains poorly understood. Here we use genome-wide CRISPR-Cas9 suppressor screens to identify the oxidative organelles peroxisomes as critical contributors to ferroptosis sensitivity in human renal and ovarian carcinoma cells. Using lipidomic profiling we show that peroxisomes contribute to ferroptosis by synthesizing polyunsaturated ether phospholipids (PUFA-ePLs), which act as substrates for lipid peroxidation that, in turn, results in the induction of ferroptosis. Carcinoma cells that are initially sensitive to ferroptosis can switch to a ferroptosis-resistant state in vivo in mice, which is associated with extensive downregulation of PUFA-ePLs. We further find that the pro-ferroptotic role of PUFA-ePLs can be extended beyond neoplastic cells to other cell types, including neurons and cardiomyocytes. Together, our work reveals roles for the peroxisome-ether-phospholipid axis in driving susceptibility to and evasion from ferroptosis, highlights PUFA-ePL as a distinct functional lipid class that is dynamically regulated during cell-state transitions, and suggests multiple regulatory nodes for therapeutic interventions in diseases that involve ferroptosis.

Selective inhibition of the BD2 bromodomain of BET proteins in prostate cancer.

Proteins of the bromodomain and extra-terminal (BET) domain family are epigenetic readers that bind acetylated histones through their bromodomains to regulate gene transcription. Dual-bromodomain BET inhibitors (DbBi) that bind with similar affinities to the first (BD1) and second (BD2) bromodomains of BRD2, BRD3, BRD4 and BRDt have displayed modest clinical activity in monotherapy cancer trials. A reduced number of thrombocytes in the blood (thrombocytopenia) as well as symptoms of gastrointestinal toxicity are dose-limiting adverse events for some types of DbBi1-5. Given that similar haematological and gastrointestinal defects were observed after genetic silencing of Brd4 in mice6, the platelet and gastrointestinal toxicities may represent on-target activities associated with BET inhibition. The two individual bromodomains in BET family proteins may have distinct functions7-9 and different cellular phenotypes after pharmacological inhibition of one or both bromodomains have been reported10,11, suggesting that selectively targeting one of the bromodomains may result in a different efficacy and tolerability profile compared with DbBi. Available compounds that are selective to individual domains lack sufficient potency and the pharmacokinetics properties that are required for in vivo efficacy and tolerability assessment10-13. Here we carried out a medicinal chemistry campaign that led to the discovery of ABBV-744, a highly potent and selective inhibitor of the BD2 domain of BET family proteins with drug-like properties. In contrast to the broad range of cell growth inhibition induced by DbBi, the antiproliferative activity of ABBV-744 was largely, but not exclusively, restricted to cell lines of acute myeloid leukaemia and prostate cancer that expressed the full-length androgen receptor (AR). ABBV-744 retained robust activity in prostate cancer xenografts, and showed fewer platelet and gastrointestinal toxicities than the DbBi ABBV-07514. Analyses of RNA expression and chromatin immunoprecipitation followed by sequencing revealed that ABBV-744 displaced BRD4 from AR-containing super-enhancers and inhibited AR-dependent transcription, with less impact on global transcription compared with ABBV-075. These results underscore the potential value of selectively targeting the BD2 domain of BET family proteins for cancer therapy.