A Myc network accounts for similarities between embryonic stem and cancer cell transcription programs.

c-Myc (Myc) is an important transcriptional regulator in embryonic stem (ES) cells, somatic cell reprogramming, and cancer. Here, we identify a Myc-centered regulatory network in ES cells by combining protein-protein and protein-DNA interaction studies and show that Myc interacts with the NuA4 complex, a regulator of ES cell identity. In combination with regulatory network information, we define three ES cell modules (Core, Polycomb, and Myc) and show that the modules are functionally separable, illustrating that the overall ES cell transcription program is composed of distinct units. With these modules as an analytical tool, we have reassessed the hypothesis linking an ES cell signature with cancer or cancer stem cells. We find that the Myc module, independent of the Core module, is active in various cancers and predicts cancer outcome. The apparent similarity of cancer and ES cell signatures reflects, in large part, the pervasive nature of Myc regulatory networks.

Transcriptional control of a metabolic switch regulating cellular methylation reactions is part of a common response to stress in divergent bee species.

Recent global declines in bee health have elevated the need for a more complete understanding of the cellular stress mechanisms employed by diverse bee species. We recently uncovered the biomarker lethal (2) essential for life [l(2)efl] genes as part of a shared transcriptional program in response to a number of cell stressors in the western honey bee (Apis mellifera). Here, we describe another shared stress-responsive gene, glycine N-methyltransferase (Gnmt), which is known as a key metabolic switch controlling cellular methylation reactions. We observed Gnmt induction by both abiotic and biotic stressors. We also found increased levels of the GNMT reaction product sarcosine in the midgut after stress, linking metabolic changes with the observed changes in gene regulation. Prior to this study, Gnmt upregulation had not been associated with cellular stress responses in other organisms. To determine whether this novel stress-responsive gene would behave similarly in other bee species, we first characterized the cellular response to endoplasmic reticulum (ER) stress in lab-reared adults of the solitary alfalfa leafcutting bee (Megachile rotundata) and compared this with age-matched honey bees. The novel stress gene Gnmt was induced in addition to a number of canonical gene targets induced in both bee species upon unfolded protein response (UPR) activation, suggesting that stress-induced regulation of cellular methylation reactions is a common feature of bees. Therefore, this study suggests that the honey bee can serve as an important model for bee biology more broadly, although studies on diverse bee species will be required to fully understand global declines in bee populations.

Pathogen- and host-directed pharmacologic strategies for control of Vairimorpha (Nosema) spp. infection in honey bees.

Microsporidia are obligate intracellular parasites of the Fungal Kingdom that cause widespread infections in nature, with important effects on invertebrates involved in food production systems. The two microsporidian species Vairimorpha (Nosema) ceranae (and the less common Vairimorpha (Nosema) apis) can cause individual disease in honey bees and contribute to colony collapse. The efficacy, safety, and availability of fumagillin, the only drug currently approved to treat microsporidia infection in bees, is uncertain. In this review, we will discuss some of the most promising alternative strategies for the mitigation of Vairimorpha spp. with an emphasis on infection by V. ceranae, now the dominant species infecting bees. We will focus on pharmacologic interventions where the mechanism of action is known and examine both pathogen-directed and host-directed approaches. As limiting toxicity to host cells has been especially emphasized in treating bees that are already facing numerous stressors, strategies that disrupt pathogen-specific targets may be especially advantageous. Therefore, efforts to increase the knowledge and tools for facilitating the discovery of such targets and pharmacologic agents directed against them should be prioritized.

Thermal stress induces tissue damage and a broad shift in regenerative signaling pathways in the honey bee digestive tract.

Honey bee colonies in the USA have suffered from increased die-off in the last few years with a complex set of interacting stresses playing a key role. With changing climate, an increase in the frequency of severe weather events, such as heat waves, is anticipated. Understanding how these changes may contribute to stress in honey bees is crucial. Individual honey bees appear to have a high capacity to endure thermal stress. One reason for this high-level endurance is likely their robust heat shock response (HSR), which contributes to thermotolerance at the cellular level. However, less is known about other mechanisms of thermotolerance, especially those operating at the tissue level. To elucidate other determinants of resilience in this species, we used thermal stress coupled with RNAseq and identified broad transcriptional remodeling of a number of key signaling pathways in the honey bee, including those pathways known to be involved in digestive tract regeneration in the fruit fly such as the Hippo and JAK/STAT pathways. We also observed cell death and shedding of epithelial cells, which likely leads to induction of this regenerative transcriptional program. We found that thermal stress affects many of these pathways in other tissues, suggesting a shared program of damage response. This study provides important foundational characterization of the tissue damage response program in this key pollinating species. In addition, our data suggest that a robust regeneration program may also be a critical contributor to thermotolerance at the tissue level, a possibility which warrants further exploration in this and other species.

Uptake and impact of natural diet-derived small RNA in invertebrates: Implications for ecology and agriculture.

The putative transfer and gene regulatory activities of diet-derived small RNAs (sRNAs) in ingesting animals are still debated. The existence of natural uptake of diet-derived sRNA by invertebrate species could have significant implication for our understanding of ecological relationships and could synergize with efforts to use RNA interference (RNAi) technology in agriculture. Here, we synthesize information gathered from studies in invertebrates using natural or artificial dietary delivery of sRNA and from studies of sRNA in vertebrate animals and plants to review our current understanding of uptake and impact of natural diet-derived sRNA on invertebrates. Our understanding has been influenced and sometimes confounded by the diversity of invertebrates and ingested plants studied, our limited insights into how gene expression may be modulated by dietary sRNAs at the mechanistic level, and the paucity of studies focusing directly on natural uptake of sRNA. As such, we suggest 2 strategies to investigate this phenomenon more comprehensively and thus facilitate the realization of its potentially broad impact on ecology and agriculture in the future.

Negligible uptake and transfer of diet-derived pollen microRNAs in adult honey bees.

The putative transfer and gene regulatory activities of diet-derived miRNAs in ingesting animals are still debated. Importantly, no study to date has fully examined the role of dietary uptake of miRNA in the honey bee, a critical pollinator in both agricultural and natural ecosystems. After controlled pollen feeding experiments in adult honey bees, we observed that midguts demonstrated robust increases in plant miRNAs after pollen ingestion. However, we found no evidence of biologically relevant delivery of these molecules to proximal or distal tissues of recipient honey bees. Our results, therefore, support the premise that pollen miRNAs ingested as part of a typical diet are not robustly transferred across barrier epithelia of adult honey bees under normal conditions. Key future questions include whether other small RNA species in honey bee diets behave similarly and whether more specialized and specific delivery mechanisms exist for more efficient transport, particularly in the context of stressed barrier epithelia.

A fluorescent method for visualization of Nosema infection in whole-mount honey bee tissues.

Honey bees are critical pollinators in both agricultural and ecological settings. The Nosema species, ceranae and apis, are microsporidian parasites that are pathogenic to honey bees. While current methods for detecting Nosema infection have key merits, additional techniques with novel properties for studying the cell biology of Nosema infection are highly desirable. We demonstrate that whole-mount staining of honey bee midgut tissue with chitin-binding agent Fluorescent Brightener 28 and DNA dye Propidium Iodide allows for observation of Nosema infection in structurally intact tissue, providing a new tool for increasing our understanding of Nosema infection at the cellular and tissue level.

Bleomycin reduces Vairimorpha (Nosema) ceranae infection in honey bees with some evident host toxicity.

Microsporidia cause disease in many beneficial insects, including honey bees, yet few pathogen control tools are available for protecting these important organisms against infection. Some evidence suggests that microsporidia possess a reduced number of genes encoding DNA repair proteins. We hypothesized that microsporidia would thus be susceptible to treatment with DNA-damaging agents and tested this hypothesis using a novel, rapid method for achieving robust and homogenous experimental infection of large numbers of newly emerged honey bees with one of its microsporidia pathogens, Vairimorpha (Nosema) ceranae. In carrying out these experiments, we found this novel V. ceranae inoculation method to have similar efficacy as other traditional methods. We show that the DNA-damaging agent bleomycin reduces V. ceranae levels, with minimal but measurable effects on honey bee survival and increased expression of midgut cellular stress genes, including those encoding SHSP. Increased expression of UpdlC suggests the occurrence of epithelial regeneration, which may contribute to host resistance to bleomycin treatment. While bleomycin does reduce infection levels, host toxicity issues may preclude its use in the field. However, with further work, bleomycin may provide a useful tool in the research setting as a potential selection agent for genetic modification of microsporidia.IMPORTANCEMicrosporidia cause disease in many beneficial insects, yet there are few tools available for control in the field or laboratory. Based on the reported paucity of DNA repair enzymes found in microsporidia genomes, we hypothesized that these obligate intracellular parasites would be sensitive to DNA damage. In support of this, we observed that the well-characterized DNA damage agent bleomycin can reduce levels of the microsporidia Vairimorpha (Nosema) ceranae in experimental infections in honey bees. Observation of slightly reduced honey bee survival and evidence of sublethal toxicity likely preclude the use of bleomycin in the field. However, this work identifies bleomycin as a compound that merits further exploration for use in research laboratories as a potential selection agent for generating genetically modified microsporidia.

Impaired CD8 T cell memory and CD4 T cell primary responses in IL-7R alpha mutant mice.

Loss of interleukin (IL)-7 or the IL-7 receptor alpha (IL-7Ralpha, CD127) results in severe immunodeficiencies in mice and humans. To more precisely identify signals governing IL-7 function in vivo, we have disrupted the IL-7Ralpha Y449XXM motif in mice by knock-in mutagenesis (IL-7Ralpha(449F)). Thymic precursors were reduced in number in IL-7Ralpha(449F) mice, but in marked contrast to IL-7Ralpha(-/-) knockout mice, thymocytes and peripheral T cells developed normally. Strikingly, Listeria infection revealed that CD4 and CD8 T cells had different requirements for IL-7Ralpha signals. CD4 T cells failed to mount a primary response, but despite normal CD8 primary responses, maintenance of CD8 memory was impaired in IL-7Ralpha(449F) mice. Furthermore, we show that Bcl-2 is IL-7Ralpha Y449 independent and insufficient for IL-7-mediated maintenance of CD8 memory.

Context-dependent function of "GATA switch" sites in vivo.

Master transcriptional regulators of development often function through dispersed cis elements at endogenous target genes. While cis-elements are routinely studied in transfection and transgenic reporter assays, it is challenging to ascertain how they function in vivo. To address this problem in the context of the locus encoding the critical hematopoietic transcription factor Gata2, we engineered mice lacking a cluster of GATA motifs 2.8 kb upstream of the Gata2 transcriptional start site. We demonstrate that the -2.8 kb site confers maximal Gata2 expression in hematopoietic stem cells and specific hematopoietic progenitors. By contrast to our previous demonstration that a palindromic GATA motif at the neighboring -1.8 kb site maintains Gata2 repression in terminally differentiating erythroid cells, the -2.8 kb site was not required to initiate or maintain repression. These analyses reveal qualitatively distinct functions of 2 GATA motif-containing regions in vivo.

Ineffective delivery of diet-derived microRNAs to recipient animal organisms.

Cross-kingdom delivery of specific microRNAs to recipient organisms via food ingestion has been reported recently. However, it is unclear if such delivery of microRNAs occurs frequently in animal organisms after typical dietary intake. We found substantial levels of specific microRNAs in diets commonly consumed orally by humans, mice, and honey bees. Yet, after ingestion of fruit replete with plant microRNAs (MIR156a, MIR159a, and MIR169a), a cohort of healthy athletes did not carry detectable plasma levels of those molecules. Similarly, despite consumption of a diet with animal fat replete in endogenous miR-21, negligible expression of miR-21 in plasma or organ tissue was observed in miR-21 -/- recipient mice. Correspondingly, when fed vegetarian diets containing the above plant microRNAs, wild-type recipient mice expressed insignificant levels of these microRNAs. Finally, despite oral uptake of pollen containing these plant microRNAs, negligible delivery of these molecules was observed in recipient honeybees. Therefore, we conclude that horizontal delivery of microRNAs via typical dietary ingestion is neither a robust nor a frequent mechanism to maintain steady-state microRNA levels in a variety of model animal organisms, thus defining the biological limits of these molecules in vivo.

A single cis element maintains repression of the key developmental regulator Gata2.

In development, lineage-restricted transcription factors simultaneously promote differentiation while repressing alternative fates. Molecular dissection of this process has been challenging as transcription factor loci are regulated by many trans-acting factors functioning through dispersed cis elements. It is not understood whether these elements function collectively to confer transcriptional regulation, or individually to control specific aspects of activation or repression, such as initiation versus maintenance. Here, we have analyzed cis element regulation of the critical hematopoietic factor Gata2, which is expressed in early precursors and repressed as GATA-1 levels rise during terminal differentiation. We engineered mice lacking a single cis element -1.8 kb upstream of the Gata2 transcriptional start site. Although Gata2 is normally repressed in late-stage erythroblasts, the -1.8 kb mutation unexpectedly resulted in reactivated Gata2 transcription, blocked differentiation, and an aberrant lineage-specific gene expression pattern. Our findings demonstrate that the -1.8 kb site selectively maintains repression, confers a specific histone modification pattern and expels RNA Polymerase II from the locus. These studies reveal how an individual cis element establishes a normal developmental program via regulating specific steps in the mechanism by which a critical transcription factor is repressed.

Colonization of Honey Bee Digestive Tracts by Environmental Yeast Lachancea thermotolerans Is Naturally Occurring, Temperature Dependent, and Impacts the Microbiome of Newly Emerged Bees.

Honey bees are critical pollinators in both agricultural and ecological settings. Recent declines in honey bee colonies in the United States have put increased strain on agricultural pollination. Although there are many environmental stressors implicated in honey bee disease, there has been intensifying focus on the role of microbial attacks on honey bee health. Despite the long-standing appreciation for the association of fungi of various groups with honey bees and their broader environment, the effects of these interactions on honey bee health are incompletely understood. Here, we report the discovery of colonization of the honey bee digestive tract by the environmental yeast Lachancea thermotolerans. Experimental colonization of honey bee digestive tracts by L. thermotolerans revealed that this yeast species maintains high levels in the honey bee midgut only at temperatures below the typical colony temperature. In newly eclosed bees, L. thermotolerans colonization alters the microbiome, suggesting that environmental yeasts can impact its composition. Future studies should be undertaken to better understand the role of L. thermotolerans and other environmental yeasts in honey bee health. IMPORTANCE Although many fungal species are found in association with honey bees and their broader environment, the effects of these interactions on honey bee health are largely unknown. Here, we report the discovery that a yeast commonly found in the environment can be found at high levels in honey bee digestive tracts. Experimentally feeding this yeast to honey bees showed that the yeast's ability to maintain high levels in the digestive tract is influenced by temperature and can lead to alterations of the microbiome in young bees. These studies provide a foundation for future studies to better understand the role of environmental yeasts in honey bee health.

Nosema apis and N. ceranae Infection in Honey bees: A Model for Host-Pathogen Interactions in Insects.

There has been increased focus on the role of microbial attack as a potential cause of recent declines in the health of the western honey bee, Apis mellifera. The Nosema species, N. apis and N. ceranae, are microsporidian parasites that are pathogenic to honey bees, and infection by these species has been implicated as a key factor in honey bee losses. Honey bees infected with both Nosema spp. display significant changes in their biology at the cellular, tissue, and organismal levels impacting host metabolism, immune function, physiology, and behavior. Infected individuals lead to colony dysfunction and can contribute to colony disease in some circumstances. The means through which parasite growth and tissue pathology in the midgut lead to the dramatic physiological and behavioral changes at the organismal level are only partially understood. In addition, we possess only a limited appreciation of the elements of the host environment that impact pathogen growth and development. Critical for answering these questions is a mechanistic understanding of the host and pathogen machinery responsible for host-pathogen interactions. A number of approaches are already being used to elucidate these mechanisms, and promising new tools may allow for gain- and loss-of-function experiments to accelerate future progress.

Paromomycin Reduces Vairimorpha (Nosema)ceranae Infection in Honey Bees but Perturbs Microbiome Levels and Midgut Cell Function.

Paromomycin is a naturally occurring aminoglycoside antibiotic that has effects on both prokaryotic and eukaryotic microbes. However, previous reports have indicated that it has little effect on microsporidia, including Vairimorpha (Nosema) ceranae, in cell culture models. V. ceranae is one of a number of microsporidia species that cause disease in honey bees and substantial efforts to find new treatment strategies for bees that are infected with these pathogens are ongoing. When testing compounds for potential activity against V. ceranae in whole organisms, we found that paromomycin reduces the infection intensity of this parasite. Critically, the necessary doses of paromomycin have high activity against the bacteria of the honey bee microbiome and cause evident stress in bees. Microsporidia have been shown to lack an essential binding site on the ribosome that is known to allow for maximal inhibition by paromomycin. Thus, it is possible that paromomycin impacts parasite levels through non-cell autonomous effects on microsporidia infection levels via effects on the microbiome or midgut cellular function. As paromomycin treatment could cause widespread honey bee health issues in agricultural settings, it does not represent an appropriate anti-microsporidia agent for use in the field.

Sumoylation regulates interaction of FOG1 with C-terminal-binding protein (CTBP).

Erythropoietic and megakaryocytic programs are specified from multipotential progenitors by the transcription factor GATA1. FOG1, a GATA1-interaction partner, is critical for GATA1 function in several contexts by bringing multiple complexes into association with GATA1 to facilitate activation or repression of target genes. To further elucidate regulation of these associations by cellular and extracellular cues, we examined FOG1 for post-translational modifications. We found that FOG1 is SUMOylated and phosphorylated in erythroid cells in a differentiation-dependent manner. Removal of the SUMOylation sites in FOG1 does not impair nuclear localization, protein stability, or chromatin occupancy. However, SUMOylation of FOG1 modulates interactions with C-terminal binding protein family members, specifically promoting CTBP1 binding. Phosphorylation of FOG1 modulates SUMOylation and, therefore, indirectly regulates the CTBP interaction. Post-translational modification of FOG1 may contribute to control of co-occupancy by CTBP family members, the NuRD complex, and GATA1 at differentially regulated genes.

Halofuginone triggers a transcriptional program centered on ribosome biogenesis and function in honey bees.

We previously found that pharmacological inhibition of prolyl-tRNA synthetase by halofuginone has potent activity against Nosema ceranae, an important pathogen of honey bees. However, we also observed that prolyl-tRNA synthetase inhibition is toxic to bees, suggesting further work is necessary to make this a feasible therapeutic strategy. As expected, we found that pharmacological inhibition of prolyl-tRNA synthetase activity resulted in robust induction of select canonical ATF4 target genes in honey bees. However, our understanding of this and other cellular stress responses in general in honey bees is incomplete. Thus, we used RNAseq to identify novel changes in gene expression after halofuginone treatment and observed induction of genes involved in ribosome biogenesis, translation, tRNA synthesis, and ribosome-associated quality control (RQC). These results suggest that halofuginone, potentially acting through the Integrated Stress Response (ISR), promotes a transcriptional response to ribosome functional impairment in honey bees rather than the response designed to oppose amino acid limitation, which has been observed in other organisms after ISR induction. In support of this idea, we found that cycloheximide (CHX) administration also induced all tested target genes, indicating that this gene expression program could be induced by ribosome stalling in addition to tRNA synthetase inhibition. Only a subset of halofuginone-induced genes was upregulated by Unfolded Protein Response (UPR) induction, suggesting that mode of activation and cross-talk with other cellular signaling pathways significantly influence ISR function and cellular response to its activation. Future work will focus on understanding how the apparently divergent transcriptional output of the ISR in honey bees impacts the health and disease of this important pollinator species.

Honey bee sHSP are responsive to diverse proteostatic stresses and potentially promising biomarkers of honey bee stress.

The pollination services provided by the honey bee are critical in both natural and agricultural ecosystems. Honey bee colonies in the United States have suffered from an increased rate of die-off in recent years, stemming from a complex set of interacting stresses that remain poorly described. Defining specific common cellular processes and cellular stress responses impacted by multiple stressors represent a key step in understanding these synergies. Proteotoxic stresses negatively impact protein synthesis, folding, and degradation. Diverse proteotoxic stresses induce expression of genes encoding small heat shock proteins (sHSP) of the expanded lethal (2) essential for life (l(2)efl) gene family. In addition to upregulation by the Integrated Stress Response (ISR), the Heat Shock Response (HSR), and the Oxidative Stress Response (OSR), our data provide first evidence that sHSP genes are upregulated by the Unfolded Protein Response (UPR). As these genes appear to be part of a core stress response that could serve as a useful biomarker for cellular stress in honey bees, we designed and tested an RT-LAMP assay to detect increased l(2)efl gene expression in response to heat-stress. While this assay provides a powerful proof of principle, further work will be necessary to link changes in sHSP gene expression to colony-level outcomes, to adapt our preliminary assay into a Point of Care Testing (POCT) assay appropriate for use as a diagnostic tool for use in the field, and to couple assay results to management recommendations.

Proteasome Inhibition Is an Effective Treatment Strategy for Microsporidia Infection in Honey Bees.

The microsporidia Nosema ceranae is an obligate intracellular parasite that causes honey bee mortality and contributes to colony collapse. Fumagillin is presently the only pharmacological control for N. ceranae infections in honey bees. Resistance is already emerging, and alternative controls are critically needed. Nosema spp. exhibit increased sensitivity to heat shock, a common proteotoxic stress. Thus, we hypothesized that targeting the Nosema proteasome, the major protease removing misfolded proteins, might be effective against N. ceranae infections in honey bees. Nosema genome analysis and molecular modeling revealed an unexpectedly compact proteasome apparently lacking multiple canonical subunits, but with highly conserved proteolytic active sites expected to be receptive to FDA-approved proteasome inhibitors. Indeed, N. ceranae were strikingly sensitive to pharmacological disruption of proteasome function at doses that were well tolerated by honey bees. Thus, proteasome inhibition is a novel candidate treatment strategy for microsporidia infection in honey bees.

Translational isoforms of FOG1 regulate GATA1-interacting complexes.

Erythropoietic and megakaryocytic programs are directed by the transcription factor GATA1. Friend of GATA1 (FOG1), a protein interaction partner of GATA1, is critical for GATA1 function in multiple contexts. Previous work has shown that FOG1 recruits two multi-protein complexes, the nucleosome remodeling domain (NuRD) complex and a C-terminal binding protein (CTBP)-containing complex, into association with GATA1 to mediate activation and repression of target genes. To elucidate mechanisms that might differentially regulate the association of FOG1, as well as GATA1, with these two complexes, we characterized a previously unrecognized translational isoform of FOG1. We found that an N-terminally truncated version of FOG1 is produced from an internal ATG and that this isoform, designated FOG1S, lacks the nucleosome remodeling domain-binding domain, altering the complexes with which it interacts. Both isoforms interact with the C-terminal binding protein complex, which we show also contains lysine-specific demethylase 1 (LSD1). FOG1S is preferentially excluded from the nucleus by unknown mechanisms. These data reveal two novel mechanisms for the regulation of GATA1 interaction with FOG1-dependent protein complexes through the production of two translational isoforms with differential interaction profiles and independent nuclear localization controls.