Search

Abstract Background While aberrant DNA methylation is a characteristic feature of tumor cells, our knowledge of how these DNA methylation patterns are established and maintained is limited. DNA methyltransferases and ten-eleven translocation methylcytosine dioxygenases (TETs) function has been found altered in a variety of cancer types. Results Here, we report that in T cell acute lymphoblastic leukemia (T-ALL) the MYC oncogene controls the expression of TET1 and TET2 to maintain 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) patterns, which is associated with tumor cell-specific gene expression. We found that cellular senescence and tumor regression upon MYC inactivation in T-ALL was associated with genome-wide changes in 5mC and 5hmC patterns. Correlating with the changes in DNA (hydroxy)methylation, we found that T-ALL overexpress TET1, while suppressing TET2 in a MYC-dependent fashion. Consequently, MYC inactivation led to an inverse expression pattern, decreasing TET1, while increasing TET2 levels. Knockdown of TET1 or ectopic expression of TET2 in T-ALL was associated with genome-wide changes in 5mC and 5hmC enrichment and decreased cell proliferation, suggesting a tumor promoting function of TET1, and a tumor suppressing role for TET2. Among the genes and pathways controlled by TET1, we found ribosomal biogenesis and translational control of protein synthesis highly enriched. Conclusions Our finding that MYC directly deregulates the expression of TET1 and TET2 in T-ALL provides novel evidence that MYC controls DNA (hydroxy)methylation in a genome-wide fashion. It reveals a coordinated interplay between the components of the DNA (de)methylating machinery that contribute to MYC-driven tumor maintenance, highlighting the potential of specific TET enzymes for therapeutic strategies.

High-fat diet consumption and sedentary lifestyle elevates risk for obesity, non-alcoholic fatty liver disease, and cancer. Exercise training conveys health benefits in populations with or without these chronic conditions. Diet and exercise regulate gene expression by mediating epigenetic mechanisms in many tissues; however, such effects are poorly documented in the liver, a central metabolic organ. To dissect the consequences of diet and exercise on the liver epigenome, we measured DNA methylation, using reduced representation bisulfite sequencing, and transcription, using RNA-seq, in mice maintained on a fast food diet with sedentary lifestyle or exercise, compared with control diet with and without exercise. Our analyses reveal that genome-wide differential DNA methylation and expression of gene clusters are induced by diet and/or exercise. A combination of fast food and exercise triggers extensive gene alterations, with enrichment of carbohydrate/lipid metabolic pathways and muscle developmental processes. Through evaluation of putative protective effects of exercise on diet-induced DNA methylation, we show that hypermethylation is effectively prevented, especially at promoters and enhancers, whereas hypomethylation is only partially attenuated. We assessed diet-induced DNA methylation changes associated with liver cancer-related epigenetic modifications and identified significant increases at liver-specific enhancers in fast food groups, suggesting partial loss of liver cell identity. Hypermethylation at a subset of gene promoters was associated with inhibition of tissue development and promotion of carcinogenic processes. Our study demonstrates extensive reprogramming of the epigenome by diet and exercise, emphasizing the functional relevance of epigenetic mechanisms as an interface between lifestyle modifications and phenotypic alterations.

High-risk neuroblastoma remains one of the deadliest childhood cancers. Identification of metabolic pathways that drive or maintain high-risk neuroblastoma may open new avenues of therapeutic interventions. Here, we report the isolation and propagation of neuroblastoma sphere-forming cells with self-renewal and differentiation potential from tumors of the TH-MYCN mouse, an animal model of high-risk neuroblastoma with MYCN amplification. Transcriptional profiling reveals that mouse neuroblastoma sphere-forming cells acquire a metabolic program characterized by transcriptional activation of the cholesterol and serine-glycine synthesis pathways, primarily as a result of increased expression of sterol regulatory element binding factors and Atf4, respectively. This metabolic reprogramming is recapitulated in high-risk human neuroblastomas and is prognostic for poor clinical outcome. Genetic and pharmacological inhibition of the metabolic program markedly decreases the growth and tumorigenicity of both mouse neuroblastoma sphere-forming cells and human neuroblastoma cell lines. These findings suggest a therapeutic strategy for targeting the metabolic program of high-risk neuroblastoma.

The histone lysine demethylase KDM4C is often overexpressed in cancers primarily through gene amplification. The molecular mechanisms of KDM4C action in tumorigenesis are not well defined. Here, we report that KDM4C transcriptionally activates amino acid biosynthesis and transport, leading to a significant increase in intracellular amino acid levels. Examination of the serine-glycine synthesis pathway reveals that KDM4C epigenetically activates the pathway genes under steady-state and serine deprivation conditions by removing the repressive histone modification H3 lysine 9 (H3K9) trimethylation. This action of KDM4C requires ATF4, a transcriptional master regulator of amino acid metabolism and stress responses. KDM4C activates ATF4 transcription and interacts with ATF4 to target serine pathway genes for transcriptional activation. We further present evidence for KDM4C in transcriptional coordination of amino acid metabolism and cell proliferation. These findings suggest a molecular mechanism linking KDM4C-mediated H3K9 demethylation and ATF4-mediated transactivation in reprogramming amino acid metabolism for cancer cell proliferation.

The complete mitochondrial genome of sand-hopper Trinorchestia longiramus was analyzed in this study, which is the first for the genus within the family Talitridae. The mitogenome sequence is 15,401 bp in length containing two ribosomal RNA genes, 22 transfer RNA genes, 13 protein-coding genes, and a control region as found in most amphipods. The gene order showed that T. longiramus has a unique control region location compared to other amphipods. Phylogenetic analysis using the maximum likelihood method positioned T. longiramus within the monophyletic clades of the family Talitridae.

Hydroxyurea (HU), the first of two drugs approved by the US Food and Drug Administration for treating patients with sickle cell disease (SCD), produces anti-sickling effect by re-activating fetal γ-globin gene to enhance production of fetal hemoglobin. However, approximately 30% of the patients do not respond to HU therapy. The molecular basis of non-responsiveness to HU is not clearly understood. To address this question, we examined HU-induced changes in the RNA and protein levels of transcription factors NF-Y, GATA-1, -2, BCL11A, TR4, MYB and NF-E4 that assemble the γ-globin promoter complex and regulate transcription of γ-globin gene. In erythroblasts cultured from peripheral blood CD34+ cells of patients with SCD, we found that HU-induced changes in the protein but not the RNA levels of activator GATA-2 and repressors GATA-1, BCL11A and TR4 correlated with HU-induced changes in fetal hemoglobin (HbF) levels in the peripheral blood of HU high and low responders. However, HU did not significantly induce changes in the protein or RNA levels of activators NF-Y and NF-E4. Based on HU-induced changes in the protein levels of GATA-2, -1 and BCL11A, we calculated an Index of Hydroxyurea Responsiveness (IndexHU-3). Compared to the HU-induced fold changes in the individual transcription factor protein levels, the numerical values of IndexHU-3 statistically correlated best with the HU-induced peripheral blood HbF levels of the patients. Thus, IndexHU-3 can serve as an appropriate indicator for inherent HU responsiveness of patients with SCD.

Global patterns of DNA methylation, mediated by the DNA methyltransferases (DNMTs), are disrupted in all cancers by mechanisms that remain largely unknown, hampering their development as therapeutic targets. Combinatorial acute depletion of all DNMTs in a pluripotent human tumor cell line, followed by epigenome and transcriptome analysis, revealed DNMT functions in fine detail. DNMT3B occupancy regulates methylation during differentiation, whereas an unexpected interplay was discovered in which DNMT1 and DNMT3B antithetically regulate methylation and hydroxymethylation in gene bodies, a finding confirmed in other cell types. DNMT3B mediated non-CpG methylation, whereas DNMT3L influenced the activity of DNMT3B toward non-CpG versus CpG site methylation. Altogether, these data reveal functional targets of each DNMT, suggesting that isoform selective inhibition would be therapeutically advantageous.

DNA methylation, mediated by the combined action of three DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), is essential for mammalian development and is a major contributor to cellular transformation. To elucidate how DNA methylation is targeted, we mapped the genome-wide localization of all DNMTs and methylation, and examined the relationships among these markers, histone modifications, and nucleosome structure in a pluripotent human tumor cell line in its undifferentiated and differentiated states. Our findings reveal a strong link between DNMTs and transcribed loci, and that DNA methylation is not a simple sum of DNMT localization patterns. A comparison of the epigenomes of normal and cancerous stem cells, and pluripotent and differentiated states shows that the presence of at least two DNMTs is strongly associated with loci targeted for DNA hypermethylation. Taken together, these results shed important light on the determinants of DNA methylation and how it may become disrupted in cancer cells.

Induction of differentiation is a therapeutic strategy in neuroblastoma, a common pediatric cancer of the sympathetic nervous system. The homeobox protein HOXC9 is a key regulator of neuroblastoma differentiation. To gain a molecular understanding of the function of HOXC9 in promoting differentiation of neuroblastoma cells, we conducted a genome-wide analysis of the HOXC9-induced differentiation program by microarray gene expression profiling and chromatin immunoprecipitation in combination with massively parallel sequencing (ChIP-seq). Here we describe in detail the experimental system, methods, and quality control for the generation of the microarray and ChIP-seq data associated with our recent publication [1].

Despite sharing much of their genomes, males and females are often highly dimorphic, reflecting at least in part the resolution of sexual conflict in response to sexually antagonistic selection. Sexual dimorphism arises owing to sex differences in gene expression, and steroid hormones are often invoked as a proximate cause of sexual dimorphism. Experimental elevation of androgens can modify behavior, physiology, and gene expression, but knowledge of the role of hormones remains incomplete, including how the sexes differ in gene expression in response to hormones. We addressed these questions in a bird species with a long history of behavioral endocrinological and ecological study, the dark-eyed junco (Junco hyemalis), using a custom microarray. Focusing on two brain regions involved in sexually dimorphic behavior and regulation of hormone secretion, we identified 651 genes that differed in expression by sex in medial amygdala and 611 in hypothalamus. Additionally, we treated individuals of each sex with testosterone implants and identified many genes that may be related to previously identified phenotypic effects of testosterone treatment. Some of these genes relate to previously identified effects of testosterone-treatment and suggest that the multiple effects of testosterone may be mediated by modifying the expression of a small number of genes. Notably, testosterone-treatment tended to alter expression of different genes in each sex: only 4 of the 527 genes identified as significant in one sex or the other were significantly differentially expressed in both sexes. Hormonally regulated gene expression is a key mechanism underlying sexual dimorphism, and our study identifies specific genes that may mediate some of these processes.

The parasitoid wasp Nasonia vitripennis is an emerging genetic model for functional analysis of DNA methylation. Here, we characterize genome-wide methylation at a base-pair resolution, and compare these results to gene expression across five developmental stages and to methylation patterns reported in other insects. An accurate assessment of DNA methylation across the genome is accomplished using bisulfite sequencing of adult females from a highly inbred line. One-third of genes show extensive methylation over the gene body, yet methylated DNA is not found in non-coding regions and rarely in transposons. Methylated genes occur in small clusters across the genome. Methylation demarcates exon-intron boundaries, with elevated levels over exons, primarily in the 5' regions of genes. It is also elevated near the sites of translational initiation and termination, with reduced levels in 5' and 3' UTRs. Methylated genes have higher median expression levels and lower expression variation across development stages than non-methylated genes. There is no difference in frequency of differential splicing between methylated and non-methylated genes, and as yet no established role for methylation in regulating alternative splicing in Nasonia. Phylogenetic comparisons indicate that many genes maintain methylation status across long evolutionary time scales. Nasonia methylated genes are more likely to be conserved in insects, but even those that are not conserved show broader expression across development than comparable non-methylated genes. Finally, examination of duplicated genes shows that those paralogs that have lost methylation in the Nasonia lineage following gene duplication evolve more rapidly, show decreased median expression levels, and increased specialization in expression across development. Methylation of Nasonia genes signals constitutive transcription across developmental stages, whereas non-methylated genes show more dynamic developmental expression patterns. We speculate that loss of methylation may result in increased developmental specialization in evolution and acquisition of methylation may lead to broader constitutive expression.

The microsporidium Nosema ceranae is a newly prevalent parasite of the European honey bee (Apis mellifera). Although this parasite is presently spreading across the world into its novel host, the mechanisms by it which affects the bees and how bees respond are not well understood. We therefore performed an extensive characterization of the parasite effects at the molecular level by using genetic and biochemical tools. The transcriptome modifications at the midgut level were characterized seven days post-infection with tiling microarrays. Then we tested the bee midgut response to infection by measuring activity of antioxidant and detoxification enzymes (superoxide dismutases, glutathione peroxidases, glutathione reductase, and glutathione-S-transferase). At the gene-expression level, the bee midgut responded to N. ceranae infection by an increase in oxidative stress concurrent with the generation of antioxidant enzymes, defense and protective response specifically observed in the gut of mammals and insects. However, at the enzymatic level, the protective response was not confirmed, with only glutathione-S-transferase exhibiting a higher activity in infected bees. The oxidative stress was associated with a higher transcription of sugar transporter in the gut. Finally, a dramatic effect of the microsporidia infection was the inhibition of genes involved in the homeostasis and renewal of intestinal tissues (Wnt signaling pathway), a phenomenon that was confirmed at the histological level. This tissue degeneration and prevention of gut epithelium renewal may explain early bee death. In conclusion, our integrated approach not only gives new insights into the pathological effects of N. ceranae and the bee gut response, but also demonstrate that the honey bee gut is an interesting model system for studying host defense responses.

Follicular lymphoma (FL) is a form of non-Hodgkin's lymphoma (NHL) that arises from germinal center (GC) B-cells. Despite the significant advances in immunotherapy, FL is still not curable. Beyond transcriptional profiling and genomics datasets, there currently is no epigenome-scale dataset or integrative biology approach that can adequately model this disease and therefore identify novel mechanisms and targets for successful prevention and treatment of FL.We performed methylation-enriched genome-wide bisulfite sequencing of FL cells and normal CD19(+) B-cells using 454 sequencing technology. The methylated DNA fragments were enriched with methyl-binding proteins, treated with bisulfite, and sequenced using the Roche-454 GS FLX sequencer. The total number of bases covered in the human genome was 18.2 and 49.3 million including 726,003 and 1.3 million CpGs in FL and CD19(+) B-cells, respectively. 11,971 and 7,882 methylated regions of interest (MRIs) were identified respectively. The genome-wide distribution of these MRIs displayed significant differences between FL and normal B-cells. A reverse trend in the distribution of MRIs between the promoter and the gene body was observed in FL and CD19(+) B-cells. The MRIs identified in FL cells also correlated well with transcriptomic data and ChIP-on-Chip analyses of genome-wide histone modifications such as tri-methyl-H3K27, and tri-methyl-H3K4, indicating a concerted epigenetic alteration in FL cells.This study is the first to provide a large scale and comprehensive analysis of the DNA methylation sequence composition and distribution in the FL epigenome. These integrated approaches have led to the discovery of novel and frequent targets of aberrant epigenetic alterations. The genome-wide bisulfite sequencing approach developed here can be a useful tool for profiling DNA methylation in clinical samples.

Motivation: Chromatin accessibility plays a key role in epigenetic regulation of gene activation and silencing. Open chromatin regions allow regulatory elements such as transcription factors and polymerases to bind for gene expression while closed chromatin regions prevent the activity of transcriptional machinery. Recently, Methyltransferase Accessibility Protocol for individual templates-Bisulfite Genome Sequencing (MAPit-BGS) and nucleosome occupancy and methylome sequencing (NOMe-seq) have been developed for simultaneously profiling chromatin accessibility and DNA methylation on single molecules. Therefore, there is a great demand in developing computational methods to identify chromatin accessibility from MAPit-BGS and NOMe-seq. Results: In this article, we present CAME (Chromatin Accessibility and Methylation), a seedextension based approach that identifies chromatin accessibility from NOMe-seq. The efficiency and effectiveness of CAME were demonstrated through comparisons with other existing techniques on both simulated and real data, and the results show that our method not only can precisely identify chromatin accessibility but also outperforms other methods. [ABSTRACT FROM AUTHOR]

Background: Nasonia vitripennis is an emerging insect model system with haplodiploid genetics. It holds a key position within the insect phylogeny for comparative, evolutionary and behavioral genetic studies. The draft genomes for N. vitripennis and two sibling species were published in 2010, yet a considerable amount of transcriptiome data have since been produced thereby enabling improvements to the original (OGS1.2) annotated gene set. We describe and apply the EvidentialGene method used to produce an updated gene set (OGS2). We also carry out comparative analyses showcasing the usefulness of the revised annotated gene set. Results: The revised annotation (OGS2) now consists of 24,388 genes with supporting evidence, compared to 18,850 for OGS1.2. Improvements include the nearly complete annotation of untranslated regions (UTR) for 97 % of the genes compared to 28 % of genes for OGS1.2. The fraction of RNA-Seq validated introns also grow from 85 to 98 % in this latest gene set. The EST and RNA-Seq expression data provide support for several non-protein coding loci and 7712 alternative transcripts for 4146 genes. Notably, we report 180 alternative transcripts for the gene lola. Nasonia now has among the most complete insect gene set; only 27 conserved single copy orthologs in arthropods are missing from OGS2. Its genome also contains 2.1-fold more duplicated genes and 1.4-fold more single copy genes than the Drosophila melanogaster genome. The Nasonia gene count is larger than those of other sequenced hymenopteran species, owing both to improvements in the genome annotation and to unique genes in the wasp lineage. We identify 1008 genes and 171 gene families that deviate significantly from other hymenopterans in their rates of protein evolution and duplication history, respectively. We also provide an analysis of alternative splicing that reveals that genes with no annotated isoforms are characterized by shorter transcripts, fewer introns, faster protein evolution and higher probabilities of duplication than genes having alternative transcripts. Conclusions: Genome-wide expression data greatly improves the annotation of the N. vitripennis genome, by increasing the gene count, reducing the number of missing genes and providing more comprehensive data on splicing and gene structure. The improved gene set identifies lineage-specific genomic features tied to Nasonia's biology, as well as numerous novel genes. [ABSTRACT FROM AUTHOR]

Background: Internal circadian (circa, about; dies, day) clocks enable organisms to maintain adaptive timing of their daily behavioral activities and physiological functions. Eukaryotic clocks consist of core transcription-translation feedback loops that generate a cycle and post-translational modifiers that maintain that cycle at about 24 h. We use the pitcher-plant mosquito, Wyeomyia smithii (subfamily Culicini, tribe Sabethini), to test whether evolutionary divergence of the circadian clock genes in this species, relative to other insects, has involved primarily genes in the core feedback loops or the post-translational modifiers. Heretofore, there is no reference transcriptome or genome sequence for any mosquito in the tribe Sabethini, which includes over 375 mainly circumtropical species. Methods: We sequenced, assembled and annotated the transcriptome of W. smithii containing nearly 95 % of conserved single-copy orthologs in animal genomes. We used the translated contigs and singletons to determine the average rates of circadian clock-gene divergence in W. smithii relative to three other mosquito genera, to Drosophila, to the butterfly, Danaus, and to the wasp, Nasonia. Results: Over 1.08 million cDNA sequence reads were obtained consisting of 432.5 million nucleotides. Their assembly produced 25,904 contigs and 54,418 singletons of which 62 % and 28 % are annotated as protein-coding genes, respectively, sharing homology with other animal proteomes. Discussion: The W. smithii transcriptome includes all nine circadian transcription-translation feedback-loop genes and all eight post-translational modifier genes we sought to identify (Fig. 1). After aligning translated W. smithii contigs and singletons from this transcriptome with other insects, we determined that there was no significant difference in the average divergence of W. smithii from the six other taxa between the core feedback-loop genes and post-translational modifiers. Conclusions: The characterized transcriptome is sufficiently complete and of sufficient quality to have uncovered all of the insect circadian clock genes we sought to identify (Fig. 1). Relative divergence does not differ between core feedback-loop genes and post-translational modifiers of those genes in a Sabethine species (W. smithii) that has experienced a continual northward dispersal into temperate regions of progressively longer summer day lengths as compared with six other insect taxa. An associated microarray platform derived from this work will enable the investigation of functional genomics of circadian rhythmicity, photoperiodic time measurement, and diapause along a photic and seasonal geographic gradient. [ABSTRACT FROM AUTHOR]

Motivation: Tag density plots are very important to intuitively reveal biological phenomena from capture-based sequencing data by visualizing the normalized read depth in a region. Results: We have developed iTagPlot to compute tag density across functional features in parallel using multicores and a grid engine and to interactively explore it in a graphical user interface. It allows us to stratify features by defining groups based on biological function and measurement, summary statistics and unsupervised clustering. [ABSTRACT FROM AUTHOR]

Background Although Daphnia is increasingly recognized as a model for ecological genomics and biomedical research, there is, as of yet, no high-resolution genetic map for the genus. Such a map would provide an important tool for mapping phenotypes and assembling the genome. Here we estimate the genome size of Daphnia magna and describe the construction of an SNP array based linkage map. We then test the suitability of the map for life history and behavioural trait mapping. The two parent genotypes used to produce the map derived from D. magna populations with and without fish predation, respectively and are therefore expected to show divergent behaviour and life-histories. Results Using flow cytometry we estimated the genome size of D. magna to be about 238 mb. We developed an SNP array tailored to type SNPs in a D. magna F2 panel and used it to construct a D. magna linkage map, which included 1,324 informative markers. The map produced ten linkage groups ranging from 108.9 to 203.6 cM, with an average distance between markers of 1.13 cM and a total map length of 1,483.6 cM (Kosambi corrected). The physical length per cM is estimated to be 160 kb. Mapping infertility genes, life history traits and behavioural traits on this map revealed several significant QTL peaks and showed a complex pattern of underlying genetics, with different traits showing strongly different genetic architectures. Conclusions The new linkage map of D. magna constructed here allowed us to characterize genetic differences among parent genotypes from populations with ecological differences. The QTL effect plots are partially consistent with our expectation of local adaptation under contrasting predation regimes. Furthermore, the new genetic map will be an important tool for the Daphnia research community and will contribute to the physical map of the D. magna genome project and the further mapping of phenotypic traits. The clones used to produce the linkage map are maintained in a stock collection and can be used for mapping QTLs of traits that show variance among the F2 clones. [ABSTRACT FROM AUTHOR]

Background Cellular differentiation is characterized by the acquisition of specialized structures and functions, cell cycle exit, and global attenuation of the DNA damage response. It is largely unknown how these diverse cellular events are coordinated at the molecular level during differentiation. We addressed this question in a model system of neuroblastoma cell differentiation induced by HOXC9. Results We conducted a genome-wide analysis of the HOXC9-induced neuronal differentiation program. Microarray gene expression profiling revealed that HOXC9-induced differentiation was associated with transcriptional regulation of 2,370 genes, characterized by global upregulation of neuronal genes and downregulation of cell cycle and DNA repair genes. Remarkably, genome-wide mapping by ChIP-seq demonstrated that HOXC9 bound to 40% of these genes, including a large number of genes involved in neuronal differentiation, cell cycle progression and the DNA damage response. Moreover, we showed that HOXC9 interacted with the transcriptional repressor E2F6 and recruited it to the promoters of cell cycle genes for repressing their expression. Conclusions Our results demonstrate that HOXC9 coordinates diverse cellular processes associated with differentiation by directly activating and repressing the transcription of distinct sets of genes. [ABSTRACT FROM AUTHOR]

Background: With the advent of next-generation sequencing (NGS) technologies, full cDNA shotgun sequencing has become a major approach in the study of transcriptomes, and several different protocols in 454 sequencing have been invented. As each protocol uses its own short DNA tags or adapters attached to the ends of cDNA fragments for labeling or sequencing, different contaminants may lead to mis-assembly and inaccurate sequence products. Results: We have designed and implemented a new program for raw sequence cleaning in a graphical user interface and a batch script. The cleaning process consists of several modules including barcode trimming, sequencing adapter trimming, amplification primer trimming, poly-A tail trimming, vector screening and low quality region trimming. These modules can be combined based on various sequencing applications. Conclusions: ESTclean is a software package not only for cleaning cDNA sequences, but also for helping to develop sequencing protocols by providing summary tables and figures for sequencing quality control in a graphical user interface. It outperforms in cleaning read sequences from complicated sequencing protocols which use barcodes and multiple amplification primers. [ABSTRACT FROM AUTHOR]

The microsporidium Nosema ceranae is a newly prevalent parasite of the European honey bee (Apis mellifera). Although this parasite is presently spreading across the world into its novel host, the mechanisms by it which affects the bees and how bees respond are not well understood. We therefore performed an extensive characterization of the parasite effects at the molecular level by using genetic and biochemical tools. The transcriptome modifications at the midgut level were characterized seven days post-infection with tiling microarrays. Then we tested the bee midgut response to infection by measuring activity of antioxidant and detoxification enzymes (superoxide dismutases, glutathione peroxidases, glutathione reductase, and glutathione-S-transferase). At the gene-expression level, the bee midgut responded to N. ceranae infection by an increase in oxidative stress concurrent with the generation of antioxidant enzymes, defense and protective response specifically observed in the gut of mammals and insects. However, at the enzymatic level, the protective response was not confirmed, with only glutathione-S-transferase exhibiting a higher activity in infected bees. The oxidative stress was associated with a higher transcription of sugar transporter in the gut. Finally, a dramatic effect of the microsporidia infection was the inhibition of genes involved in the homeostasis and renewal of intestinal tissues (Wnt signaling pathway), a phenomenon that was confirmed at the histological level. This tissue degeneration and prevention of gut epithelium renewal may explain early bee death. In conclusion, our integrated approach not only gives new insights into the pathological effects of N. ceranae and the bee gut response, but also demonstrate that the honey bee gut is an interesting model system for studying host defense responses. [ABSTRACT FROM AUTHOR]

Background: Though genomic-level data are becoming widely available, many of the metazoan species sequenced are laboratory systems whose natural history is not well documented. In contrast, the wide array of species with very well-characterized natural history have, until recently, lacked genomics tools. It is now possible to address significant evolutionary genomics questions by applying high-throughput sequencing to discover the majority of genes for ecologically tractable species, and by subsequently developing microarray platforms from which to investigate gene regulatory networks that function in natural systems. We used GS-FLX Titanium Sequencing (Roche/454-Sequencing) of two normalized libraries of pooled RNA samples to characterize a transcriptome of the dark-eyed junco (Junco hyemalis), a North American sparrow that is a classically studied species in the fields of photoperiodism, speciation, and hormone-mediated behavior. Results: From a broad pool of RNA sampled from tissues throughout the body of a male and a female junco, we sequenced a total of 434 million nucleotides from 1.17 million reads that were assembled de novo into 31,379 putative transcripts representing 22,765 gene sets covering 35.8 million nucleotides with 12-fold average depth of coverage. Annotation of roughly half of the putative genes was accomplished using sequence similarity, and expression was confirmed for the majority with a preliminary microarray analysis. Of 716 core bilaterian genes, 646 (90 %) were recovered within our characterized gene set. Gene Ontology, orthoDB orthology groups, and KEGG Pathway annotation provide further functional information about the sequences, and 25,781 potential SNPs were identified. Conclusions: The extensive sequence information returned by this effort adds to the growing store of genomic data on diverse species. The extent of coverage and annotation achieved and confirmation of expression, show that transcriptome sequencing provides useful information for ecological model systems that have historically lacked genomic tools. The junco-specific microarray developed here is allowing investigations of gene expression responses to environmental and hormonal manipulations - extending the historic work on natural history and hormonemediated phenotypes in this system. [ABSTRACT FROM AUTHOR]