Telomere chromatin structure is pivotal for maintaining genome stability by regulating the binding of telomere-associated proteins and inhibiting the DNA damage response. In Saccharomyces cerevisiae, silent information regulator (Sir) proteins bind to terminal repeats and to subtelomeric X-elements, resulting in transcriptional silencing. Herein, we show that sir2 mutant strains display a specific loss of a nucleosome residing in the X-elements and that this deficiency is remarkably consistent between different telomeres. The X-elements contain several binding sites for the transcription factor Reb1 and we found that Sir2 and Reb1 compete for stabilizing/destabilizing this nucleosome, i.e. inactivation of Reb1 in a sir2 background reinstated the lost nucleosome. The telomeric-repeat-containing RNAs (TERRAs) originate from subtelomeric regions and extend into the terminal repeats. Both Sir2 and Reb1 repress TERRAs and in a sir2 reb1 double mutant, TERRA levels increased synergistically, showing that Sir2 and Reb1 act in different pathways for repressing TERRAs. We present evidence that Reb1 restricts TERRAs by terminating transcription. Mapping the 5′-ends of TERRAs from several telomeres revealed that the Sir2-stabilized nucleosome is the first nucleosome downstream from the transcriptional start site for TERRAs. Finally, moving an X-element to a euchromatic locus changed nucleosome occupancy and positioning, demonstrating that X-element nucleosome structure is dependent on the local telomere environment.

Plants have evolved strategies to avoid shade and optimize the capture of sunlight. While some species are tolerant to shade, plants such as Arabidopsis thaliana are shade-intolerant and induce elongation of their hypocotyl to outcompete neighboring plants. We report the identification of a developmental module acting downstream of shade perception controlling vascular patterning. We show that Arabidopsis plants react to shade by increasing the number and types of water-conducting tracheary elements in the vascular cylinder to maintain vascular density constant. Mutations in genes affecting vascular patterning impair the production of additional xylem and also show defects in the shade-induced hypocotyl elongation response. Comparative analysis of the shade-induced transcriptomes revealed differences between wild type and vascular patterning mutants and it appears that the latter mutants fail to induce sets of genes encoding biosynthetic and cell wall modifying enzymes. Our results thus set the stage for a deeper understanding of how growth and patterning are coordinated in a dynamic environment.

Filamentous fungi display allorecognition genes that trigger regulated cell death (RCD) when strains of unlike genotype fuse. Podospora anserina is one of several model species for the study of this allorecognition process termed heterokaryon or vegetative incompatibility. Incompatibility restricts transmission of mycoviruses between isolates. In P. anserina, genetic analyses have identified nine incompatibility loci, termed het loci. Here we set out to clone the genes controlling het-B incompatibility. het-B displays two incompatible alleles, het-B1 and het-B2. We find that the het-B locus encompasses two adjacent genes, Bh and Bp that exist as highly divergent allelic variants (Bh1/Bh2 and Bp1/Bp2) in the incompatible haplotypes. Bh encodes a protein with an N-terminal HET domain, a cell death inducing domain bearing homology to Toll/interleukin-1 receptor (TIR) domains and a C-terminal domain with a predicted lectin fold. The Bp product is homologous to PII-like proteins, a family of small trimeric proteins acting as sensors of adenine nucleotides in bacteria. We show that although the het-B system appears genetically allelic, incompatibility is in fact determined by the non-allelic Bh1/Bp2 interaction while the reciprocal Bh2/Bp1 interaction plays no role in incompatibility. The highly divergent C-terminal lectin fold domain of BH determines recognition specificity. Population studies and genome analyses indicate that het-B is under balancing selection with trans-species polymorphism, highlighting the evolutionary significance of the two incompatible haplotypes. In addition to emphasizing anew the central role of TIR-like HET domains in fungal RCD, this study identifies novel players in fungal allorecognition and completes the characterization of the entire het gene set in that species.

Positive Transcription Elongation Factor b (P-TEFb) is a kinase consisting of Cdk9 and Cyclin T that releases RNA Polymerase II (Pol II) into active elongation. It can assemble into a larger Super Elongation Complex (SEC) consisting of additional elongation factors. Here, we use a miRNA-based approach to knock down the maternal contribution of P-TEFb and SEC components in early Drosophila embryos. P-TEFb or SEC depletion results in loss of cells from the embryo posterior and in cellularization defects. Interestingly, the expression of many patterning genes containing promoter-proximal paused Pol II is relatively normal in P-TEFb embryos. Instead, P-TEFb and SEC are required for expression of some non-paused, rapidly transcribed genes in pre-cellular embryos, including the cellularization gene Serendipity-a. We also demonstrate that another P-TEFb regulated gene, terminus, has an essential function in embryo development. Similar morphological and gene expression phenotypes were observed upon knock down of Mediator subunits, providing in vivo evidence that P-TEFb, the SEC and Mediator collaborate in transcription control. Surprisingly, P-TEFb depletion does not affect the ratio of Pol II at the promoter versus the 3' end, despite affecting global Pol II Ser2 phosphorylation levels. Instead, Pol II occupancy is reduced at P-TEFb down-regulated genes. We conclude that a subset of non-paused, pre-cellular genes are among the most susceptible to reduced P-TEFb, SEC and Mediator levels in Drosophila embryos.

Hybridization of closely related plant species is frequently connected to endosperm arrest and seed failure, for reasons that remain to be identified. In this study, we investigated the molecular events accompanying seed failure in hybrids of the closely related species pair Capsella rubella and C. grandiflora. Mapping of QTL for the underlying cause of hybrid incompatibility in Capsella identified three QTL that were close to pericentromeric regions. We investigated whether there are specific changes in heterochromatin associated with interspecific hybridizations and found a strong reduction of chromatin condensation in the endosperm, connected with a strong loss of CHG and CHH methylation and random loss of a single chromosome. Consistent with reduced DNA methylation in the hybrid endosperm, we found a disproportionate deregulation of genes located close to pericentromeric regions, suggesting that reduced DNA methylation allows access of transcription factors to targets located in heterochromatic regions. Since the identified QTL were also associated with pericentromeric regions, we propose that relaxation of heterochromatin in response to interspecies hybridization exposes and activates loci leading to hybrid seed failure.

RNA surveillance factors are involved in heterochromatin regulation in yeast and plants, but less is known about the possible roles of ribonucleases in the heterochromatin of animal cells. Here we show that RRP6, one of the catalytic subunits of the exosome, is necessary for silencing heterochromatic repeats in the genome of Drosophila melanogaster. We show that a fraction of RRP6 is associated with heterochromatin, and the analysis of the RRP6 interaction network revealed physical links between RRP6 and the heterochromatin factors HP1a, SU(VAR)3-9 and RPD3. Moreover, genome-wide studies of RRP6 occupancy in cells depleted of SU(VAR)3-9 demonstrated that SU(VAR)3-9 contributes to the tethering of RRP6 to a subset of heterochromatic loci. Depletion of the exosome ribonucleases RRP6 and DIS3 stabilizes heterochromatic transcripts derived from transposons and repetitive sequences, and renders the heterochromatin less compact, as shown by micrococcal nuclease and proximity-ligation assays. Such depletion also increases the amount of HP1a bound to heterochromatic transcripts. Taken together, our results suggest that SU(VAR)3-9 targets RRP6 to a subset of heterochromatic loci where RRP6 degrades chromatin-associated non-coding RNAs in a process that is necessary to maintain the packaging of the heterochromatin.

Complex chromosomal rearrangements (CCRs) are rearrangements involving more than two chromosomes or more than two breakpoints. Whole genome sequencing (WGS) allows for outstanding high resolution characterization on the nucleotide level in unique sequences of such rearrangements, but problems remain for mapping breakpoints in repetitive regions of the genome, which are known to be prone to rearrangements. Hence, multiple complementary WGS experiments are sometimes needed to solve the structures of CCRs. We have studied three individuals with CCRs: Case 1 and Case 2 presented with de novo karyotypically balanced, complex interchromosomal rearrangements (46,XX,t(2;8;15)(q35;q24.1;q22) and 46,XY,t(1;10;5)(q32;p12;q31)), and Case 3 presented with a de novo, extremely complex intrachromosomal rearrangement on chromosome 1. Molecular cytogenetic investigation revealed cryptic deletions in the breakpoints of chromosome 2 and 8 in Case 1, and on chromosome 10 in Case 2, explaining their clinical symptoms. In Case 3, 26 breakpoints were identified using WGS, disrupting five known disease genes. All rearrangements were subsequently analyzed using optical maps, linked-read WGS, and short-read WGS. In conclusion, we present a case series of three unique de novo CCRs where we by combining the results from the different technologies fully solved the structure of each rearrangement. The power in combining short-read WGS with long-molecule sequencing or optical mapping in these unique de novo CCRs in a clinical setting is demonstrated. Author summary Unexpected complexities are common findings in the breakpoints of karyotypically balanced complex chromosomal rearrangements (CCRs). Such findings are of clinical importance, as they may be the cause of mendelian phenotypes in the rearrangement carrier. Whole genome sequencing (WGS) allows for high resolution characterization of CCRs, but problems remain for mapping breakpoints located in repetitive regions of the genome, which are known to be prone to rearrangements. In our study, we use multiple complementary WGS experiments to solve the structures of three CCRs originally identified by karyotyping. In all cases, the genomic structure of the derivative chromosomes was resolved and a molecular genetic explanation of the clinical symptoms of the patients was obtained. Furthermore, we compare the performance, sensitivity and resolution of four different WGS techniques for solving these CCRs in a clinical diagnostic laboratory set.

Despite the success of genome-wide association studies in medical genetics, the underlying genetics of many complex diseases remains enigmatic. One plausible reason for this could be the failure to account for the presence of genetic interactions in current analyses. Exhaustive investigations of interactions are typically infeasible because the vast number of possible interactions impose hard statistical and computational challenges. There is, therefore, a need for computationally efficient methods that build on models appropriately capturing interaction. We introduce a new methodology where we augment the interaction hypothesis with a set of simpler hypotheses that are tested, in order of their complexity, against a saturated alternative hypothesis representing interaction. This sequential testing provides an efficient way to reduce the number of non-interacting variant pairs before the final interaction test. We devise two different methods, one that relies on a priori estimated numbers of marginally associated variants to correct for multiple tests, and a second that does this adaptively. We show that our methodology in general has an improved statistical power in comparison to seven other methods, and, using the idea of closed testing, that it controls the family-wise error rate. We apply our methodology to genetic data from the PRO-CARDIS coronary artery disease case/control cohort and discover three distinct interactions. While analyses on simulated data suggest that the statistical power may suffice for an exhaustive search of all variant pairs in ideal cases, we explore strategies for a priori selecting subsets of variant pairs to test. Our new methodology facilitates identification of new disease-relevant interactions from existing and future genome-wide association data, which may involve genes with previously unknown association to the disease. Moreover, it enables construction of interaction networks that provide a systems biology view of complex diseases, serving as a basis for more comprehensive understanding of disease pathophysiology and its clinical consequences.

Canavan disease is a severe progressive neurodegenerative disorder that is characterized by swelling and spongy degeneration of brain white matter. The disease is genetically linked to polymorphisms in the aspartoacylase (ASPA) gene, including the substitution C152W. ASPA C152W is associated with greatly reduced protein levels in cells, yet biophysical experiments suggest a wild-type like thermal stability. Here, we use ASPA C152W as a model to investigate the degradation pathway of a disease-causing protein variant. When we expressed ASPA C152W in Saccharomyces cerevisiae, we found a decreased steady state compared to wild-type ASPA as a result of increased proteasomal degradation. However, molecular dynamics simulations of ASPA C152W did not substantially deviate from wild-type ASPA, indicating that the native state is structurally preserved. Instead, we suggest that the C152W substitution interferes with the de novo folding pathway resulting in increased proteasomal degradation before reaching its stable conformation. Systematic mapping of the protein quality control components acting on misfolded and aggregation-prone species of C152W, revealed that the degradation is highly dependent on the molecular chaperone Hsp70, its co-chaperone Hsp110 as well as several quality control E3 ubiquitin-protein ligases, including Ubr1. In addition, the disaggregase Hsp104 facilitated refolding of aggregated ASPA C152W, while Cdc48 mediated degradation of insoluble ASPA protein. In human cells, ASPA C152W displayed increased proteasomal turnover that was similarly dependent on Hsp70 and Hsp110. Our findings underscore the use of yeast to determine the protein quality control components involved in the degradation of human pathogenic variants in order to identify potential therapeutic targets. Author summary Canavan disease is a fatal neurodegenerative disorder which is genetically linked to polymorphisms in the aspartoacylase (ASPA) gene. Although the molecular mechanism of most disease-causing substitutions remains to be examined, some variants have been suggested to cause the loss-of-function phenotype by perturbing the structural stability of ASPA. So far the cellular fate of these variants have not been examined. Here we examine the stability and degradation pathways of the disease-causing ASPA variant C152W. In yeast cells, ASPA C152W showed decreased steady-state protein levels as a result of increased proteasomal turnover. Our molecular dynamics simulations showed that the C152W substitution did not globally perturb the native structure of ASPA. Instead we propose that ASPA C152W is targeted by the protein quality control system during de novo folding. Specifically, we found that the molecular chaperone Hsp70, its co-chaperone Hsp110, and the E3 ubiquitin-protein ligase Ubr1 promote degradation of ASPA C152W. When we expressed ASPA C152W in cultured human cells, we found that Hsp70 and Hsp110 similarly mediated degradation. Therefore, we propose that Hsp110 should be further examined as a potential therapeutic target in Canavan disease and other protein misfolding diseases.

Feeding is essential for animal survival and reproduction and is regulated by both internal states and external stimuli. However, little is known about how internal states influence the perception of external sensory cues that regulate feeding behavior. Here, we investigated the neuronal and molecular mechanisms behind nutritional state-mediated regulation of gustatory perception in control of feeding behavior in the brown planthopper and Drosophila. We found that feeding increases the expression of the cholecystokinin-like peptide, sulfakinin (SK), and the activity of a set of SK-expressing neurons. Starvation elevates the transcription of the sugar receptor Gr64f and SK negatively regulates the expression of Gr64f in both insects. Interestingly, we found that one of the two known SK receptors, CCKLR-17D3, is expressed by some of Gr64f-expressing neurons in the proboscis and proleg tarsi. Thus, we have identified SK as a neuropeptide signal in a neuronal circuitry that responds to food intake, and regulates feeding behavior by diminishing gustatory receptor gene expression and activity of sweet sensing GRNs. Our findings demonstrate one nutritional state-dependent pathway that modulates sweet perception and thereby feeding behavior, but our experiments cannot exclude further parallel pathways. Importantly, we show that the underlying mechanisms are conserved in the two distantly related insect species.

Animals need to continuously adjust their water metabolism to the internal and external conditions. Homeostasis of body fluids thus requires tight regulation of water intake and excretion, and a balance between ingestion of water and solid food. Here, we investigated how these processes are coordinated in Drosophila melanogaster. We identified the first thirst-promoting and anti-diuretic hormone of Drosophila, encoded by the gene Ion transport peptide (ITP). This endocrine regulator belongs to the CHH (crustacean hyperglycemic hormone) family of peptide hormones. Using genetic gain- and loss-of-function experiments, we show that ITP signaling acts analogous to the human vasopressin and renin-angiotensin systems; expression of ITP is elevated by dehydration of the fly, and the peptide increases thirst while repressing excretion, promoting thus conservation of water resources. ITP responds to both osmotic and desiccation stress, and dysregulation of ITP signaling compromises the fly’s ability to cope with these stressors. In addition to the regulation of thirst and excretion, ITP also suppresses food intake. Altogether, our work identifies ITP as an important endocrine regulator of thirst and excretion, which integrates water homeostasis with feeding of Drosophila.

The capacity of a cell to maintain proteostasis progressively declines during aging. Virtually all age-associated neurodegenerative disorders associated with aggregation of neurotoxic proteins are linked to defects in the cellular proteostasis network, including insufficient lysosomal hydrolysis. Here, we report that proteotoxicity in yeast and Drosophila models for Parkinson’s disease can be prevented by increasing the bioavailability of Ca2+, which adjusts intracellular Ca2+ handling and boosts lysosomal proteolysis. Heterologous expression of human α-synuclein (αSyn), a protein critically linked to Parkinson’s disease, selectively increases total cellular Ca2+ content, while the levels of manganese and iron remain unchanged. Disrupted Ca2+ homeostasis results in inhibition of the lysosomal protease cathepsin D and triggers premature cellular and organismal death. External administration of Ca2+ reduces αSyn oligomerization, stimulates cathepsin D activity and in consequence restores survival, which critically depends on the Ca2+/calmodulin-dependent phosphatase calcineurin. In flies, increasing the availability of Ca2+ discloses a neuroprotective role of αSyn upon manganese overload. In sum, we establish a molecular interplay between cathepsin D and calcineurin that can be activated by Ca2+ administration to counteract αSyn proteotoxicity.

Upon nutrient depletion, bacteria stop proliferating and undergo physiological and morphological changes to ensure their survival. Yet, how these processes are coordinated in response to distinct starvation conditions is poorly understood. Here we compare the cellular responses of Caulobacter crescentus to carbon (C), nitrogen (N) and phosphorus (P) starvation conditions. We find that DNA replication initiation and abundance of the replication initiator DnaA are, under all three starvation conditions, regulated by a common mechanism involving the inhibition of DnaA translation. By contrast, cell differentiation from a motile swarmer cell to a sessile stalked cell is regulated differently under the three starvation conditions. During C and N starvation, production of the signaling molecules (p)ppGpp is required to arrest cell development in the motile swarmer stage. By contrast, our data suggest that low (p)ppGpp levels under P starvation allow P-starved swarmer cells to differentiate into sessile stalked cells. Further, we show that limited DnaA availability, and consequently absence of DNA replication initiation, is the main reason that prevents P-starved stalked cells from completing the cell cycle. Together, our findings demonstrate that C. crescentus decouples cell differentiation from DNA replication initiation under certain starvation conditions, two otherwise intimately coupled processes. We hypothesize that arresting the developmental program either as motile swarmer cells or as sessile stalked cells improves the chances of survival of C. crescentus during the different starvation conditions.

The bacterial cell cycle has been extensively studied under standard growth conditions. How it is modulated in response to environmental changes remains poorly understood. Here, we demonstrate that the freshwater bacterium Caulobacter crescentus blocks cell division and grows to filamentous cells in response to stress conditions affecting the cell membrane. Our data suggest that stress switches the membrane-bound cell cycle kinase CckA to its phosphatase mode, leading to the rapid dephosphorylation, inactivation and proteolysis of the master cell cycle regulator CtrA. The clearance of CtrA results in downregulation of division and morphogenesis genes and consequently a cell division block. Upon shift to non-stress conditions, cells quickly restart cell division and return to normal cell size. Our data indicate that the temporary inhibition of cell division through the regulated inactivation of CtrA constitutes a growth advantage under stress. Taken together, our work reveals a new mechanism that allows bacteria to alter their mode of proliferation in response to environmental cues by controlling the activity of a master cell cycle transcription factor. Furthermore, our results highlight the role of a bifunctional kinase in this process that integrates the cell cycle with environmental information.

CBP and the related p300 protein are widely used transcriptional co-activators in metazoans that interact with multiple transcription factors. Whether CBP/p300 occupies the genome equally with all factors or preferentially binds together with some factors is not known. We therefore compared Drosophila melanogaster CBP (nejire) ChIP-seq peaks with regions bound by 40 different transcription factors in early embryos, and we found high co-occupancy with the Rel-family protein Dorsal. Dorsal is required for CBP occupancy in the embryo, but only at regions where few other factors are present. CBP peaks in mutant embryos lacking nuclear Dorsal are best correlated with TGF-beta/Dpp-signaling and Smad-protein binding. Differences in CBP occupancy in mutant embryos reflect gene expression changes genome-wide, but CBP also occupies some non-expressed genes. The presence of CBP at silent genes does not result in histone acetylation. We find that Polycomb-repressed H3K27me3 chromatin does not preclude CBP binding, but restricts histone acetylation at CBP-bound genomic sites. We conclude that CBP occupancy in Drosophila embryos preferentially overlaps factors controlling dorsoventral patterning and that CBP binds silent genes without causing histone hyperacetylation.

Recent studies have detailed a remarkable degree of genetic and linguistic diversity in Northern Island Melanesia. Here we utilize that diversity to examine two models of genetic and linguistic coevolution. The first model predicts that genetic and linguistic correspondences formed following population splits and isolation at the time of early range expansions into the region. The second is analogous to the genetic model of isolation by distance, and it predicts that genetic and linguistic correspondences formed through continuing genetic and linguistic exchange between neighboring populations. We tested the predictions of the two models by comparing observed and simulated patterns of genetic variation, genetic and linguistic trees, and matrices of genetic, linguistic, and geographic distances. The data consist of 751 autosomal microsatellites and 108 structural linguistic features collected from 33 Northern Island Melanesian populations. The results of the tests indicate that linguistic and genetic exchange have erased any evidence of a splitting and isolation process that might have occurred early in the settlement history of the region. The correlation patterns are also inconsistent with the predictions of the isolation by distance coevolutionary process in the larger Northern Island Melanesian region, but there is strong evidence for the process in the rugged interior of the largest island in the region (New Britain). There we found some of the strongest recorded correlations between genetic, linguistic, and geographic distances. We also found that, throughout the region, linguistic features have generally been less likely to diffuse across population boundaries than genes. The results from our study, based on exceptionally fine-grained data, show that local genetic and linguistic exchange are likely to obscure evidence of the early history of a region, and that language barriers do not particularly hinder genetic exchange. In contrast, global patterns may emphasize more ancient demographic events, including population splits associated with the early colonization of major world regions.

DNA methylation is a key regulator of eukaryote genomes, and is of particular relevance in the regulation of gene expression on the sex chromosomes, with a key role in dosage compensation in mammalian XY systems. In the case of birds, dosage compensation is largely absent, with it being restricted to two small Male Hyper-Methylated (MHM) regions on the Z chromosome. To investigate how variation in DNA methylation is regulated on the Z chromosome we utilised a wild x domestic advanced intercross in the chicken, with both hypothalamic methylomes and transcriptomes assayed in 124 individuals. The relatively large numbers of individuals allowed us to identify additional genomic MHM regions on the Z chromosome that were significantly differentially methylated between the sexes. These regions appear to down-regulate local gene expression in males, but not remove it entirely (unlike the lncRNAs identified in the initial MHM regions). These MHM regions were further tested and the most balanced genes appear to show decreased expression in males, whilst methylation appeared to be far more correlated with gene expression in the less balanced, as compared to the most balanced genes. In addition, quantitative trait loci (QTL) that regulate variation in methylation on the Z chromosome, and those loci that regulate methylation on the autosomes that derive from the Z chromosome were mapped. Trans-effect hotspots were also identified that were based on the autosomes but affected the Z, and also one that was based on the Z chromosome but that affected both autosomal and sex chromosome DNA methylation regulation. We show that both cis and trans loci that originate from the Z chromosome never exhibit an interaction with sex, whereas trans loci originating from the autosomes but affecting the Z chromosome always display such an interaction. Our results highlight how additional MHM regions are actually present on the Z chromosome, and they appear to have smaller-scale effects on gene expression in males. Quantitative variation in methylation is also regulated both from the autosomes to the Z chromosome, and from the Z chromosome to the autosomes.

Bacteria can arrest their own growth and proliferation upon nutrient depletion and under various stressful conditions to ensure their survival. However, the molecular mechanisms responsible for suppressing growth and arresting the cell cycle under such conditions remain incompletely understood. Here, we identify post-transcriptional mechanisms that help enforce a cell-cycle arrest in Caulobacter crescentus following nutrient limitation and during entry into stationary phase by limiting the accumulation of DnaA, the conserved replication initiator protein. DnaA is rapidly degraded by the Lon protease following nutrient limitation. However, the rate of DnaA degradation is not significantly altered by changes in nutrient availability. Instead, we demonstrate that decreased nutrient availability downregulates dnaA translation by a mechanism involving the 5' untranslated leader region of the dnaA transcript; Lon-dependent proteolysis of DnaA then outpaces synthesis, leading to the elimination of DnaA and the arrest of DNA replication. Our results demonstrate how regulated translation and constitutive degradation provide cells a means of precisely and rapidly modulating the concentration of key regulatory proteins in response to environmental inputs.

Neurons and other cells display a large variation in size in an organism. Thus, a fundamental question is how growth of individual cells and their organelles is regulated. Is size scaling of individual neurons regulated post-mitotically, independent of growth of the entire CNS? Although the role of insulin/IGF-signaling (IIS) in growth of tissues and whole organisms is well established, it is not known whether it regulates the size of individual neurons. We therefore studied the role of IIS in the size scaling of neurons in the Drosophila CNS. By targeted genetic manipulations of insulin receptor (dInR) expression in a variety of neuron types we demonstrate that the cell size is affected only in neuroendocrine cells specified by the bHLH transcription factor DIMMED (DIMM). Several populations of DIMM-positive neurons tested displayed enlarged cell bodies after overexpression of the dInR, as well as PI3 kinase and Akt1 (protein kinase B), whereas DIMM-negative neurons did not respond to dInR manipulations. Knockdown of these components produce the opposite phenotype. Increased growth can also be induced by targeted overexpression of nutrient-dependent TOR (target of rapamycin) signaling components, such as Rheb (small GTPase), TOR and S6K (S6 kinase). After Dimm-knockdown in neuroendocrine cells manipulations of dInR expression have significantly less effects on cell size. We also show that dInR expression in neuroendocrine cells can be altered by up or down-regulation of Dimm. This novel dInR-regulated size scaling is seen during postembryonic development, continues in the aging adult and is diet dependent. The increase in cell size includes cell body, axon terminations, nucleus and Golgi apparatus. We suggest that the dInR-mediated scaling of neuroendocrine cells is part of a plasticity that adapts the secretory capacity to changing physiological conditions and nutrient-dependent organismal growth.

The tubular networks of the Drosophila respiratory system and our vasculature show distinct branching patterns and tube shapes in different body regions. These local variations are crucial for organ function and organismal fitness. Organotypic patterns and tube geometries in branched networks are typically controlled by variations of extrinsic signaling but the impact of intrinsic factors on branch patterns and shapes is not well explored. Here, we show that the intersection of extrinsic hedgehog(hh) and WNT/wingless (wg) signaling with the tube-intrinsic Hox code of distinct segments specifies the tube pattern and shape of the Drosophila airways. In the cephalic part of the airways, hh signaling induces expression of the transcription factor (TF) knirps (kni) in the anterior dorsal trunk (DTa1). kni represses the expression of another TF spalt major (salm), making DTa1 a narrow and long tube. In DTa branches of more posterior metameres, Bithorax Complex (BX-C) Hox genes autonomously divert hh signaling from inducing kni, thereby allowing DTa branches to develop as salm-dependent thick and short tubes. Moreover, the differential expression of BX-C genes is partly responsible for the anterior-to-posterior gradual increase of the DT tube diameter through regulating the expression level of Salm, a transcriptional target of WNT/wg signaling. Thus, our results highlight how tube intrinsic differential competence can diversify tube morphology without changing availabilities of extrinsic factors.

There are longstanding questions about the origins and ancestry of the Picts of early medieval Scotland (ca. 300–900 CE), prompted in part by exotic medieval origin myths, their enigmatic symbols and inscriptions, and the meagre textual evidence. The Picts, first mentioned in the late 3^rd century CE resisted the Romans and went on to form a powerful kingdom that ruled over a large territory in northern Britain. In the 9^th and 10^th centuries Gaelic language, culture and identity became dominant, transforming the Pictish realm into Alba, the precursor to the medieval kingdom of Scotland. To date, no comprehensive analysis of Pictish genomes has been published, and questions about their biological relationships to other cultural groups living in Britain remain unanswered. Here we present two high-quality Pictish genomes (2.4 and 16.5X coverage) from central and northern Scotland dated from the 5^th-7^th century which we impute and co-analyse with >8,300 previously published ancient and modern genomes. Using allele frequency and haplotype-based approaches, we can firmly place the genomes within the Iron Age gene pool in Britain and demonstrate regional biological affinity. We also demonstrate the presence of population structure within Pictish groups, with Orcadian Picts being genetically distinct from their mainland contemporaries. When investigating Identity-By-Descent (IBD) with present-day genomes, we observe broad affinities between the mainland Pictish genomes and the present-day people living in western Scotland, Wales, Northern Ireland and Northumbria, but less with the rest of England, the Orkney islands and eastern Scotland—where the political centres of Pictland were located. The pre-Viking Age Orcadian Picts evidence a high degree of IBD sharing across modern Scotland, Wales, Northern Ireland, and the Orkney islands, demonstrating substantial genetic continuity in Orkney for the last ~2,000 years. Analysis of mitochondrial DNA diversity at the Pictish cemetery of Lundin Links (n = 7) reveals absence of direct common female ancestors, with implications for broader social organisation. Overall, our study provides novel insights into the genetic affinities and population structure of the Picts and direct relationships between ancient and present-day groups of the UK.

Indonesia is the world’s fourth most populous country, host to striking levels of human diversity, regional patterns of admixture, and varying degrees of introgression from both Neanderthals and Denisovans. However, it has been largely excluded from the human genomics sequencing boom of the last decade. To serve as a benchmark dataset of molecular phenotypes across the region, we generated genome-wide CpG methylation and gene expression measurements in over 100 individuals from three locations that capture the major genomic and geographical axes of diversity across the Indonesian archipelago. Investigating between- and within-island differences, we find up to 10.55% of tested genes are differentially expressed between the islands of Sumba and New Guinea. Variation in gene expression is closely associated with DNA methylation, with expression levels of 9.80% of genes correlating with nearby promoter CpG methylation, and many of these genes being differentially expressed between islands. Genes identified in our differential expression and methylation analyses are enriched in pathways involved in immunity, highlighting Indonesia's tropical role as a source of infectious disease diversity and the strong selective pressures these diseases have exerted on humans. Finally, we identify robust within-island variation in DNA methylation and gene expression, likely driven by fine-scale environmental differences across sampling sites. Together, these results strongly suggest complex relationships between DNA methylation, transcription, archaic hominin introgression and immunity, all jointly shaped by the environment. This has implications for the application of genomic medicine, both in critically understudied Indonesia and globally, and will allow a better understanding of the interacting roles of genomic and environmental factors shaping molecular and complex phenotypes.

Clustered copy number variants (CNVs) as detected by chromosomal microarray analysis (CMA) are often reported as germline chromothripsis. However, such cases might need further investigations by massive parallel whole genome sequencing (WGS) in order to accurately define the underlying complex rearrangement, predict the occurrence mechanisms and identify additional complexities. Here, we utilized WGS to delineate the rearrangement structure of 21 clustered CNV carriers first investigated by CMA and identified a total of 83 breakpoint junctions (BPJs). The rearrangements were further sub-classified depending on the patterns observed: I) Cases with only deletions (n = 8) often had additional structural rearrangements, such as insertions and inversions typical to chromothripsis; II) cases with only duplications (n = 7) or III) combinations of deletions and duplications (n = 6) demonstrated mostly interspersed duplications and BPJs enriched with microhomology. In two cases the rearrangement mutational signatures indicated both a breakage-fusion-bridge cycle process and haltered formation of a ring chromosome. Finally, we observed two cases with Alu- and LINE-mediated rearrangements as well as two unrelated individuals with seemingly identical clustered CNVs on 2p25.3, possibly a rare European founder rearrangement. In conclusion, through detailed characterization of the derivative chromosomes we show that multiple mechanisms are likely involved in the formation of clustered CNVs and add further evidence for chromoanagenesis mechanisms in both simple and highly complex chromosomal rearrangements. Finally, WGS characterization adds positional information, important for a correct clinical interpretation and deciphering mechanisms involved in the formation of these rearrangements.

Reshuffling of genetic variation occurs both by independent assortment of chromosomes and by homologous recombination. Such reshuffling can generate novel allele combinations and break linkage between advantageous and deleterious variants which increases both the potential and the efficacy of natural selection. Here we used high-density linkage maps to characterize global and regional recombination rate variation in two populations of the wood white butterfly (Leptidea sinapis) that differ considerably in their karyotype as a consequence of at least 27 chromosome fissions and fusions. The recombination data were compared to estimates of genetic diversity and measures of selection to assess the relationship between chromosomal rearrangements, crossing over, maintenance of genetic diversity and adaptation. Our data show that the recombination rate is influenced by both chromosome size and number, but that the difference in the number of crossovers between karyotypes is reduced as a consequence of a higher frequency of double crossovers in larger chromosomes. As expected from effects of selection on linked sites, we observed an overall positive association between recombination rate and genetic diversity in both populations. Our results also revealed a significant effect of chromosomal rearrangements on the rate of intergenic diversity change between populations, but limited effects on polymorphisms in coding sequence. We conclude that chromosomal rearrangements can have considerable effects on the recombination landscape and consequently influence both maintenance of genetic diversity and efficiency of selection in natural populations. Author summaryReshuffling genetic variation is fundamental for maintaining genetic diversity and creating novel allelic combinations. The two main processes involved are the independent assortment of chromosomes and homologous recombination. The number and size of chromosomes can influence the amount of pairwise reshuffling and local recombination patterns. However, studying this in natural populations is challenging. In this study, we used the wood white butterfly, which exhibits an extreme within-species karyotype difference. Extensive fusions and fissions have resulted in almost twice as many chromosomes in the southern populations compared to the northeast populations. This unique system allowed us to assess the relationship between karyotype differences, pairwise reshuffling, recombination rate variation and subsequent effects on diversity and linked selection. We found that a higher number of chromosomes results in higher number of crossovers, although the difference was less than expected due to multiple recombination events occurring on longer chromosomes. Both populations showed an association between recombination rate and genome-wide patterns of genetic diversity and efficacy of selection. We provide evidence that chromosomal rearrangements have considerable effects on the recombination landscape and thereby influence the maintenance of genetic diversity in populations.

Mitochondrial defects can cause a variety of human diseases and protective mechanisms exist to maintain mitochondrial functionality. Imbalances in mitochondrial proteostasis trigger a transcriptional program, termed mitochondrial unfolded protein response (mtUPR). However, the temporal sequence of events in mtUPR is unclear and the consequences on mitochondrial protein import are controversial. Here, we have quantitatively analyzed all main import pathways into mitochondria after different time spans of mtUPR induction. Kinetic analyses reveal that protein import into all mitochondrial subcompartments strongly increases early upon mtUPR and that this is accompanied by rapid remodelling of the mitochondrial signature lipid cardiolipin. Genetic inactivation of cardiolipin synthesis precluded stimulation of protein import and compromised cellular fitness. At late stages of mtUPR upon sustained stress, mitochondrial protein import efficiency declined. Our work clarifies the enigma of protein import upon mtUPR and identifies sequential mtUPR stages, in which an early increase in protein biogenesis to restore mitochondrial proteostasis is followed by late stages characterized by a decrease in import capacity upon prolonged stress induction.

The transition to selfing in Capsella rubella accompanies its recent divergence from the ancestral outcrossing C. grandiflora species about 100,000 years ago. Whether the change in mating system was accompanied by the evolution of additional reproductive barriers that enforced species divergence remained unknown. Here, we show that C. rubella and C. grandiflora are reproductively separated by an endosperm-based, non-reciprocal postzygotic hybridization barrier. While hybridizations of C. rubella maternal plants with C. grandiflora pollen donors resulted in complete seed abortion caused by endosperm cellularization failure, the reciprocal hybridization resulted in the formation of small seeds with precociously cellularized endosperm. Strikingly, the transcriptomic response of both hybridizations mimicked respectively the response of paternal and maternal excess hybridizations in Arabidopsis thaliana, suggesting unbalanced genome strength causes hybridization failure in both species. These results provide strong support for the theory that crosses between plants of different mating systems will be unbalanced, with the outcrosser behaving like a plant of increased ploidy, evoking a response that resembles an interploidy-type seed failure. Seed incompatilibity of C. rubella pollinated by C. grandiflora followed the Bateson-Dobzhansky-Muller model, involving negative genetic interaction of multiple paternal C. grandiflora loci with at least one maternal C. rubella locus. Given that both species only recently diverged, our data suggest that a fast evolving mechanism underlies the post-zygotic hybridization barrier(s) separating both species.

Living in dynamic environments such as the social domain, where interaction with others determines the reproductive success of individuals, requires the ability to recognize opportunities to obtain natural rewards and cope with challenges that are associated with achieving them. As such, actions that promote survival and reproduction are reinforced by the brain reward system, whereas coping with the challenges associated with obtaining these rewards is mediated by stress-response pathways, the activation of which can impair health and shorten lifespan. While much research has been devoted to understanding mechanisms underlying the way by which natural rewards are processed by the reward system, less attention has been given to the consequences of failure to obtain a desirable reward. As a model system to study the impact of failure to obtain a natural reward, we used the well-established courtship suppression paradigm in Drosophila melanogaster as means to induce repeated failures to obtain sexual reward in male flies. We discovered that beyond the known reduction in courtship actions caused by interaction with non-receptive females, repeated failures to mate induce a stress response characterized by persistent motivation to obtain the sexual reward, reduced male-male social interaction, and enhanced aggression. This frustrative-like state caused by the conflict between high motivation to obtain sexual reward and the inability to fulfill their mating drive impairs the capacity of rejected males to tolerate stressors such as starvation and oxidative stress. We further show that sensitivity to starvation and enhanced social arousal is mediated by the disinhibition of a small population of neurons that express receptors for the fly homologue of neuropeptide Y. Our findings demonstrate for the first time the existence of social stress in flies and offers a framework to study mechanisms underlying the crosstalk between reward, stress, and reproduction in a simple nervous system that is highly amenable to genetic manipulation. In this study we investigated the effects of failure to obtain reward on the behavioral actions and physiology of male flies. We exposed Drosophila males to repeated sexual encounters with non-receptive females that rejected their courtship efforts and tested the effect on their behavioral responses using a collection of behavioral paradigms. These responses encompass alterations in social behavior, increased aggression, heightened motivation to mate, and a reduced capacity to cope with stressors. We further show that the high motivational state and sensitivity to stress is mediated by the disinhibition of a small population of neurons that express receptors for the fly homologue of neuropeptide Y.

Nuclear myosin 1c (NM1) mediates RNA polymerase I (pol I) transcription activation and cell cycle progression by facilitating PCAF-mediated H3K9 acetylation, but the molecular mechanism by which NM1 is regulated remains unclear. Here, we report that at early G1 the glycogen synthase kinase (GSK) 3 beta phosphorylates and stabilizes NM1, allowing for NM1 association with the chromatin. Genomic analysis by ChIP-Seq showed that this mechanism occurs on the rDNA as active GSK3 beta selectively occupies the gene. ChIP assays and transmission electron microscopy in GSK3 beta(-/-) mouse embryonic fibroblasts indicated that at G1 rRNA synthesis is suppressed due to decreased H3K9 acetylation leading to a chromatin state incompatible with transcription. We found that GSK3 beta directly phosphorylates the endogenous NM1 on a single serine residue (Ser-1020) located within the NM1 C-terminus. In G1 this phosphorylation event stabilizes NM1 and prevents NM1 polyubiquitination by the E3 ligase UBR5 and proteasome-mediated degradation. We conclude that GSK3 beta-mediated phosphorylation of NM1 is required for pol I transcription activation.

Actin and nuclear myosin 1c (NM1) cooperate in RNA polymerase I (pol I) transcription. NM1 is also part of a multiprotein assembly, B-WICH, which is involved in transcription. This assembly contains the chromatin remodeling complex WICH with its subunits WSTF and SNF2h. We report here that NM1 binds SNF2h with enhanced affinity upon impairment of the actin-binding function. ChIP analysis revealed that NM1, SNF2h, and actin gene occupancies are cell cycle-dependent and require intact motor function. At the onset of cell division, when transcription is temporarily blocked, B-WICH is disassembled due to WSTF phosphorylation, to be reassembled on the active gene at exit from mitosis. NM1 gene knockdown and motor function inhibition, or stable expression of NM1 mutants that do not interact with actin or chromatin, overall repressed rRNA synthesis by stalling pol I at the gene promoter, led to chromatin alterations by changing the state of H3K9 acetylation at gene promoter, and delayed cell cycle progression. These results suggest a unique structural role for NM1 in which the interaction with SNF2h stabilizes B-WICH at the gene promoter and facilitates recruitment of the HAT PCAF. This leads to a permissive chromatin structure required for transcription activation.

Hsp70 chaperones are well known for their important functions in maintaining protein homeostasis during thermal stress conditions. In many bacteria the Hsp70 homolog DnaK is also required for growth in the absence of stress. The molecular reasons underlying Hsp70 essentiality remain in most cases unclear. Here, we demonstrate that DnaK is essential in the alpha-proteobacterium Caulobacter crescentus due to its regulatory function in gene expression. Using a suppressor screen we identified mutations that allow growth in the absence of DnaK. All mutations reduced the activity of the heat shock sigma factor sigma(32) , demonstrating that the DnaK-dependent inactivation of sigma(32) is a growth requirement. While most mutations occurred in the rpoH gene encoding sigma(32) , we also identified mutations affecting sigma(32) activity or stability in trans, providing important new insight into the regulatory mechanisms controlling sigma(32) activity. Most notably, we describe a mutation in the ATP dependent protease HslUV that induces rapid degradation of sigma(32) , and a mutation leading to increased levels of the house keeping sigma(70) that outcompete sigma(32) for binding to the RNA polymerase. We demonstrate that sigma(32) inhibits growth and that its unrestrained activity leads to an extensive reprogramming of global gene expression, resulting in upregulation of repair and maintenance functions and downregulation of the growth-promoting functions of protein translation, DNA replication and certain metabolic processes. While this re-allocation from proliferative to maintenance functions could provide an advantage during heat stress, it leads to growth defects under favorable conditions. We conclude that Caulobacter has coopted the DnaK chaperone system as an essential regulator of gene expression under conditions when its folding activity is dispensable.

Amino acids are among the earliest identified inducers of yeast-to-hyphal transitions in Candida albicans, an opportunistic fungal pathogen of humans. Here, we show that the morphogenic amino acids arginine, ornithine and proline are internalized and metabolized in mitochondria via a PUT1- and PUT2-dependent pathway that results in enhanced ATP production. Elevated ATP levels correlate with Ras1/cAMP/PKA pathway activation and Efg1-induced gene expression. The magnitude of amino acid-induced filamentation is linked to glucose availability; high levels of glucose repress mitochondrial function thereby dampening filamentation. Furthermore, arginine-induced morphogenesis occurs more rapidly and independently of Dur1,2-catalyzed urea degradation, indicating that mitochondrial-generated ATP, not CO2, is the primary morphogenic signal derived from arginine metabolism. The important role of the SPS-sensor of extracellular amino acids in morphogenesis is the consequence of induced amino acid permease gene expression, i.e., SPS-sensor activation enhances the capacity of cells to take up morphogenic amino acids, a requisite for their catabolism. C. albicans cells engulfed by murine macrophages filament, resulting in macrophage lysis. Phagocytosed put1-/- and put2-/- cells do not filament and exhibit reduced viability, consistent with a critical role of mitochondrial proline metabolism in virulence. Author summary Candida albicans is an opportunistic fungal pathogen that exists as a benign member of the human microbiome. Immunosuppression, or microbial dysbiosis, can predispose an individual to infection, enabling this fungus to evade innate immune cells and initiate a spectrum of pathologies, including superficial mucocutaneous or even life-threatening invasive infections. Infectious growth is attributed to an array of virulence characteristics, a major one being the ability to switch morphologies from round yeast-like to elongated hyphal cells. Here we report that mitochondrial proline catabolism is required to induce hyphal growth of C. albicans cells in phagosomes of engulfing macrophages, which is key to evade killing by macrophages. The finding that proline catabolism, also required for the utilization of arginine and ornithine, is required to sustain the energy demands of hyphal growth underscores the central role of mitochondria in fungal virulence. In contrast to existing dogma, we show that in C. albicans, mitochondrial function is subject to glucose repression, amino acid-induced signals are strictly dependent on Ras1 and the SPS-sensor is the primary sensor of extracellular amino acids. The results provide a clear example of how C. albicans cells sense and respond to host nutrients to ensure proper nutrient uptake and survival.

The X chromosome constitutes a unique genomic environment because it is present in one copy in males, but two copies in females. This simple fact has motivated several theoretical predictions with respect to how standing genetic variation on the X chromosome should differ from the autosomes. Unmasked expression of deleterious mutations in males and a lower census size are expected to reduce variation, while allelic variants with sexually antagonistic effects, and potentially those with a sex-specific effect, could accumulate on the X chromosome and contribute to increased genetic variation. In addition, incomplete dosage compensation of the X chromosome could potentially dampen the male-specific effects of random mutations, and promote the accumulation of X-linked alleles with sexually dimorphic phenotypic effects. Here we test both the amount and the type of genetic variation on the X chromosome within a population of Drosophila melanogaster, by comparing the proportion of X linked and autosomal trans-regulatory SNPs with a sexually concordant and discordant effect on gene expression. We find that the X chromosome is depleted for SNPs with a sexually concordant effect, but hosts comparatively more SNPs with a sexually discordant effect. Interestingly, the contrasting results for SNPs with sexually concordant and discordant effects are driven by SNPs with a larger influence on expression in females than expression in males. Furthermore, the distribution of these SNPs is shifted towards regions where dosage compensation is predicted to be less complete. These results suggest that intrinsic properties of dosage compensation influence either the accumulation of different types of trans-factors and/or their propensity to accumulate mutations. Our findings document a potential mechanistic basis for sex-specific genetic variation, and identify the X as a reservoir for sexually dimorphic phenotypic variation. These results have general implications for X chromosome evolution, as well as the genetic basis of sex-specific evolutionary change.

During neuronal development, beta-actin serves an important role in growth cone mediated axon guidance. Consistent with this notion, in vivo ablation of the beta-actin gene leads to abnormalities in the nervous system. However, whether beta-actin is involved in the regulation of neuronal gene programs is not known. In this study, we directly reprogramed beta-actin(+/+) WT, beta-actin(+/-) HET and beta-actin(-/-) KO mouse embryonic fibroblast (MEFs) into chemically induced neurons (CiNeurons). Using RNA-seq analysis, we profiled the transcriptome changes among the CiNeurons. We discovered that induction of neuronal gene programs was impaired in KO CiNeurons in comparison to WT ones, whereas HET CiNeurons showed an intermediate levels of induction. ChIP-seq analysis of heterochromatin markers demonstrated that the impaired expression of neuronal gene programs correlated with the elevated H3K9 and H3K27 methylation levels at gene loci in beta-actin deficient MEFs, which is linked to the loss of chromatin association of the BAF complex ATPase subunit Brg1. Together, our study shows that heterochromatin alteration in beta-actin null MEFs impedes the induction of neuronal gene programs during direct reprograming. These findings are in line with the notion that H3K9Me3-based heterochromatin forms a major epigenetic barrier during cell fate change.

Environmental factors challenge the physiological homeostasis in animals, thereby evoking stress responses. Various mechanisms have evolved to counter stress at the organism level, including regulation by neuropeptides. In recent years, much progress has been made on the mechanisms and neuropeptides that regulate responses to metabolic/nutritional stress, as well as those involved in countering osmotic and ionic stresses. Here, we identified a peptidergic pathway that links these types of regulatory functions. We uncover the neuropeptide Corazonin (Crz), previously implicated in responses to metabolic stress, as a neuroendocrine factor that inhibits the release of a diuretic hormone, CAPA, and thereby modulates the tolerance to osmotic and ionic stress. Both knockdown of Crz and acute injections of Crz peptide impact desiccation tolerance and recovery from chill-coma. Mapping of the Crz receptor (CrzR) expression identified three pairs of Capa-expressing neurons (Va neurons) in the ventral nerve cord that mediate these effects of Crz. We show that Crz acts to restore water/ion homeostasis by inhibiting release of CAPA neuropeptides via inhibition of cAMP production in Va neurons. Knockdown of CrzR in Va neurons affects CAPA signaling, and consequently increases tolerance for desiccation, ionic stress and starvation, but delays chill-coma recovery. Optogenetic activation of Va neurons stimulates excretion and simultaneous activation of Crz and CAPA-expressing neurons reduces this response, supporting the inhibitory action of Crz. Thus, Crz inhibits Va neurons to maintain osmotic and ionic homeostasis, which in turn affects stress tolerance. Earlier work demonstrated that systemic Crz signaling restores nutrient levels by promoting food search and feeding. Here we additionally propose that Crz signaling also ensures osmotic homeostasis by inhibiting release of CAPA neuropeptides and suppressing diuresis. Thus, Crz ameliorates stress-associated physiology through systemic modulation of both peptidergic neurosecretory cells and the fat body in Drosophila.

Behavior and physiology are orchestrated by neuropeptides acting as central neuromodulators and circulating hormones. An outstanding question is how these neuropeptides function to coordinate complex and competing behaviors. In Drosophila, the neuropeptide leucokinin (LK) modulates diverse functions, but mechanisms underlying these complex interactions remain poorly understood. As a first step towards understanding these mechanisms, we delineated LK circuitry that governs various aspects of post-feeding physiology and behavior. We found that impaired LK signaling in Lk and Lk receptor (Lkr) mutants affects diverse but coordinated processes, including regulation of stress, water homeostasis, feeding, locomotor activity, and metabolic rate. Next, we sought to define the populations of LK neurons that contribute to the different aspects of this physiology. We find that the calcium activity in abdominal ganglia LK neurons (ABLKs), but not in the two sets of brain neurons, increases specifically following water consumption, suggesting that ABLKs regulate water homeostasis and its associated physiology. To identify targets of LK peptide, we mapped the distribution of Lkr expression, mined a brain single-cell transcriptome dataset for genes coexpressed with Lkr, and identified synaptic partners of LK neurons. Lkrexpression in the brain insulin-producing cells (IPCs), gut, renal tubules and chemosensory cells, correlates well with regulatory roles detected in the Lkand Lkr mutants. Furthermore, these mutants and flies with targeted knockdown of Lkr in IPCs displayed altered expression of insulin-like peptides (DILPs) and transcripts in IPCs and increased starvation resistance. Thus, some effects of LK signaling appear to occur via DILP action. Collectively, our data suggest that the three sets of LK neurons have different targets, but modulate the establishment of postprandial homeostasis by regulating distinct physiological processes and behaviors such as diuresis, metabolism, organismal activity and insulin signaling. These findings provide a platform for investigating feeding-related neuroendocrine regulation of vital behavior and physiology.