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A small number of peptide growth factor ligands are used repeatedly in development and homeostasis to drive programs of cell differentiation and function. Cells and tissues must integrate inputs from these diverse signals correctly, while failure to do so leads to pathology, reduced fitness, or death. Previous work using the nematode C. elegans identified an interaction between the bone morphogenetic protein (BMP) and insulin/IGF-1-like signaling (IIS) pathways in the regulation of lipid homeostasis. The molecular components required for this interaction, however, were not fully understood. Here we report that INS-4, one of 40 insulin-like peptides (ILPs), is regulated by BMP signaling to modulate fat accumulation. Furthermore, we find that the IIS transcription factor DAF-16/FoxO, but not SKN-1/Nrf, acts downstream of BMP signaling in lipid homeostasis. Interestingly, BMP activity alters sensitivity of these two transcription factors to IIS-promoted cytoplasmic retention in opposite ways. Finally, we probe the extent of BMP and IIS interactions by testing additional IIS functions including dauer formation, aging, and autophagy induction. Coupled with our previous work and that of other groups, we conclude that BMP and IIS pathways have at least three modes of interaction: independent, epistatic, and antagonistic. The molecular interactions we identify provide new insight into mechanisms of signaling crosstalk and potential therapeutic targets for IIS-related pathologies such as diabetes and metabolic syndrome., Author summary Systemic homeostasis depends on the coordinated action of different cells and tissues throughout the body in response to internal and external cues. This coordination is achieved in part by cellular communication through the production, release, and response to molecular signals in a process known as cell signaling. These signals and their response pathways are highly conserved among animal species; therefore, we are able to use genetic tools in a simple animal model to identify broadly relevant mechanisms of function. Here, we demonstrate that two conserved signaling pathways, the bone morphogenetic protein (BMP) and insulin pathways, crosstalk to control lipid storage. We identify molecular components that mediate this regulation and determine their sites of action. Furthermore, we show that these two signaling pathways have multiple modes of interaction in a variety of developmental and physiological contexts. Due to the devastating human diseases caused by misregulation of these pathways, including cancer, cardiovascular disease, diabetes, and metabolic syndrome, understanding the mechanisms of crosstalk is critical.

Obesity and its associated metabolic syndrome are a leading cause of morbidity and mortality. Given the disease’s heavy burden on patients and the healthcare system, there has been increased interest in identifying pharmacological targets for the treatment and prevention of obesity. Towards this end, genome-wide association studies (GWAS) have identified hundreds of human genetic variants associated with obesity. The next challenge is to experimentally define which of these variants are causally linked to obesity, and could therefore become targets for the treatment or prevention of obesity. Here we employ high-throughput in vivo RNAi screening to test for causality 293 C. elegans orthologs of human obesity-candidate genes reported in GWAS. We RNAi screened these 293 genes in C. elegans subject to two different feeding regimens: (1) regular diet, and (2) high-fructose diet, which we developed and present here as an invertebrate model of diet-induced obesity (DIO). We report 14 genes that promote obesity and 3 genes that prevent DIO when silenced in C. elegans. Further, we show that knock-down of the 3 DIO genes not only prevents excessive fat accumulation in primary and ectopic fat depots but also improves the health and extends the lifespan of C. elegans overconsuming fructose. Importantly, the direction of the association between expression variants in these loci and obesity in mice and humans matches the phenotypic outcome of the loss-of-function of the C. elegans ortholog genes, supporting the notion that some of these genes would be causally linked to obesity across phylogeny. Therefore, in addition to defining causality for several genes so far merely correlated with obesity, this study demonstrates the value of model systems compatible with in vivo high-throughput genetic screening to causally link GWAS gene candidates to human diseases., Author summary Human GWAS have identified hundreds of genetic variants associated with human obesity. The genes being regulated by these variants at the protein or expression level represent potential anti-obesity targets. However, for the vast majority of these genes, it is unclear whether they cause obesity or are coincidentally associated with the disease. Here we use a high-throughput genetic screening strategy to test in vivo in Caenorhabditis elegans the potential causal role of human-obesity GWAS hits. Further, we combined the results of the genetic screen with analyses of mouse and human GWAS databases. As a result, we present 17 genes that promote or prevent C. elegans obesity, and the early onset of organismal deterioration and death associated with obesity. Further, the sign of the correlation between the expression levels of the human genes and their associated clinical traits matches, for the most part, the phenotypic effects of knocking down these genes in C. elegans, suggesting conserved causality and pharmacological potential for these genes.

Insect metamorphosis is triggered by the production, secretion and degradation of 20-hydroxyecdysone (ecdysone). In addition to its role in developmental regulation, increasing evidence suggests that ecdysone is involved in innate immunity processes, such as phagocytosis and the induction of antimicrobial peptide (AMP) production. AMP regulation includes systemic responses as well as local responses at surface epithelia that contact with the external environment. At pupariation, Drosophila melanogaster increases dramatically the expression of three AMP genes, drosomycin (drs), drosomycin-like 2 (drsl2) and drosomycin-like 5 (drsl5). We show that the systemic action of drs at pupariation is dependent on ecdysone signalling in the fat body and operates via the ecdysone downstream target, Broad. In parallel, ecdysone also regulates local responses, specifically through the activation of drsl2 expression in the gut. Finally, we confirm the relevance of this ecdysone dependent AMP expression for the control of bacterial load by showing that flies lacking drs expression in the fat body have higher bacterial persistence over metamorphosis. In contrast, local responses may be redundant with the systemic effect of drs since reduction of ecdysone signalling or of drsl2 expression has no measurable negative effect on bacterial load control in the pupa. Together, our data emphasize the importance of the association between ecdysone signalling and immunity using in vivo studies and establish a new role for ecdysone at pupariation, which impacts developmental success by regulating the immune system in a stage-dependent manner. We speculate that this co-option of immune effectors by the hormonal system may constitute an anticipatory mechanism to control bacterial numbers in the pupa, at the core of metamorphosis evolution., Author summary Developmental transitions in insects are regulated by the hormone ecdysone. Triggering metamorphosis constitutes a key feature of holometabolan insects and its evolution has required the establishment of new cross-talks between multiple organ systems and processes. Before metamorphosis, concomitantly with the initiation of a dramatic remodelling of larval tissues including the bacteria-containing gut, D. melanogaster increases the expression of three antimicrobial peptides (AMPs). In this study, we establish that such peak of antimicrobial peptide expression is part of the ecdysone pulse-dependent trigger of the metamorphic moult. We show that this immune response is deployed both locally and systemically, the latter being vital for the control of bacterial numbers in the pupal case. We establish a new role for ecdysone in the co-option of immunity by the metamorphosis programme and speculate that it may constitute an important element in the evolution of this developmental novelty.

Coordinated regulation of stress response pathways is crucial for cellular homeostasis. However, crosstalk between the different stress pathways and the physiological significance of this crosstalk remain poorly understood. In this study, using the model organism C. elegans, we discovered that suppression of the transcription factor LET-607/CREBH, a regulator of cellular defense and proteostatic responses, triggers adaptive induction of DAF-16-dependent stress responses. Suppression of LET-607 improves stress resistance and extends C. elegans lifespan in a DAF-16-dependent manner. We identified the sphingomyelin synthase SMS-5 to be a central mediator in the communication between LET-607 and DAF-16. SMS-5 reduces the contents of unsaturated phosphatidylcholine (PC), which activates DAF-16 through ITR-1-dependent calcium signaling and calcium-sensitive kinase PKC-2. Our data reveal the significance of crosstalk between different stress pathways in animal fitness and identify LET-607/CREBH and specific PC as regulators of DAF-16 and longevity., Author summary In order to cope with stresses, cells have evolved complex and elegant adaptive mechanisms, which are also referred to as stress responses. Central to these responses are core transcription factors. It is widely hypothesized that interruption of one key stress response pathway could compromise overall cellular function and survival. In order to avoid such an issue, stress response pathways communicate with each other. A defect in one pathway may adaptively activate other pathways, thus restoring homeostasis and increasing fitness. However, how these pathways communicate is largely unexplored. In this study, we unraveled crosstalk between the LET-607 and DAF-16 pathways in C. elegans. Suppression of LET-607, a regulator of defense and proteostatic responses, was shown to adaptively activate DAF-16, which is a crucial regulator of general stress responses. This crosstalk was shown to be vital for animal fitness, as suppression of LET-607 extends lifespan in a DAF-16-dependent manner. Intriguingly, loss of LET-607 results in increased levels of the sphingomyelin synthase SMS-5, which metabolizes membrane lipid PC. Consequently, the reduction in PC causes activation of DAF-16 via membrane-located calcium channel ITR-1 and calcium-sensitive kinase PKC-2. This study identifies a novel crosstalk between stress response pathways, which is potentially significant in animal longevity.

Orienting the causal relationship between pairs of traits is a fundamental task in scientific research with significant implications in practice, such as in prioritizing molecular targets and modifiable risk factors for developing therapeutic and interventional strategies for complex diseases. A recent method, called Steiger’s method, using a single SNP as an instrument variable (IV) in the framework of Mendelian randomization (MR), has since been widely applied. We report the following new contributions. First, we propose a single SNP-based alternative, overcoming a severe limitation of Steiger’s method in simply assuming, instead of inferring, the existence of a causal relationship. We also clarify a condition necessary for the validity of the methods in the presence of hidden confounding. Second, to improve statistical power, we propose combining the results from multiple, and possibly correlated, SNPs as multiple instruments. Third, we develop three goodness-of-fit tests to check modeling assumptions, including those required for valid IVs. Fourth, by relaxing one of the three IV assumptions in MR, we propose several methods, including an Egger regression-like approach and its multivariable version (analogous to multivariable MR), to account for horizontal pleiotropy of the SNPs/IVs, which is often unavoidable in practice. All our methods can simultaneously infer both the existence and (if so) the direction of a causal relationship, largely expanding their applicability over that of Steiger’s method. Although we focus on uni-directional causal relationships, we also briefly discuss an extension to bi-directional relationships. Through extensive simulations and an application to infer the causal directions between low density lipoprotein (LDL) cholesterol, or high density lipoprotein (HDL) cholesterol, and coronary artery disease (CAD), we demonstrate the superior performance and advantage of our proposed methods over Steiger’s method and bi-directional MR. In particular, after accounting for horizontal pleiotropy, our method confirmed the well known causal direction from LDL to CAD, while other methods, including bi-directional MR, might fail., Author summary In spite of its importance, due to technical challenges, orienting causal relationships between pairs of traits has been largely under-studied. Mendelian randomization (MR) Steiger’s method has become increasingly used in the last two years. Here we point out several limitations with MR Steiger’s method and propose alternative approaches. First, MR Steiger’s method is based on using only one single SNP as the instrument variable (IV), for which we propose a correlation ratio-based method, called Causal Direction-Ratio, or simply CD-Ratio. An advantage of CD-Ratio is its inference of both the existence and (if so) the direction of a causal relationship, in contrast to MR Steiger’s prior assumption of the existence and its poor performance if the assumption is violated. Furthermore, CD-Ratio can be extended to combine the results from multiple, possibly correlated, SNPs with improved statistical power. Second, we propose two methods, called CD-Egger and CD-GLS, for multiple and possibly correlated SNPs while allowing horizontal pleiotropy. Third, we propose three goodness-of-fit tests to check modeling assumptions for the three proposed methods. Finally, we introduce multivariable CD-Egger, analogous to multivariable MR, as a more robust approach, and an extension of CD-Ratio to cases with possibly bi-directional causal relationships. Our numerical studies demonstrated superior performance of our proposed methods over MR Steiger and bi-directional MR. Our proposed methods, along with freely available software, are expected to be useful in practice for causal inference.

The control of chronic inflammation is dependent on the possibility of limiting bystander activation of autoreactive and potentially pathogenic T cells. We have identified a non-sense loss of function single nucleotide polymorphism in the C-type lectin receptor, Clec4b, and have shown that it controls chronic autoimmune arthritis in rat models of rheumatoid arthritis. Clec4b is specifically expressed in CD4+ myeloid cells, mainly classical dendritic cells (DCs), and is defined by the markers CD4+/MHCIIhi/CD11b/c+. We found that Clec4b limited the activation of arthritogenic CD4+αβT cells and the absence of Clec4b allowed development of arthritis already 5 days after adjuvant injection. Clec4b sufficient CD4+ myeloid dendritic cells successfully limited the arthritogenic T cell expansion immediately after activation both in vitro and in vivo. We conclude that Clec4b expressed on CD4+ myeloid dendritic cells regulate the expansion of auto-reactive and potentially pathogenic T cells during an immune response, demonstrating an early checkpoint control mechanism to avoid autoimmunity leading to chronic inflammation., Author summary To identify early disease regulatory mechanisms in autoimmune diseases such as rheumatoid arthritis (RA) is challenging not only because of the genetic and environmental complexity but also because of the critical autoimmune time-period that precedes the clinical diagnosis. Therefore, we set out to study the complex disease pathways in a more restricted setting. Through genetic segregation of rat crosses, followed by the selection of recombinants to produce minimal congenic strains, we have identified a single nucleotide polymorphism regulating the expression of Clec4b2 that in turn controls the development of arthritis. The Clec4b gene is normally expressed in a population of antigen-presenting cells that can limit enhanced activation of bystander autoreactive T cells during an immune-priming response. This previously unknown type of immune regulation reveals the existence of a mechanism protecting against autoimmune dieases by the avoidance of bystander activation of autoreactive T cells during a normal immune response to foreign antigen.

Non-alcoholic fatty liver disease (NAFLD) is a metabolic disorder characterized by excess lipid accumulation in the liver without significant consumption of alcohol. The transmembrane 6 superfamily member 2 (TM6SF2) E167K missense variant strongly associates with NAFLD in humans. The E167K mutation destabilizes TM6SF2, resulting in hepatic lipid accumulation and low serum lipid levels. However, the molecular mechanism by which TM6SF2 regulates lipid metabolism remains unclear. By using tandem affinity purification in combination with mass spectrometry, we found that apolipoprotein B (APOB), ER lipid raft protein (ERLIN) 1 and 2 were TM6SF2-interacting proteins. ERLINs and TM6SF2 mutually bound and stabilized each other. TM6SF2 bound and stabilized APOB via two luminal loops. ERLINs did not interact with APOB directly but still increased APOB stability through stabilizing TM6SF2. This APOB stabilization was hampered by the E167K mutation that reduced the protein expression of TM6SF2. In mice, knockout of Tm6sf2 and knockdown of Tm6sf2 or Erlins decreased hepatic APOB protein level, causing lipid accumulation in the liver and lowering lipid levels in the serum. We conclude that defective APOB stabilization, as a result of ERLINs or TM6SF2 deficiency or E167K mutation, is a key factor contributing to NAFLD., Author summary Non-alcoholic fatty liver disease (NAFLD) is a very common liver disorder that occurs in people who do not drink too much alcohol. It initiates from extra fat storage in the liver and can advance to hepatitis, fibrosis, liver failure and liver cancer. NAFLD is often associated with other health problems such as obesity, diabetes, and hyperlipidemia. The TM6SF2 gene variant is a strong risk factor for NAFLD in humans. However, the mechanism by which loss of TM6SF2 protein causes NAFLD is unclear. Here, we demonstrate that TM6SF2 forms a complex with ERLINs and APOB. ERLINs and TM6SF2 stabilize each other, and TM6SF2 stabilizes APOB. In mice, ablating the expression of ERLINs or TM6SF2 lowers APOB protein level, causing lipid accumulation in the liver while decreasing lipid levels in the blood. These phenotypes resemble the symptoms of NAFLD patients carrying TM6SF2 mutations. We conclude that TM6SF2 promotes APOB stability via complex formation and that defective APOB stabilization is one of the underlying causes of NAFLD.

Phosphatidylserine (PS), synthesized in the endoplasmic reticulum (ER) by phosphatidylserine synthase (PSS), is transported to the plasma membrane (PM) and mitochondria through distinct routes. The in vivo functions of PS at different subcellular locations and the coordination between different PS transport routes are not fully understood. Here, we report that Drosophila PSS regulates cell growth, lipid storage and mitochondrial function. In pss RNAi, reduced PS depletes plasma membrane Akt, contributing to cell growth defects; the metabolic shift from phospholipid synthesis to neutral lipid synthesis results in ectopic lipid accumulation; and the reduction of mitochondrial PS impairs mitochondrial protein import and mitochondrial integrity. Importantly, reducing PS transport from the ER to PM by loss of PI4KIIIα partially rescues the mitochondrial defects of pss RNAi. Together, our results uncover a balance between different PS transport routes and reveal that PSS regulates cellular homeostasis through distinct metabolic mechanisms., Author summary Phosphatidylserine (PS), a membrane phospholipid synthesized in the endoplasmic reticulum (ER) by the enzyme phosphatidylserine synthase (PSS), is transported to the plasma membrane (PM) and mitochondria through different paths. The cellular functions of PS at different places in the cell and the mechanisms that coordinate the different PS transport paths are not fully understood. Here, we identified that PSS regulates cell growth, lipid storage and mitochondrial function in the fruit fly larval salivary gland. We showed that loss of pss function has three effects: (1) reduced levels of PS lead to reduced levels of plasma membrane Akt, a key component in the insulin pathway, which is important for cell growth; (2) it causes a shift from phospholipid synthesis to neutral lipid synthesis, which results in excess lipid accumulation; and (3) it reduces the level of mitochondrial PS, which impairs mitochondrial protein import and mitochondrial morphology. We also found that reducing the transport of PS from the ER to PM partially rescues the mitochondrial defects caused by loss of pss function. Together, our results reveal that PSS regulates cellular homeostasis through distinct metabolic changes, and uncover a balance between different PS transport pathways.

Metarhizium is a group of insect-pathogenic fungi that can produce insecticidal metabolites, such as destruxins. Interestingly, the acridid-specific fungus Metarhizium acridum (MAC) can kill locusts faster than the generalist fungus Metarhizium robertsii (MAA) even without destruxin. However, the underlying mechanisms of different pathogenesis between host-generalist and host-specialist fungi remain unknown. This study compared transcriptomes and metabolite profiles to analyze the difference in responsiveness of locusts to MAA and MAC infections. Results confirmed that the detoxification and tryptamine catabolic pathways were significantly enriched in locusts after MAC infection compared with MAA infection and that high levels of tryptamine could kill locusts. Furthermore, tryptamine was found to be capable of activating the aryl hydrocarbon receptor of locusts (LmAhR) to produce damaging effects by inducing reactive oxygen species production and immune suppression. Therefore, reducing LmAhR expression by RNAi or inhibitor (SR1) attenuates the lethal effects of tryptamine on locusts. In addition, MAA, not MAC, possessed the monoamine oxidase (Mao) genes in tryptamine catabolism. Hence, deleting MrMao-1 could increase the virulence of generalist MAA on locusts and other insects. Therefore, our study provides a rather feasible way to design novel mycoinsecticides by deleting a gene instead of introducing any exogenous gene or domain., Author summary Mycoinsecticides are widely used in place of chemical pesticides to protect crops from pest damage. Metarhizium spp. fungi specifically live inside the body cavity of insects and can produce insecticidal metabolites, such as beauvericin, destruxins, and taxol. The variable virulence between host-generalist fungus Metarhizium robertsii (MAA) and host-specialist fungus Metarhizium acridum (MAC) to locusts was studied. We found that MAC is more virulent than MAA on locusts, and MAC-infected locusts display higher amounts of tryptamine than do MAA-infected locusts. Furthermore, MAC cannot produce destruxins, but can produce abundant tryptamine to kill locusts when accumulated owing to the absence of a gene for tryptamine catabolism in the MAC genome. Tryptamine activates the aryl hydrocarbon receptor of locusts (LmAhR) that produces damaging effects by regulating reactive oxygen species production and suppressing the immune system of locusts. Therefore, the deletion of MrMao-1 in the generalist fungus MAA significantly improves the virulence of the fungus to locusts and other insect species. The resulting new insights into the core metabolism of high virulence of host-specialist fungus can provide an improved basis for designing mycoinsecticide strains.

Insects depend on the innate immune response for defense against a wide array of pathogens. Central to Drosophila immunity are antimicrobial peptides (AMPs), released into circulation when pathogens trigger either of the two widely studied signal pathways, Toll or IMD. The Toll pathway responds to infection by Gram-positive bacteria and fungi while the IMD pathway is activated by Gram-negative bacteria. During activation of the IMD pathway, the NF-κB-like transcription factor Relish is phosphorylated and then cleaved, which is crucial for IMD-dependent AMP gene induction. Here we show that loss-of-function mutants of the unconventional histone variant H2Av upregulate IMD-dependent AMP gene induction in germ-free Drosophila larvae and adults. After careful dissection of the IMD pathway, we found that Relish has an epistatic relationship with H2Av. In the H2Av mutant larvae, SUMOylation is down-regulated, suggesting a possible role of SUMOylation in the immune phenotype. Eventually we demonstrated that Relish is mostly SUMOylated on amino acid K823. Loss of the potential SUMOylation site leads to significant auto-activation of Relish in vivo. Further work indicated that H2Av regulates Relish SUMOylation after physically interacting with Su(var)2-10, the E3 component of the SUMOylation pathway. Biochemical analysis suggested that SUMOylation of Relish prevents its cleavage and activation. Our findings suggest a new mechanism by which H2Av can negatively regulate, and thus prevent spontaneous activation of IMD-dependent AMP production, through facilitating SUMOylation of the NF-κB like transcription factor Relish., Author summary Toll and IMD signaling pathways should be involved in the production of antimicrobial peptides in animals upon infection. Immunity responses are energy consuming. Thus, these two pathways are fine-tuned. Animal H2A variant histones are involved in many physiological functions. In Drosophila, the production of antibacterial peptides is out of control in the mutant of H2A variant (H2Av810). After careful examination, we found that Relish, the transcription factor of the IMD pathway, was activated in this mutant. Eventually we demonstrate that Relish can be SUMOylated with the involvement of H2Av. Loss of the main SUMOylation site in Relish induces it to auto-activate following over-expression. Therefore, H2Av is a negative regulator of the IMD signaling pathway by maintaining the normal level of Relish SUMOylation in Drosophila.

The intracellular level of fatty aldehydes is tightly regulated by aldehyde dehydrogenases to minimize the formation of toxic lipid and protein adducts. Importantly, the dysregulation of aldehyde dehydrogenases has been implicated in neurologic disorder and cancer in humans. However, cellular responses to unresolved, elevated fatty aldehyde levels are poorly understood. Here, we report that ALH-4 is a C. elegans aldehyde dehydrogenase that specifically associates with the endoplasmic reticulum, mitochondria and peroxisomes. Based on lipidomic and imaging analysis, we show that the loss of ALH-4 increases fatty aldehyde levels and reduces fat storage. ALH-4 deficiency in the intestine, cell-nonautonomously induces NHR-49/NHR-79-dependent hypodermal peroxisome proliferation. This is accompanied by the upregulation of catalases and fatty acid catabolic enzymes, as indicated by RNA sequencing. Such a response is required to counteract ALH-4 deficiency since alh-4; nhr-49 double mutant animals are sterile. Our work reveals unexpected inter-tissue communication of fatty aldehyde levels and suggests pharmacological modulation of peroxisome proliferation as a therapeutic strategy to tackle pathology related to excess fatty aldehydes., Author summary Fatty aldehydes are generated during the turnover of membrane lipids and when cells are under oxidative stress. Because excess fatty aldehydes form toxic adducts with proteins and lipids, their levels are tightly controlled by a family of aldehyde dehydrogenases whose dysfunction has been implicated in genetic disease and cancer in humans. Here, we characterize mutant C. elegans that lack a conserved, membrane-associated aldehyde dehydrogenase ALH-4. Despite elevated levels of fatty aldehydes, these mutant worms survive by increasing the abundance of peroxisomes, which are important organelles for lipid metabolism. Such peroxisome proliferative response depends on the activation of transcription factors NHR-49 and NHR-79, via putative endocrine signals. Accordingly, the fertility of alh-4 mutant worms relies on NHR-49. Our work suggests a latent mechanism that may be activated during aldehyde dehydrogenase deficiency.

Fat stored in the form of lipid droplets has long been considered a defining characteristic of cytoplasm. However, recent studies have shown that nuclear lipid droplets occur in multiple cells and tissues, including in human patients with fatty liver disease. The function(s) of stored fat in the nucleus has not been determined, and it is possible that nuclear fat is beneficial in some situations. Conversely, nuclear lipid droplets might instead be deleterious by disrupting nuclear organization or triggering aggregation of hydrophobic proteins. We show here that nuclear lipid droplets occur normally in C. elegans intestinal cells and germ cells, but appear to be associated with damage only in the intestine. Lipid droplets in intestinal nuclei can be associated with novel bundles of microfilaments (nuclear actin) and membrane tubules that might have roles in damage repair. To increase the normal, low frequency of nuclear lipid droplets in wild-type animals, we used a forward genetic screen to isolate mutants with abnormally large or abundant nuclear lipid droplets. Genetic analysis and cloning of three such mutants showed that the genes encode the lipid regulator SEIP-1/seipin, the inner nuclear membrane protein NEMP-1/Nemp1/TMEM194A, and a component of COPI vesicles called COPA-1/α-COP. We present several lines of evidence that the nuclear lipid droplet phenotype of copa-1 mutants results from a defect in retrieving mislocalized membrane proteins that normally reside in the endoplasmic reticulum. The seip-1 mutant causes most germ cells to have nuclear lipid droplets, the largest of which occupy more than a third of the nuclear volume. Nevertheless, the nuclear lipid droplets do not trigger apoptosis, and the germ cells differentiate into gametes that produce viable, healthy progeny. Thus, our results suggest that nuclear lipid droplets are detrimental to intestinal nuclei, but have no obvious deleterious effect on germ nuclei., Author summary Several human disorders such as obesity are associated with abnormal fat storage. Cells normally store fat in cytoplasmic organelles called lipid droplets. However, recent studies have shown that fat can also form inside of the cell nucleus, and the effects of nuclear fat are not known. Here we use the cell biology and genetics of the model organism C. elegans to study the causes and consequences of nuclear fat. We show that intestinal cells can contain nuclear fat, particularly during high-low-high changes in cytoplasmic fat that involve de novo fat synthesis. Nuclear fat is associated with multiple changes in intestinal nuclei that appear to represent damage and repair. Germ nuclei that normally differentiate into oocytes can also contain nuclear fat. In germ cells, however, even high levels of nuclear fat appear to cause little or no damage. Our results suggest that intestinal nuclei and germ cell nuclei might have different responses to nuclear fat in part because they differ in chromosomal organization at the nuclear envelope.

Arthropod-specific juvenile hormones control numerous essential functions in development and reproduction. In the dengue-fever mosquito Aedes aegypti, in addition to its role in immature stages, juvenile hormone III (JH) governs post-eclosion (PE) development in adult females, a phase required for competence acquisition for blood feeding and subsequent egg maturation. During PE, JH through its receptor Methoprene-tolerant (Met) regulate the expression of many genes, causing either activation or repression. Met-mediated gene repression is indirect, requiring involvement of intermediate repressors. Hairy, which functions downstream of Met in the JH gene-repression hierarchy, is one such factor. Krüppel-homolog 1, a zinc-finger transcriptional factor, is directly regulated by Met and has been implicated in both activation and repression of JH-regulated genes. However, the interaction between Hairy and Kr-h1 in the JH-repression hierarchy is not well understood. Our RNAseq-based transcriptomic analysis of the Kr-h1-depleted mosquito fat body revealed that 92% of Kr-h1 repressed genes are also repressed by Met, supporting the existence of a hierarchy between Met and Kr-h1 as previously demonstrated in various insects. Notably, 130 genes are co-repressed by both Kr-h1 and Hairy, indicating regulatory complexity of the JH-mediated PE gene repression. A mosquito Kr-h1 binding site in genes co-regulated by this factor and Hairy was identified computationally. Moreover, this was validated using electrophoretic mobility shift assays. A complete phenocopy of the effect of Met RNAi depletion on target genes could only be observed after Kr-h1 and Hairy double RNAi knockdown, suggesting a synergistic action between these two factors in target gene repression. This was confirmed using a cell-culture-based luciferase reporter assay. Taken together, our results indicate that Hairy and Kr-h1 not only function as intermediate downstream factors, but also act together in a synergistic fashion in the JH/Met gene repression hierarchy., Author summary Juvenile hormone (JH) plays an essential role in preparing Aedes aegypti female mosquitoes for blood feeding, egg development, and pathogen transmission. JH acting through its receptor Methoprene-tolerant (Met) regulates the expression of large gene cohorts. JH mediated gene repression, unlike activation that is directly mediated by Met, is indirect and requires intermediate transcriptional repressors Hairy and Krüppel-homolog 1 (Kr-h1). Here, we demonstrate that Hairy and Kr-h1 can act synergistically in the JH-Met gene repression pathway in Aedes female mosquitoes. These interact directly with regulatory regions of the genes that have both Hairy and Kr-h1 binding sites. Thus, this study has significantly advanced our understanding of the complexity of the JH-mediated gene expression pathway. This research yields valuable information about the JH control of reproductive development of the mosquito A. aegypti, one of the most important vectors of human diseases.

Increasing seed oil content is one of the most important breeding goals for soybean due to a high global demand for edible vegetable oil. However, genetic improvement of seed oil content has been difficult in soybean because of the complexity of oil metabolism. Determining the major variants and molecular mechanisms conferring oil accumulation is critical for substantial oil enhancement in soybean and other oilseed crops. In this study, we evaluated the seed oil contents of 219 diverse soybean accessions across six different environments and dissected the underlying mechanism using a high-resolution genome-wide association study (GWAS). An environmentally stable quantitative trait locus (QTL), GqOil20, significantly associated with oil content was identified, accounting for 23.70% of the total phenotypic variance of seed oil across multiple environments. Haplotype and expression analyses indicate that an oleosin protein-encoding gene (GmOLEO1), colocated with a leading single nucleotide polymorphism (SNP) from the GWAS, was significantly correlated with seed oil content. GmOLEO1 is predominantly expressed during seed maturation, and GmOLEO1 is localized to accumulated oil bodies (OBs) in maturing seeds. Overexpression of GmOLEO1 significantly enriched smaller OBs and increased seed oil content by 10.6% compared with those of control seeds. A time-course transcriptomics analysis between transgenic and control soybeans indicated that GmOLEO1 positively enhanced oil accumulation by affecting triacylglycerol metabolism. Our results also showed that strong artificial selection had occurred in the promoter region of GmOLEO1, which resulted in its high expression in cultivated soybean relative to wild soybean, leading to increased seed oil accumulation. The GmOLEO1 locus may serve as a direct target for both genetic engineering and selection for soybean oil improvement., Author summary Soybean seed oil is an important quality trait targeted during domestication and breeding. However, the molecular mechanism of soybean oil regulation is largely unknown due to its complex genetic architecture and environmental sensitivity. In this paper, we integrated GWAS across multiple environments, haplotype analysis, genetic transformation, and diversity analysis to study the genetic architecture of oil content and the underlying mechanism in soybean. This combined analysis enabled us to identify an environmentally stable QTL (GqOil20) and functionally verified that GmOLEO1 positively regulates total seed oil accumulation in soybean seeds. In addition, we found that GmOLEO1 showed a higher level of expression in cultivated soybean seeds than in wild soybean seeds, possibly as the result of the positive selection of the promoter, resulting in seed oil accumulation. Moreover, we identified an elite GmOLEO1 haplotype that correlated strongly with high oil content in soybean, holding great potential for assisting oil improvement in soybean breeding. Our study provided a new genetic resource for oil content improvement in soybean and other oilseed crops.

The Drosophila protocadherins Dachsous and Fat regulate growth and tissue polarity by modulating the levels, membrane localization and polarity of the atypical myosin Dachs. Localization to the apical junctional membrane is critical for Dachs function, and the adapter protein Vamana/Dlish and palmitoyl transferase Approximated are required for Dachs membrane localization. However, how Dachs levels are regulated is poorly understood. Here we identify the early girl gene as playing an essential role in Fat signaling by limiting the levels of Dachs protein. early girl mutants display overgrowth of the wings and reduced cross vein spacing, hallmark features of mutations affecting Fat signaling. Genetic experiments reveal that it functions in parallel with Fat to regulate Dachs. early girl encodes an E3 ubiquitin ligase, physically interacts with Dachs, and regulates its protein stability. Concomitant loss of early girl and approximated results in accumulation of Dachs and Vamana in cytoplasmic punctae, suggesting that it also regulates their trafficking to the apical membrane. Our findings establish a crucial role for early girl in Fat signaling, involving regulation of Dachs and Vamana, two key downstream effectors of this pathway., Author summary During development, organs grow to achieve a consistent final size. The evolutionarily conserved Hippo signaling network plays a central role in organ size control, and when dysregulated can be associated with cancer and other diseases. Fat signaling is one of several upstream pathways that impinge on Hippo signaling to regulate organ growth. We describe here identification of the Drosophila early girl gene as a new component of the Fat signaling pathway. We show that Early girl controls Fat signaling by regulating the levels of the Dachs protein. However Early girl differs from other Fat signaling regulators in that it doesn’t influence planar cell polarity or control the polarity of Dachs localization. early girl encodes a conserved protein that is predicted to influence protein stability, and it can physically associate with Dachs. We also discovered that Early girl acts together with another protein, called Approximated, to regulate the sub-cellular localization of Dachs and a Dachs-interacting protein called Vamana. Altogether, our observations establish Early girl as an essential component of Fat signaling that acts to regulate the levels and localization of Dachs and Vamana.

Diapause, a programmed developmental arrest primarily induced by seasonal environmental changes, is very common in the animal kingdom, and found in vertebrates and invertebrates alike. Diapause provides an adaptive advantage to animals, as it increases the odds of surviving adverse conditions. In insects, individuals perceive photoperiodic cues and modify endocrine signaling to direct reproductive diapause traits, such as ovary arrest and increased fat accumulation. However, it remains unclear as to which endocrine factors are involved in this process and how they regulate the onset of reproductive diapause. Here, we found that the long day-mediated drop in the concentration of the steroid hormone ecdysone is essential for the preparation of photoperiodic reproductive diapause in Colaphellus bowringi, an economically important cabbage beetle. The diapause-inducing long-day condition reduced the expression of ecdysone biosynthetic genes, explaining the drop in the titer of 20-hydroxyecdysone (20E, the active form of ecdysone) in female adults. Application of exogenous 20E induced vitellogenesis and ovarian development but reduced fat accumulation in the diapause-destined females. Knocking down the ecdysone receptor (EcR) in females destined for reproduction blocked reproductive development and induced diapause traits. RNA-seq and hormone measurements indicated that 20E stimulates the production of juvenile hormone (JH), a key endocrine factor in reproductive diapause. To verify this, we depleted three ecdysone biosynthetic enzymes via RNAi, which confirmed that 20E is critical for JH biosynthesis and reproductive diapause. Importantly, impairing Met function, a component of the JH intracellular receptor, partially blocked the 20E-regulated reproductive diapause preparation, indicating that 20E regulates reproductive diapause in both JH-dependent and -independent manners. Finally, we found that 20E deficiency decreased ecdysis-triggering hormone signaling and reduced JH production, thereby inducing diapause. Together, these results suggest that 20E signaling is a pivotal regulator that coordinates reproductive plasticity in response to environmental inputs., Author summary Developmental arrest pervades organismal development and physiology where it facilitates an enormous range of adaptive responses to stressful conditions. Many animals exhibit various forms of developmental arrest that ensures survival under the most adverse environments. Reproductive diapause occurs when adults temporarily suspend reproduction in response to environmental stress and has been documented for a variety of invertebrates, particularly insects. Endocrine signals play a central role in translating environmental cues such as photoperiod into reproductive diapause-related physiology and behavior. However, it has been an unresolved issue as to which endocrine factors can respond to photoperiodic inputs and regulate diapause outputs. In this study, we found that a decrease in ecdysone levels is critical for reproductive diapause to occur. Also, ecdysone could interact with juvenile hormone to regulate the occurrence of reproductive diapause in response to photoperiodic cues. Our findings provide new insight into endocrine mechanisms of photoperiodic reproductive diapause and an example of phenotypic plasticity in animals.

Hypercholesterolemia is a causal and modifiable risk factor for atherosclerotic cardiovascular disease. A critical pathway regulating cholesterol homeostasis involves the receptor-mediated endocytosis of low-density lipoproteins into hepatocytes, mediated by the LDL receptor. We applied genome-scale CRISPR screening to query the genetic determinants of cellular LDL uptake in HuH7 cells cultured under either lipoprotein-rich or lipoprotein-starved conditions. Candidate LDL uptake regulators were validated through the synthesis and secondary screening of a customized library of gRNA at greater depth of coverage. This secondary screen yielded significantly improved performance relative to the primary genome-wide screen, with better discrimination of internal positive controls, no identification of negative controls, and improved concordance between screen hits at both the gene and gRNA level. We then applied our customized gRNA library to orthogonal screens that tested for the specificity of each candidate regulator for LDL versus transferrin endocytosis, the presence or absence of genetic epistasis with LDLR deletion, the impact of each perturbation on LDLR expression and trafficking, and the generalizability of LDL uptake modifiers across multiple cell types. These findings identified several previously unrecognized genes with putative roles in LDL uptake and suggest mechanisms for their functional interaction with LDLR., Author summary The level of cholesterol circulating in the blood in low-density lipoproteins (LDL) is an important determinant of overall risk for cardiovascular diseases, including heart attack and stroke. This level is regulated by the removal of LDL from circulation into liver cells. While many molecules involved in LDL uptake have been characterized, we hypothesized that other currently unrecognized genetic interactions are also involved in this process. We therefore applied CRISPR-mediated genome editing to systematically test the contribution of every gene in the human genome to the uptake of LDL by a liver-derived cell line. We synthesized a secondary CRISPR library targeting the top candidate genes from this initial genome-wide screen to confirm their role in LDL uptake and to test their influence on other cellular functions. Our findings confirm the role of genes previously known to participate in LDL uptake and also provide novel insight into the overall regulation of this process.

During infection, cellular resources are allocated toward the metabolically-demanding processes of synthesizing and secreting effector proteins that neutralize and kill invading pathogens. In Drosophila, these effectors are antimicrobial peptides (AMPs) that are produced in the fat body, an organ that also serves as a major lipid storage depot. Here we asked how activation of Toll signaling in the larval fat body perturbs lipid homeostasis to understand how cells meet the metabolic demands of the immune response. We find that genetic or physiological activation of fat body Toll signaling leads to a tissue-autonomous reduction in triglyceride storage that is paralleled by decreased transcript levels of the DGAT homolog midway, which carries out the final step of triglyceride synthesis. In contrast, Kennedy pathway enzymes that synthesize membrane phospholipids are induced. Mass spectrometry analysis revealed elevated levels of major phosphatidylcholine and phosphatidylethanolamine species in fat bodies with active Toll signaling. The ER stress mediator Xbp1 contributed to the Toll-dependent induction of Kennedy pathway enzymes, which was blunted by deleting AMP genes, thereby reducing secretory demand elicited by Toll activation. Consistent with ER stress induction, ER volume is expanded in fat body cells with active Toll signaling, as determined by transmission electron microscopy. A major functional consequence of reduced Kennedy pathway induction is an impaired immune response to bacterial infection. Our results establish that Toll signaling induces a shift in anabolic lipid metabolism to favor phospholipid synthesis and ER expansion that may serve the immediate demand for AMP synthesis and secretion but with the long-term consequence of insufficient nutrient storage., Author summary Fighting infection requires that immune cells synthesize antimicrobial peptides and antibodies and carry out cellular processes like phagocytosis to destroy microbes and clear infected cells. During infection, metabolic processes support and direct immune function. Here, we use the fruit fly Drosophila melanogaster as a model system to understand the interaction between immunity and lipid metabolism. In Drosophila larvae, infection leads to tremendous production of antimicrobial peptides that destroy invading microbes. These peptides are made in the fat body, an organ that is also the site of fat storage. Activating the immune response reduces lipid storage but increases the production of phospholipids that form the membranes of organelles such as the endoplasmic reticulum. This organelle is the starting point for synthesis and secretion of antimicrobial peptides, and its volume is increased in response to immune activation. Shifting lipid metabolism to membrane phospholipid synthesis supports the immune response. However, this comes at the expense of the ability to withstand other types of stress such as desiccation at later stages in life. These findings are important because they suggest that some of the metabolic changes induced by fighting an infection may become detrimental if they are maintained over long periods of time.

Endurance exercise has broadly protective effects across organisms, increasing metabolic fitness and reducing incidence of several age-related diseases. Drosophila has emerged as a useful model for studying changes induced by chronic endurance exercise, as exercising flies experience improvements to various aspects of fitness at the cellular, organ and organismal level. The activity of octopaminergic neurons is sufficient to induce the conserved cellular and physiological changes seen following endurance training. All 4 octopamine receptors are required in at least one target tissue, but only one, Octβ1R, is required for all of them. Here, we perform tissue- and adult-specific knockdown of alpha- and beta-adrenergic octopamine receptors in several target tissues. We find that reduced expression of Octβ1R in adult muscles abolishes exercise-induced improvements in endurance, climbing speed, flight, cardiac performance and fat-body catabolism in male Drosophila. Importantly, Octβ1R and OAMB expression in the heart is also required cell-nonautonomously for adaptations in other tissues, such as skeletal muscles in legs and adult fat body. These findings indicate that activation of distinct octopamine receptors in skeletal and cardiac muscle are required for Drosophila exercise adaptations, and suggest that cell non-autonomous factors downstream of octopaminergic activation play a key role., Author summary Repeated endurance exercise produces gradual adaptations in animals and humans that improve many aspects of health. One important component of the exercise response is adrenergic signaling. Norepinephrine secretion is known to be upregulated during an exercise bout, and affects multiple organ systems. We previously demonstrated that activation of neurons that secrete the invertebrate version of norepinephrine, octopamine, are necessary for fruit flies to respond to chronic exercise by increasing their endurance, speed and cardiac performance. Here, we use tissue-specific genetics to show which octopamine receptors are required in each tissue for exercise to exert its positive effects on long-term health. We find that all the various receptors are required in at least one organ system, and that one receptor, Octβ1R, is required in all of them. We also report that, despite the high similarity between the various receptors, they have distinct responses, and cannot always substitute for one another. We also find that activation of octopamine receptors in skeletal or cardiac muscle have tissue-non-autonomous effects on adaptations in other tissues. These results help to further understand the complex interplay between neuronal signaling and responses in various organs during chronic exercise.

Antimicrobial peptides act as a host defense mechanism and regulate the commensal microbiome. To obtain a comprehensive view of genes contributing to long-term memory we performed mRNA sequencing from single Drosophila heads following behavioral training that produces long-lasting memory. Surprisingly, we found that Diptericin B, an immune peptide with antimicrobial activity, is upregulated following behavioral training. Deletion and knock down experiments revealed that Diptericin B and another immune peptide, Gram-Negative Bacteria Binding Protein like 3, regulate long-term but not short-term memory or instinctive behavior in Drosophila. Interestingly, removal of DptB in the head fat body and GNBP-like3 in neurons results in memory deficit. That putative antimicrobial peptides influence memory provides an example of how some immune peptides may have been repurposed to influence the function of nervous system., Author summary It is becoming evident that the nervous system and immune system share not only some of the same molecular logic but also the same components. Here, we report a novel and unanticipated example of how immune genes influence nervous system function. Exploring how Drosophila form long-lasting memories of certain experiences, we have found that antimicrobial peptides that fight bacteria in the body, are expressed in the head, and control whether an animal will form long-term memory of a food source or an unsuccessful mating experience. Antimicrobial peptides are detected in the brain of many species and has often been associated with dysfunction of the nervous system. This and other recent works, provide an explanation to why antimicrobial peptides may be expressed in the head: they regulate normal functions of the brain. Both eating, and mating engage the immune system in preparation of exposure to external agents including bacteria. We speculate antimicrobial peptides were upregulated in the body to deal with immune challenges and over evolutionary time some of them are co-adopted to activate signaling pathways to convey specific information to the nervous system.

Signaling pathways that control the activities in non-photosynthetic plastids, important sites of plant metabolism, are largely unknown. Previously, we demonstrated that WRKY2 and WRKY34 transcription factors play an essential role in pollen development downstream of mitogen-activated protein kinase 3 (MPK3) and MPK6 in Arabidopsis. Here, we report that GLUCOSE-6-PHOSPHATE/PHOSPHATE TRANSLOCATOR 1 (GPT1) is a key target gene of WRKY2/WRKY34. GPT1 transports glucose-6-phosphate (Glc6P) into plastids for starch and/or fatty acid biosynthesis depending on the plant species. Loss of function of WRKY2/WRKY34 results in reduced GPT1 expression, and concomitantly, reduced accumulation of lipid bodies in mature pollen, which leads to compromised pollen viability, germination, pollen tube growth, and male transmission in Arabidopsis. Pollen-specific overexpression of GPT1 rescues the pollen defects of wrky2 wrky34 double mutant. Furthermore, gain-of-function activation of MPK3/MPK6 enhances GPT1 expression; whereas GPT1 expression is reduced in mkk4 mkk5 double mutant. Together, this study revealed a cytoplasmic/nuclear signaling pathway capable of coordinating the metabolic activities in plastids. High-level expression of GPT1 at late stages of pollen development drives Glc6P from cytosol into plastids, where Glc6P is used for fatty acid biosynthesis, an important step of lipid body biogenesis. The accumulation of lipid bodies during pollen maturation is essential to pollen fitness and successful reproduction., Author summary Plastids are important sites of plant metabolism including fatty acid and starch biosynthesis. At present, how the activities in the plastids are coordinated with those in the cytoplasm and the signaling pathway(s) involved are largely unknown. Previously, we demonstrated that WRKY2 and WRKY34 transcription factors play an essential role in pollen development downstream of mitogen-activated protein kinase 3 (MPK3) and MPK6 in Arabidopsis. Here, we report that GLUCOSE-6-PHOSPHATE/PHOSPHATE TRANSLOCATOR 1 (GPT1) is a key target gene of WRKY2/WRKY34. GPT1 is localized on the membrane of plastids and transports glucose-6-phosphate (Glc6P) into plastids for starch and/or fatty acid biosynthesis depending on the plant species. Genetic analyses demonstrated that WRKY2/WRKY34 and their upstream MPK3/MPK6 are involved in regulating GPT1 expression, therefore, the accumulation of lipid bodies in mature pollen, which is critical to pollen viability, pollen germination, pollen tube growth, and male transmission in Arabidopsis. This study revealed a cytoplasmic/nuclear signaling pathway capable of coordinating the metabolic activities in plastids. High-level expression of GPT1 at late stages of pollen development drives Glc6P from cytosol into plastids, where Glc6P is used for fatty acid biosynthesis, an important step of lipid body biogenesis. The accumulation of lipid bodies during pollen maturation is essential to pollen fitness and successful reproduction.

Tri-methylation on histone H3 lysine 4 (H3K4me3) is associated with active gene expression but its regulatory role in transcriptional activation is unclear. Here we used Caenorhabditis elegans to investigate the connection between H3K4me3 and gene expression regulation during aging. We uncovered around 30% of H3K4me3 enriched regions to show significant and reproducible changes with age. We further showed that these age-dynamic H3K4me3 regions largely mark gene-bodies and are acquired during adult stages. We found that these adult-specific age-dynamic H3K4me3 regions are correlated with gene expression changes with age. In contrast, H3K4me3 marking established during developmental stages remained largely stable with age, even when the H3K4me3 associated genes exhibited RNA expression changes during aging. Importantly, the genes associated with changes in H3K4me3 and RNA levels with age are enriched for functional groups commonly implicated in aging biology. Therefore, our findings suggested divergent roles of H3K4me3 in gene expression regulation during aging, with important implications on aging-dependent pathophysiologies., Author summary Histone modifications, the specific chemical modifications on histone proteins, are key for regulating the packing of DNA, and thus have important influence on diverse biological processes. An intensely studied function of histone modifications is their contribution to regulating gene expression. Recent studies in diverse model organisms demonstrated that the global alterations of particular histone modifications, for instance H3K4me3, extend the lifespan of the organism. However, the underlying molecular mechanisms remain largely unclear. In this study, we monitored whether and how the genome-wide pattern of the histone modification H3K4me3 changes during aging in the somatic cells of the model organism C. elegans. We identified interesting and non-conventional patterns of H3K4me3, which span gene-bodies and are acquired during adulthood, that are particularly prone to changes with aging. This is contrasted to the well-studied H3K4me3 patterns that span transcriptional start sites and 5’ promoter regions and are established early during development, which remain stable with age. Consistent with the close association between H3K4me3 marking and active transcription, we observed that the age-dynamic H3K4me3 markings are highly correlated with corresponding RNA expression changes. Importantly, the genes that are associated with both H3K4me3 and RNA expression changes with age are over-represented for functional groups commonly implicated in aging biology. In summary, our findings revealed a lesser known pattern of H3K4me3 modification that can have important biological roles in aging.

Human GWAS of obesity have been successful in identifying loci associated with adiposity, but for the most part, these are non-coding SNPs whose function, or even whose gene of action, is unknown. To help identify the genes on which these human BMI loci may be operating, we conducted a high throughput screen in Drosophila melanogaster. Starting with 78 BMI loci from two recently published GWAS meta-analyses, we identified fly orthologs of all nearby genes (± 250KB). We crossed RNAi knockdown lines of each gene with flies containing tissue-specific drivers to knock down (KD) the expression of the genes only in the brain and the fat body. We then raised the flies on a control diet and compared the amount of fat/triglyceride in the tissue-specific KD group compared to the driver-only control flies. 16 of the 78 BMI GWAS loci could not be screened with this approach, as no gene in the 500-kb region had a fly ortholog. Of the remaining 62 GWAS loci testable in the fly, we found a significant fat phenotype in the KD flies for at least one gene for 26 loci (42%) even after correcting for multiple comparisons. By contrast, the rate of significant fat phenotypes in RNAi KD found in a recent genome-wide Drosophila screen (Pospisilik et al. (2010) is ~5%. More interestingly, for 10 of the 26 positive regions, we found that the nearest gene was not the one that showed a significant phenotype in the fly. Specifically, our screen suggests that for the 10 human BMI SNPs rs11057405, rs205262, rs9925964, rs9914578, rs2287019, rs11688816, rs13107325, rs7164727, rs17724992, and rs299412, the functional genes may NOT be the nearest ones (CLIP1, C6orf106, KAT8, SMG6, QPCTL, EHBP1, SLC39A8, ADPGK /ADPGK-AS1, PGPEP1, KCTD15, respectively), but instead, the specific nearby cis genes are the functional target (namely: ZCCHC8, VPS33A, RSRC2; SPDEF, NUDT3; PAGR1; SETD1, VKORC1; SGSM2, SRR; VASP, SIX5; OTX1; BANK1; ARIH1; ELL; CHST8, respectively). The study also suggests further functional experiments to elucidate mechanism of action for genes evolutionarily conserved for fat storage., Author summary Human Genome Wide Association Studies have successfully found thousands of novel genetic variants associated with many diseases. While these undoubtedly point to new biology, the field has been slowed in exploiting these new findings to reach a better understanding of exactly how they confer increased risk. Many, if not most, appear to be regulatory not coding variants, so their immediate consequence is not obvious. A real rate limiting step is even identifying which gene these variants might be regulating, and in what tissues they are operating to increase disease risk. In the absence of any other information, a first order assumption is that they may be more likely to be regulating a nearby gene, and such variants are often initially annotated by the “nearest” gene until their function is more definitively validated. Exploiting the idea that many genes may have conserved function across species, we conducted a high-throughput screen of fruit-fly orthologs of human genes nearby 78 well validated GWAS variants for human obesity, in order to more precisely identify the gene(s) of action. We systematically knocked down the function of each of these nearby genes in the brain and fat-body of the flies, raised them on a standard diet, and compared their percent body fat with control flies, in order to validate which genes showed a fat response. 43% of the time when fly orthologs existed in the region, we were able to identify the causal gene. Interestingly, nearly half the time (46%), it was not the nearest gene but another nearby one that regulated fat.

The Th-inducing POK (Th-POK, also known as ZBTB7B or cKrox) transcription factor is a key regulator of lineage commitment of immature T cell precursors. It is yet unclear the physiological functions of Th-POK besides helper T cell differentiation. Here we show that Th-POK is restrictedly expressed in the luminal epithelial cells in the mammary glands that is upregulated at late pregnancy and lactation. Lineage restrictedly expressed Th-POK exerts distinct biological functions in the mammary epithelial cells and T cells in a tissue-specific manner. Th-POK is not required for mammary epithelial cell fate determination. Mammary gland morphogenesis in puberty and alveologenesis in pregnancy are phenotypically normal in the Th-POK-deficient mice. However, Th-POK-deficient mice are defective in triggering the onset of lactation upon parturition with large cellular lipid droplets retained within alveolar epithelial cells. As a result, Th-POK knockout mice are unable to efficiently secret milk lipid and to nurse the offspring. Such defect is mainly attributed to the malfunctioned mammary epithelial cells, but not the tissue microenvironment in the Th-POK deficient mice. Th-POK directly regulates expression of insulin receptor substrate-1 (IRS-1) and insulin-induced Akt-mTOR-SREBP signaling. Th-POK deficiency compromises IRS-1 expression and Akt-mTOR-SREBP signaling in the lactating mammary glands. Conversely, insulin induces Th-POK expression. Thus, Th-POK functions as an important feed-forward regulator of insulin signaling in mammary gland lactation., Author summary Th-POK, aka cKrox or ZBTB7B, is a zinc finger transcription factor that specifies the cell fate of immature T cell precursors towards the CD4 lineage. It is yet largely unknown if Th-POK participates in the regulation of other biological processes. It is also not known if Th-POK functions beyond cell fate determination. In this study, we found that Th-POK is expressed in the luminal, but not the basal epithelial cells in the mammary glands. Despite the lineage restricted expression in the mammary glands, Th-POK is dispensable for mammary epithelial cell lineage specification and mammary gland development. Rather, Th-POK regulates the onset of lactation and milk lipid production via mammary epithelial cell autonomous mechanisms, suggesting that Th-POK exerts distinct functions in a tissue specific manner. Th-POK regulates the expression of insulin receptor sunstrate-1 and insulin-induced mTOR-SREBP pathway activation and lipid biosynthesis in the mammary alveolar cells at lactation. Thus, Th-POK functions as an important metabolic regulator in the lactating mammary glands, in addition to its well documented cell fate specification functions in T cell development.

Dietary fatty acids can be incorporated directly into phospholipids. This poses a specific challenge to cellular membranes since their composition, hence properties, could greatly vary with different diets. That vast variations in diets are tolerated therefore implies the existence of regulatory mechanisms that monitor and regulate membrane compositions. Here we show that the adiponectin receptor AdipoR2, and its C. elegans homolog PAQR-2, are essential to counter the membrane rigidifying effects of exogenously provided saturated fatty acids. In particular, we use dietary supplements or mutated E. coli as food, together with direct measurements of membrane fluidity and composition, to show that diets containing a high ratio of saturated to monounsaturated fatty acids cause membrane rigidity and lethality in the paqr-2 mutant. We also show that mammalian cells in which AdipoR2 has been knocked-down by siRNA are unable to prevent the membrane-rigidifying effects of palmitic acid. We conclude that the PAQR-2 and AdipoR2 proteins share an evolutionarily conserved function that maintains membrane fluidity in the presence of exogenous saturated fatty acids., Author summary Our cells and their internal organelles are bound by membranes composed primarily of phospholipids, i.e. polar molecules containing two fatty acids attached to a hydrophilic head group. The types of fatty acids in phospholipids greatly influence membrane properties: saturated fatty acids make the membranes rigid while unsaturated fatty acids promote fluidity. The fact that dietary fats can be incorporated into cellular membranes poses a serious challenge to the cells: how to regulate membrane composition to compensate for dietary variations? For the present study we used bacteria mutants with different fat compositions as food sources for the nematode C. elegans, together with assays to determine membrane rigidity and composition, and discovered that the C. elegans membrane protein PAQR-2 is responsible for detecting membrane rigidification by dietary saturated fatty acids and to promote fatty acid desaturation to restore membrane fluidity. We also studied the human homolog of PAQR-2, a protein called AdipoR2, and showed that it too is essential to prevent membrane rigidification by saturated fatty acids. AdipoR2 and PAQR-2 therefore serve a critical function in cells by acting as evolutionarily conserved regulators of membrane properties.

The general transcription factor TBP (TATA-box binding protein) and its associated factors (TAFs) together form the TFIID complex, which directs transcription initiation. Through RNAi and mutant analysis, we identified a specific TBP family protein, TRF2, and a set of TAFs that regulate lipid droplet (LD) size in the Drosophila larval fat body. Among the three Drosophila TBP genes, trf2, tbp and trf1, only loss of function of trf2 results in increased LD size. Moreover, TRF2 and TAF9 regulate fatty acid composition of several classes of phospholipids. Through RNA profiling, we found that TRF2 and TAF9 affects the transcription of a common set of genes, including peroxisomal fatty acid β-oxidation-related genes that affect phospholipid fatty acid composition. We also found that knockdown of several TRF2 and TAF9 target genes results in large LDs, a phenotype which is similar to that of trf2 mutants. Together, these findings provide new insights into the specific role of the general transcription machinery in lipid homeostasis., Author summary Lipid droplets (LD) are main lipid storage structures in most cells. The size of LDs varies greatly in different cell types or different metabolic states to accommodate cellular functions and metabolism demands. How cells regulate the lipid storage and LD dynamics is not fully understood. Here, we identified that general transcription factors, including a specific TBP (TATA-box binding protein) family protein TRF2 (TBP-related factor 2) and several TAFs (TBP-associated factors), regulate LD size in the fruitfly larval fat body. Moreover, quantitated lipid analysis reveals that TRF2 and TAF9 affect the fatty acid composition of several classes of phospholipids. We showed that TRF2 and TAF9 regulate transcription of several target genes, including peroxisomal fatty acid β-oxidation-related genes which likely mediate the effect of TRF2 and TAF9 on phospholipid fatty acid composition. We also found that overexpression of some target genes restores the LD phenotype in trf2 mutants. Our findings therefore reveal specific roles of general transcription factors in lipid homeostasis.

Given the relevance of beige adipocytes in adult humans, a better understanding of the molecular circuits involved in beige adipocyte biogenesis has provided new insight into human brown adipocyte biology. Genetic mutations in SLC39A13/ZIP13, a member of zinc transporter family, are known to reduce adipose tissue mass in humans; however, the underlying mechanisms remains unknown. Here, we demonstrate that the Zip13-deficient mouse shows enhanced beige adipocyte biogenesis and energy expenditure, and shows ameliorated diet-induced obesity and insulin resistance. Both gain- and loss-of-function studies showed that an accumulation of the CCAAT/enhancer binding protein-β (C/EBP-β) protein, which cooperates with dominant transcriptional co-regulator PR domain containing 16 (PRDM16) to determine brown/beige adipocyte lineage, is essential for the enhanced adipocyte browning caused by the loss of ZIP13. Furthermore, ZIP13-mediated zinc transport is a prerequisite for degrading the C/EBP-β protein to inhibit adipocyte browning. Thus, our data reveal an unexpected association between zinc homeostasis and beige adipocyte biogenesis, which may contribute significantly to the development of new therapies for obesity and metabolic syndrome., Author summary Inducible brown fat-like cells, named beige adipocytes have recently been a topic of great interest, mainly because they are induced in response to external cues, and are closely associated with adult human brown adipocyte. Therefore, the identification of selective molecular circuits involved in beige adipocyte biogenesis and thermogenesis will enable the selective induction of white adipocyte browning as a therapy for obesity. Here, we show that zinc homeostasis, which is controlled by ZIP13, a protein associated with human disease, is essential for the accurate regulation of beige adipocyte differentiation. Inhibition of ZIP13 function enhances beige adipocyte biogenesis and thermogenesis, highlighting the potential of ZIP13 as a therapeutic target for obesity and metabolic syndrome.

Many of the lipids found on the cuticles of insects function as pheromones and communicate information about age, sex, and reproductive status. In Drosophila, the composition of the information-rich lipid profile is dynamic and changes over the lifetime of an individual. However, the molecular basis of this change is not well understood. To identify genes that control cuticular lipid production in Drosophila, we performed a RNA interference screen and used Direct Analysis in Real Time and gas chromatography mass spectrometry to quantify changes in the chemical profiles. Twelve putative genes were identified whereby transcriptional silencing led to significant differences in cuticular lipid production. Amongst them, we characterized a gene which we name spidey, and which encodes a putative steroid dehydrogenase that has sex- and age-dependent effects on viability, pheromone production, and oenocyte survival. Transcriptional silencing or overexpression of spidey during embryonic development results in pupal lethality and significant changes in levels of the ecdysone metabolite 20-hydroxyecdysonic acid and 20-hydroxyecdysone. In contrast, inhibiting gene expression only during adulthood resulted in a striking loss of oenocyte cells and a concomitant reduction of cuticular hydrocarbons, desiccation resistance, and lifespan. Oenocyte loss and cuticular lipid levels were partially rescued by 20-hydroxyecdysone supplementation. Taken together, these results identify a novel regulator of pheromone synthesis and reveal that ecdysteroid signaling is essential for the maintenance of cuticular lipids and oenocytes throughout adulthood., Author Summary Pheromones are used by many animals to control social behaviors such as mate choice and kin recognition. The pheromone profile of insects is dynamic and can change depending on environmental, physiological, and social conditions. While many genes responsible for the biosynthesis of insect pheromones have been identified, much less is known about how pheromone production is systemically regulated over the lifetime of an animal. In this work, we identify 12 genes in Drosophila melanogaster that play a role in pheromone production. We characterized the function of one gene, which we name spidey, and which encodes a steroid dehydrogenase. Silencing spidey expression during the larval stage results in the rapid inactivation of an essential insect steroid, 20-hydroxyecdysone, and developmental arrest. In adults, spidey is needed for maintaining the viability of oenocytes, specialized cells that produce pheromones and also regulate energy homeostasis. Our work reveals a novel role for ecdysteroids in the adult animal and uncovers a regulatory mechanism for oenocyte activity. Potentially, ecdysteroid signaling serves as a mechanism by which environmental or social conditions shape pheromone production. Exploitation of this conserved pathway could be useful for interfering with the mating behavior and lifespan of disease-bearing insects or agricultural pests.

Elimination of the proliferating germline extends lifespan in C. elegans. This phenomenon provides a unique platform to understand how complex metazoans retain metabolic homeostasis when challenged with major physiological perturbations. Here, we demonstrate that two conserved transcription regulators essential for the longevity of germline-less adults, DAF-16/FOXO3A and TCER-1/TCERG1, concurrently enhance the expression of multiple genes involved in lipid synthesis and breakdown, and that both gene classes promote longevity. Lipidomic analyses revealed that key lipogenic processes, including de novo fatty acid synthesis, triglyceride production, desaturation and elongation, are augmented upon germline removal. Our data suggest that lipid anabolic and catabolic pathways are coordinately augmented in response to germline loss, and this metabolic shift helps preserve lipid homeostasis. DAF-16 and TCER-1 also perform essential inhibitory functions in germline-ablated animals. TCER-1 inhibits the somatic gene-expression program that facilitates reproduction and represses anti-longevity genes, whereas DAF-16 impedes ribosome biogenesis. Additionally, we discovered that TCER-1 is critical for optimal fertility in normal adults, suggesting that the protein acts as a switch supporting reproductive fitness or longevity depending on the presence or absence of the germline. Collectively, our data offer insights into how organisms adapt to changes in reproductive status, by utilizing the activating and repressive functions of transcription factors and coordinating fat production and degradation., Author Summary The balance between production and breakdown of fats is critical for health, especially during reproduction-related changes such as onset of puberty or menopause. However, little is known about how animals retain a balanced metabolism when undergoing major life events. Here, we have used a C. elegans mutant that successfully adapts to loss of reproductive cells to address this question. Our data suggest that the conserved proteins DAF-16/FOXO3A and TCER-1/TCERG1 mediate a coordinated increase in fat synthesis and degradation when the reproductive cells are lost. This coupling likely helps the animal to manage the lipids that would have been deposited in eggs as yolk, thus preventing metabolic disarray. These proteins also inhibit processes that would have normally supported reproduction. Together the activities of these transcription regulators allow the mutant to convert a debilitating loss of fertility into improved health and longevity. We also report that TCER-1 promotes reproductive health in normal adults, whereas when procreation is impeded, it switches roles to repress fertility and enhance lipid equilibrium. These observations offer insights into how complex organisms coordinate their metabolism to suit their reproductive needs.

The sessile plants have evolved diverse intrinsic mechanisms to control their proper development under variable environments. In contrast to plastic vegetative development, reproductive traits like floral identity often show phenotypic robustness against environmental variations. However, it remains obscure about the molecular basis of this phenotypic robustness. In this study, we found that eg1 (extra glume1) mutants of rice (Oryza savita L.) showed floral phenotypic variations in different growth locations resulting in a breakdown of floral identity robustness. Physiological and biochemical analyses showed that EG1 encodes a predominantly mitochondria-localized functional lipase and functions in a high temperature-dependent manner. Furthermore, we found that numerous environmentally responsive genes including many floral identity genes are transcriptionally repressed in eg1 mutants and OsMADS1, OsMADS6 and OsG1 genetically act downstream of EG1 to maintain floral robustness. Collectively, our results demonstrate that EG1 promotes floral robustness against temperature fluctuation by safeguarding the expression of floral identify genes through a high temperature-dependent mitochondrial lipid pathway and uncovers a novel mechanistic insight into floral developmental control., Author Summary Various mechanisms have evolved to ensure proper organ formation under variable environments in order to complete one organism’s life cycle. In angiosperms, vegetative and reproductive organs show a differential plastic development between varied environments, with a low plasticity or high robustness for flower formation, but little is known about its intrinsic mechanism. Here we report that gene EG1 (EXTRA GLUME1) can enhance the floral robustness against temperature fluctuation in rice. EG1 encodes a predominantly mitochondria-localized functional lipase and its loss of function disrupts floral development in a high temperature-dependent manner. In consistent, both EG1 and its lipase activity are positively induced by high temperature. Transcriptomic and genetic analyses revealed that EG1 functions upstream of several floral identity genes, eg, OsMADS1, OsMADS6 and OsG1. Taken together, our results uncover a novel mitochondria-mediated lipid metabolic pathway to promote floral developmental robustness. Our findings may help to genetically improve floral traits of rice to maintain a stable yield when planted in different locations and/or under heat stress conditions.

Cell growth and proliferation depend upon many different aspects of lipid metabolism. One key signaling pathway that is utilized in many different anabolic contexts involves Phosphatidylinositide 3-kinase (PI3K) and its membrane lipid products, the Phosphatidylinositol (3,4,5)-trisphosphates. It remains unclear, however, which other branches of lipid metabolism interact with the PI3K signaling pathway. Here, we focus on specialized fat metabolizing cells in Drosophila called larval oenocytes. In the presence of dietary nutrients, oenocytes undergo PI3K-dependent cell growth and contain very few lipid droplets. In contrast, during starvation, oenocytes decrease PI3K signaling, shut down cell growth and accumulate abundant lipid droplets. We now show that PI3K in larval oenocytes, but not in fat body cells, functions to suppress lipid droplet accumulation. Several enzymes of fatty acid, triglyceride and hydrocarbon metabolism are required in oenocytes primarily for lipid droplet induction rather than for cell growth. In contrast, a very long chain fatty-acyl-CoA reductase (FarO) and a putative lipid dehydrogenase/reductase (Spidey, also known as Kar) not only promote lipid droplet induction but also inhibit oenocyte growth. In the case of Spidey/Kar, we show that the growth suppression mechanism involves inhibition of the PI3K signaling pathway upstream of Akt activity. Together, the findings in this study show how Spidey/Kar and FarO regulate the balance between the cell growth and lipid storage of larval oenocytes., Author Summary Lipids play diverse roles in health and disease. Some types of lipids function as metabolic fuels for energy homeostasis, whereas others act as components of cell membranes or serve as signals regulating cell behaviors. Much, however, remains to be discovered about the molecular connections between different categories of lipids. Phosphatidylinositide 3-kinase (PI3K) is an enzyme that synthesizes phosphatidylinositide lipids, which act as signals essential for growth during normal development and cancer. Using genetics in the fruit fly, Drosophila, we identify new regulatory links between phosphatidylinositides and lipid oxidoreductases in specialized fat-metabolizing cells called oenocytes. We find that an enzyme metabolizing very long chain fatty acids (VLCFAs) and also a putative lipid dehydrogenase/reductase both act to prevent the inappropriate overgrowth of oenocytes. In the case of the latter enzyme, it suppresses cell growth by inhibiting phosphatidylinositide signaling. Future studies will determine whether similar lipid enzymes regulate PI3K signaling in other cell and tissue types during normal development and tumorigenesis.