Sex-specific chromatin landscapes in an ultra-compact chordate genome.

BACKGROUND: In multicellular organisms, epigenome dynamics are associated with transitions in the cell cycle, development, germline specification, gametogenesis and inheritance. Evolutionarily, regulatory space has increased in complex metazoans to accommodate these functions. In tunicates, the sister lineage to vertebrates, we examine epigenome adaptations to strong secondary genome compaction, sex chromosome evolution and cell cycle modes. RESULTS: Across the 70 MB Oikopleura dioica genome, we profiled 19 histone modifications, and RNA polymerase II, CTCF and p300 occupancies, to define chromatin states within two homogeneous tissues with distinct cell cycle modes: ovarian endocycling nurse nuclei and mitotically proliferating germ nuclei in testes. Nurse nuclei had active chromatin states similar to other metazoan epigenomes, with large domains of operon-associated transcription, a general lack of heterochromatin, and a possible role of Polycomb PRC2 in dosage compensation. Testis chromatin states reflected transcriptional activity linked to spermatogenesis and epigenetic marks that have been associated with establishment of transgenerational inheritance in other organisms. We also uncovered an unusual chromatin state specific to the Y-chromosome, which combined active and heterochromatic histone modifications on specific transposable elements classes, perhaps involved in regulating their activity. CONCLUSIONS: Compacted regulatory space in this tunicate genome is accompanied by reduced heterochromatin and chromatin state domain widths. Enhancers, promoters and protein-coding genes have conserved epigenomic features, with adaptations to the organization of a proportion of genes in operon units. We further identified features specific to sex chromosomes, cell cycle modes, germline identity and dosage compensation, and unusual combinations of histone PTMs with opposing consensus functions.

ESCRT-II/Vps25 constrains digit number by endosome-mediated selective modulation of FGF-SHH signaling.

Sorting and degradation of receptors and associated signaling molecules maintain homeostasis of conserved signaling pathways during cell specification and tissue development. Yet, whether machineries that sort signaling proteins act preferentially on different receptors and ligands in different contexts remains mysterious. Here, we show that Vacuolar protein sorting 25, Vps25, a component of ESCRT-II (Endosomal Sorting Complex Required for Transport II), directs preferential endosome-mediated modulation of FGF signaling in limbs. By ENU-induced mutagenesis, we isolated a polydactylous mouse line carrying a hypomorphic mutation of Vps25 (Vps25(ENU)). Unlike Vps25-null embryos we generated, Vps25(ENU/ENU) mutants survive until late gestation. Their limbs display FGF signaling enhancement and consequent hyperactivation of the FGF-SHH feedback loop causing polydactyly, whereas WNT and BMP signaling remain unperturbed. Notably, Vps25(ENU/ENU) Mouse Embryonic Fibroblasts exhibit aberrant FGFR trafficking and degradation; however, SHH signaling is unperturbed. These studies establish that the ESCRT-II machinery selectively limits FGF signaling in vertebrate skeletal patterning.

Interferon regulatory factor 6 promotes differentiation of the periderm by activating expression of Grainyhead-like 3.

IFN regulatory factor 6 (IRF6) is a transcription factor that, in mammals, is required for the differentiation of skin, breast epithelium, and oral epithelium. However, the transcriptional targets that mediate these effects are currently unknown. In zebrafish and frog embryos, Irf6 is necessary for differentiation of the embryonic superficial epithelium, or periderm. Here we use microarrays to identify genes that are expressed in the zebrafish periderm and whose expression is inhibited by a dominant-negative variant of Irf6 (dnIrf6). These methods identify Grainyhead-like 3 (Grhl3), an ancient regulator of the epidermal permeability barrier, as acting downstream of Irf6. In human keratinocytes, IRF6 binds conserved elements near the GRHL3 [corrected] promoter. We show that one of these elements has enhancer activity in human keratinocytes and zebrafish periderm, suggesting that Irf6 directly stimulates Grhl3 expression in these tissues. Simultaneous inhibition of grhl1 and grhl3 disrupts periderm differentiation in zebrafish, and, intriguingly, forced grhl3 expression restores periderm markers in both zebrafish injected with dnIrf6 and frog embryos depleted of Irf6. Finally, in Irf6-deficient mouse embryos, Grhl3 expression in the periderm and oral epithelium is virtually absent. These results indicate that Grhl3 is a key effector of Irf6 in periderm differentiation.

OikoBase: a genomics and developmental transcriptomics resource for the urochordate Oikopleura dioica.

We report the development of OikoBase (http://oikoarrays.biology.uiowa.edu/Oiko/), a tiling array-based genome browser resource for Oikopleura dioica, a metazoan belonging to the urochordates, the closest extant group to vertebrates. OikoBase facilitates retrieval and mining of a variety of useful genomics information. First, it includes a genome browser which interrogates 1260 genomic sequence scaffolds and features gene, transcript and CDS annotation tracks. Second, we annotated gene models with gene ontology (GO) terms and InterPro domains which are directly accessible in the browser with links to their entries in the GO (http://www.geneontology.org/) and InterPro (http://www.ebi.ac.uk/interpro/) databases, and we provide transcript and peptide links for sequence downloads. Third, we introduce the transcriptomics of a comprehensive set of developmental stages of O. dioica at high resolution and provide downloadable gene expression data for all developmental stages. Fourth, we incorporate a BLAST tool to identify homologs of genes and proteins. Finally, we include a tutorial that describes how to use OikoBase as well as a link to detailed methods, explaining the data generation and analysis pipeline. OikoBase will provide a valuable resource for research in chordate development, genome evolution and plasticity and the molecular ecology of this important marine planktonic organism.

Expression of human paraoxonase 1 decreases superoxide levels and alters bacterial colonization in the gut of Drosophila melanogaster.

Paraoxonases (PON) are a family of proteins (PON1, 2 and 3) with multiple enzymatic activities. PON1 interferes with homoserine lactone-mediated quorum sensing in bacteria and with reactive oxygen species (ROS) in humans and mice. PON1 gene mutations have been linked to multiple traits, including aging, and diseases of the cardiovascular, nervous and gastrointestinal system. The overlapping enzymatic activities in the PON family members and high linkage disequilibrium rates within their polymorphisms confound animal and human studies of PON1 function. In contrast, arthropods such as Drosophila melanogaster have no PON homologs, resulting in an ideal model to study interactions between PON genotype and host phenotypes. We hypothesized that expression of PON1 in D. melanogaster would alter ROS. We found that PON1 alters expression of multiple oxidative stress genes and decreases superoxide anion levels in normal and germ-free D. melanogaster. We also found differences in the composition of the gut microbiota, with a remarkable increase in levels of Lactobacillus plantarum and associated changes in expression of antimicrobial and cuticle-related genes. PON1 expression directly decreased superoxide anion levels and altered bacterial colonization of the gut and its gene expression profile, highlighting the complex nature of the interaction between host genotype and gut microbiota. We speculate that the interaction between some genotypes and human diseases may be mediated by the presence of certain gut bacteria that can induce specific immune responses in the gut and other host tissues.

Conservation and divergence of chemical defense system in the tunicate Oikopleura dioica revealed by genome wide response to two xenobiotics.

BACKGROUND: Animals have developed extensive mechanisms of response to xenobiotic chemical attacks. Although recent genome surveys have suggested a broad conservation of the chemical defensome across metazoans, global gene expression responses to xenobiotics have not been well investigated in most invertebrates. Here, we performed genome survey for key defensome genes in Oikopleura dioica genome, and explored genome-wide gene expression using high density tiling arrays with over 2 million probes, in response to two model xenobiotic chemicals - the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) the pharmaceutical compound Clofibrate (Clo). RESULTS: Oikopleura genome surveys for key genes of the chemical defensome suggested a reduced repertoire. Not more than 23 cytochrome P450 (CYP) genes could be identified, and neither CYP1 family genes nor their transcriptional activator AhR was detected. These two genes were present in deuterostome ancestors. As in vertebrates, the genotoxic compound BaP induced xenobiotic biotransformation and oxidative stress responsive genes. Notable exceptions were genes of the aryl hydrocarbon receptor (AhR) signaling pathway. Clo also affected the expression of many biotransformation genes and markedly repressed genes involved in energy metabolism and muscle contraction pathways. CONCLUSIONS: Oikopleura has the smallest number of CYP genes among sequenced animal genomes and lacks the AhR signaling pathway. However it appears to have basic xenobiotic inducible biotransformation genes such as a conserved genotoxic stress response gene set. Our genome survey and expression study does not support a role of AhR signaling pathway in the chemical defense of metazoans prior to the emergence of vertebrates.

Genomic and proteomic studies on the effects of the insect growth regulator diflubenzuron in the model beetle species Tribolium castaneum.

Several benzoylphenyl urea-derived insecticides such as diflubenzuron (DFB, Dimilin) are in wide use to control various insect pests. Although this class of compounds is known to disrupt molting and to affect chitin content, their precise mode of action is still not understood. To gain a broader insight into the mechanism underlying the insecticidal effects of benzoylphenyl urea compounds, we conducted a comprehensive study with the model beetle species and stored product pest Tribolium castaneum (red flour beetle) utilizing genomic and proteomic approaches. DFB was added to a wheat flour-based diet at various concentrations and fed to larvae and adults. We observed abortive molting, hatching defects and reduced chitin amounts in the larval cuticle, the peritrophic matrix and eggs. Electron microscopic examination of the larval cuticle revealed major structural changes and a loss of lamellate structure of the procuticle. We used a genomic tiling array for determining relative expression levels of about 11,000 genes predicted by the GLEAN algorithm. About 6% of all predicted genes were more than 2-fold up- or down-regulated in response to DFB treatment. Genes encoding enzymes involved in chitin metabolism were unexpectedly unaffected, but many genes encoding cuticle proteins were affected. In addition, several genes presumably involved in detoxification pathways were up-regulated. Comparative 2D gel electrophoresis of proteins extracted from the midgut revealed 388 protein spots, of which 7% were significantly affected in their levels by DFB treatment as determined by laser densitometry. Mass spectrometric identification revealed that UDP-N-acetylglucosamine pyrophosphorylase and glutathione synthetase were up-regulated. In summary, the red flour beetle turned out to be a good model organism for investigating the global effects of bioactive materials such as insect growth regulators and other insecticides. The results of this study recapitulate all of the different DFB-induced symptoms in a single model insect, which have been previously found in several different insect species, and further illustrate that DFB treatment causes a wide range of effects at the molecular level.

Temporal coordination of gene networks by Zelda in the early Drosophila embryo.

In past years, much attention has focused on the gene networks that regulate early developmental processes, but less attention has been paid to how multiple networks and processes are temporally coordinated. Recently the discovery of the transcriptional activator Zelda (Zld), which binds to CAGGTAG and related sequences present in the enhancers of many early-activated genes in Drosophila, hinted at a mechanism for how batteries of genes could be simultaneously activated. Here we use genome-wide binding and expression assays to identify Zld target genes in the early embryo with the goal of unraveling the gene circuitry regulated by Zld. We found that Zld binds to genes involved in early developmental processes such as cellularization, sex determination, neurogenesis, and pattern formation. In the absence of Zld, many target genes failed to be activated, while others, particularly the patterning genes, exhibited delayed transcriptional activation, some of which also showed weak and/or sporadic expression. These effects disrupted the normal sequence of patterning-gene interactions and resulted in highly altered spatial expression patterns, demonstrating the significance of a timing mechanism in early development. In addition, we observed prevalent overlap between Zld-bound regions and genomic "hotspot" regions, which are bound by many developmental transcription factors, especially the patterning factors. This, along with the finding that the most over-represented motif in hotspots, CAGGTA, is the Zld binding site, implicates Zld in promoting hotspot formation. We propose that Zld promotes timely and robust transcriptional activation of early-gene networks so that developmental events are coordinated and cell fates are established properly in the cellular blastoderm embryo.

Plasticity of animal genome architecture unmasked by rapid evolution of a pelagic tunicate.

Genomes of animals as different as sponges and humans show conservation of global architecture. Here we show that multiple genomic features including transposon diversity, developmental gene repertoire, physical gene order, and intron-exon organization are shattered in the tunicate Oikopleura, belonging to the sister group of vertebrates and retaining chordate morphology. Ancestral architecture of animal genomes can be deeply modified and may therefore be largely nonadaptive. This rapidly evolving animal lineage thus offers unique perspectives on the level of genome plasticity. It also illuminates issues as fundamental as the mechanisms of intron gain.

BeetleBase in 2010: revisions to provide comprehensive genomic information for Tribolium castaneum.

BeetleBase (http://www.beetlebase.org) has been updated to provide more comprehensive genomic information for the red flour beetle Tribolium castaneum. The database contains genomic sequence scaffolds mapped to 10 linkage groups (genome assembly release Tcas_3.0), genetic linkage maps, the official gene set, Reference Sequences from NCBI (RefSeq), predicted gene models, ESTs and whole-genome tiling array data representing several developmental stages. The database was reconstructed using the upgraded Generic Model Organism Database (GMOD) modules. The genomic data is stored in a PostgreSQL relatational database using the Chado schema and visualized as tracks in GBrowse. The updated genetic map is visualized using the comparative genetic map viewer CMAP. To enhance the database search capabilities, the BLAST and BLAT search tools have been integrated with the GMOD tools. BeetleBase serves as a long-term repository for Tribolium genomic data, and is compatible with other model organism databases.

Dissociation of serum dehydroepiandrosterone and dehydroepiandrosterone sulfate in septic shock.

CONTEXT: Dehydroepiandrosterone (DHEA) replacement in sepsis has been advocated because of the sepsis-associated decrease in serum DHEA sulfate (DHEAS). However, experimental sepsis in rodents leads to down-regulation of DHEA sulfotransferase, which inactivates DHEA to DHEAS, theoretically resulting in higher DHEA levels. OBJECTIVE: The objective of the study was to test whether serum DHEA and DHEAS are dissociated in septic shock and to determine their association with circulating cortisol in the context of severity of disease and mortality. DESIGN, SETTING, AND PARTICIPANTS: This was a cross-sectional study consisting of 181 patients with septic shock, 31 patients with acute trauma, and 60 healthy controls. MAIN OUTCOME MEASURES: Serum cortisol, DHEA, and DHEAS were measured before and 60 min after ACTH stimulation. RESULTS: Serum cortisol was increased and DHEAS was decreased in both septic shock and trauma patients (all P < 0.001). However, compared with healthy controls, DHEA was significantly increased in sepsis but decreased after trauma (all P < 0.001). In sepsis, neither cortisol nor DHEA increased significantly after ACTH. Most severely ill patients had higher cortisol (P = 0.069) and lower DHEA (P = 0.076) and a significantly higher cortisol to DHEA ratio (P = 0.004). Similarly, the cortisol to DHEA ratio was significantly increased in nonsurvivors of septic shock (P = 0.026), whereas survivors did not differ from controls (P = 0.322). CONCLUSIONS: The observed dissociation of DHEA and DHEAS in septic shock contradicts the previous concept of sepsis-associated DHEA deficiency. Increased DHEA levels may maintain the balance between glucocorticoid- and DHEA-mediated immune and vascular effects. However, most severe disease and mortality is associated with an increased cortisol to DHEA ratio, which may represent a novel prognostic marker in septic shock.

Raised cortisol:DHEAS ratios in the elderly after injury: potential impact upon neutrophil function and immunity.

The detrimental effect of stress on the immune response increases with age, though the mechanisms responsible are not fully understood. The physiological response to stress is regulated in part by the adrenocortical system. Adrenal hormones dehydroepiandrosterone sulphate (DHEAS) and cortisol have opposing effects on the innate immune system, DHEAS enhances while cortisol suppresses immunity and the molar ratio of cortisol to DHEAS increases with age. We found that elderly hip fracture patients produced a robust neutrophilia after injury, but circulating neutrophils showed an impaired antibacterial response. We therefore proposed that adrenocortical hormones mediate the heightened immunosuppression seen in the elderly after injury. We examined neutrophil function and adrenocortical hormone levels in elderly (> 65 years) hip fracture patients and age-matched healthy controls. Thirteen out of 35 elderly patients acquired infections following hip fracture. Neutrophil superoxide production was lower in elderly hip fracture patients compared with controls (P < 0.005) and lower in patients who acquired infection following injury compared with those who did not (P < 0.05). Serum cortisol:DHEAS ratio was higher in elderly hip fracture patients (0.56 +/- 0.38) compared with either age-matched controls (0.36 +/- 0.21; P < 0.05) or young fracture patients (0.087 +/- 0.033; P < 0.0001). Moreover, cortisol: DHEAS was increased in elderly patients who succumbed to infection compared with those who did not (0.803 +/- 0.42 vs. 0.467 +/- 0.28; P < 0.02). In vitro cortisol significantly decreased neutrophil superoxide generation (P < 0.05) and this was prevented by coincubation with DHEAS. We propose that increased cortisol:DHEAS ratios may contribute to reduced immunity following physical stress in the elderly.

Stress responses and innate immunity: aging as a contributory factor.

Evolutionary pressure has selected individuals with traits that allow them to survive to reproduction, without consideration of the consequences for the post-child rearing years and old age. In the 21st century, society is populated increasingly by the elderly and with the falling birth rate and improved health care this trend is set to continue for the foreseeable future. To minimize the potential burden on health services one would hope that 'growing old gracefully' should also mean 'growing old healthily'. However, for too many the aging process is accompanied by increasing physical and mental frailty producing an elevated risk of physical and psychological stress in old age. Stress is a potent modulator of immune function, which in youth can be compensated for by the presence of an optimal immune response. In the elderly the immune response is blunted as a result of the decline in several components of the immune system (immune senescence) and a shifting to a chronic pro-inflammatory status (the so-called 'inflamm-aging' effect). We discuss here what is known of the effects of both stress and aging upon the innate immune system, focusing in particular upon the age-related alterations in the hypopituitary-adrenal axis. We propose a double hit model for age and stress in which the age-related increase in the cortisol/sulphated dehydroepiandrosterone ratio synergizes with elevated cortisol during stress to reduce immunity in the elderly significantly.