Candidate genes for monitoring hydrogen peroxide resistance in the salmon louse, Lepeophtheirus salmonis.

BACKGROUND: Hydrogen peroxide (H2O2) is one of the delousing agents used to control sea lice infestations in salmonid aquaculture. However, some Lepeophtheirus salmonis populations have developed resistance towards H2O2. An increased gene expression and activity of catalase, an enzyme that breaks down H2O2, have been detected in resistant lice, being therefore introduced as a resistance marker in the salmon industry. In the present study the aim was to validate the use of catalase expression as a marker and to identify new candidate genes as additional markers to catalase, related to H2O2 resistance in L. salmonis. METHODS: A sensitive and an H2O2 resistant laboratory strain (P0 generation, not exposed to H2O2 for several years) were batch crossed to generate a cohort with a wide range of H2O2 sensitivities (F2 generation). F2 adult females were then exposed to H2O2 to separate sensitive and resistant individuals. Those F2 lice, the P0 lice and field-collected resistant lice (exposed to H2O2 in the field) were used in an RNA sequencing study. RESULTS: Catalase was upregulated in resistant lice exposed to H2O2 compared to sensitive lice. This was, however, not the case for unexposed resistant P0 lice. Several other genes were found differentially expressed between sensitive and resistant lice, but most of them seemed to be related to H2O2 exposure. However, five genes were consistently up- or downregulated in the resistant lice independent of exposure history. The upregulated genes were: one gene in the DNA polymerase family, one gene encoding a Nesprin-like protein and an unannotated gene encoding a small protein. The downregulated genes encoded endoplasmic reticulum resident protein 29 and an aquaporin (Glp1_v2). CONCLUSIONS: Catalase expression seems to be induced by H2O2 exposure, since it was not upregulated in unexposed resistant lice. This may pose a challenge for its use as a resistance marker. The five new genes associated with resistance are put forward as complementary candidate genes. The most promising was Glp1_v2, an aquaglyceroporin that may serve as a passing channel for H2O2. Lower channel number can reduce the influx or distribution of H2O2 in the salmon louse, being directly involved in the resistance mechanism.

Epac1 null mice have nephrogenic diabetes insipidus with deficient corticopapillary osmotic gradient and weaker collecting duct tight junctions.

AIM: The cAMP-mediator Epac1 (RapGef3) has high renal expression. Preliminary observations revealed increased diuresis in Epac1-/- mice. We hypothesized that Epac1 could restrict diuresis by promoting transcellular collecting duct (CD) water and urea transport or by stabilizing CD paracellular junctions to reduce osmolyte loss from the renal papillary interstitium. METHODS: In Epac1-/- and Wt C57BL/6J mice, renal papillae, dissected from snap-frozen kidneys, were assayed for the content of key osmolytes. Cell junctions were analysed by transmission electron microscopy. Urea transport integrity was evaluated by urea loading with 40% protein diet, endogenous vasopressin production was manipulated by intragastric water loading and moderate dehydration and vasopressin type 2 receptors were stimulated selectively by i.p.-injected desmopressin (dDAVP). Glomerular filtration rate (GFR) was estimated as [14 C]inulin clearance. The glomerular filtration barrier was evaluated by urinary albumin excretion and microvascular leakage by the renal content of time-spaced intravenously injected 125 I- and 131 I-labelled albumin. RESULTS: Epac1-/- mice had increased diuresis and increased free water clearance under antidiuretic conditions. They had shorter and less dense CD tight junction (TJs) and attenuated corticomedullary osmotic gradient. Epac1-/- mice had no increased protein diet-induced urea-dependent osmotic diuresis, and expressed Wt levels of aquaporin-2 (AQP-2) and urea transporter A1/3 (UT-A1/3). Epac1-/- mice had no urinary albumin leakage and unaltered renal microvascular albumin extravasation. Their GFR was moderately increased, unless when treated with furosemide. CONCLUSION: Our results conform to the hypothesis that Epac1-dependent mechanisms protect against diabetes insipidus by maintaining renal papillary osmolarity and the integrity of CD TJs.

Mice depleted for Exchange Proteins Directly Activated by cAMP (Epac) exhibit irregular liver regeneration in response to partial hepatectomy.

The exchange proteins directly activated by cAMP 1 and 2 (Epac1 and Epac2) are expressed in a cell specific manner in the liver, but their biological functions in this tissue are poorly understood. The current study was undertaken to begin to determine the potential roles of Epac1 and Epac2 in liver physiology and disease. Male C57BL/6J mice in which expression of Epac1 and/or Epac2 are deleted, were subjected to partial hepatectomy and the regenerating liver was analyzed with regard to lipid accumulation, cell replication and protein expression. In response to partial hepatectomy, deletion of Epac1 and/or Epac2 led to increased hepatocyte proliferation 36 h post surgery, and the transient steatosis observed in wild type mice was virtually absent in mice lacking both Epac1 and Epac2. The expression of the protein cytochrome P4504a14, which is implicated in hepatic steatosis and fibrosis, was substantially reduced upon deletion of Epac1/2, while a number of factors involved in lipid metabolism were significantly decreased. Moreover, the number of Küpffer cells was affected, and Epac2 expression was increased in the liver of wild type mice in response to partial hepatectomy, further supporting a role for these proteins in liver function. This study establishes hepatic phenotypic abnormalities in mice deleted for Epac1/2 for the first time, and introduces Epac1/2 as regulators of hepatocyte proliferation and lipid accumulation in the regenerative process.

Fusion pore regulation by cAMP/Epac2 controls cargo release during insulin exocytosis.

Regulated exocytosis establishes a narrow fusion pore as initial aqueous connection to the extracellular space, through which small transmitter molecules such as ATP can exit. Co-release of polypeptides and hormones like insulin requires further expansion of the pore. There is evidence that pore expansion is regulated and can fail in diabetes and neurodegenerative disease. Here, we report that the cAMP-sensor Epac2 (Rap-GEF4) controls fusion pore behavior by acutely recruiting two pore-restricting proteins, amisyn and dynamin-1, to the exocytosis site in insulin-secreting beta-cells. cAMP elevation restricts and slows fusion pore expansion and peptide release, but not when Epac2 is inactivated pharmacologically or in Epac2-/- (Rapgef4-/-) mice. Consistently, overexpression of Epac2 impedes pore expansion. Widely used antidiabetic drugs (GLP-1 receptor agonists and sulfonylureas) activate this pathway and thereby paradoxically restrict hormone release. We conclude that Epac2/cAMP controls fusion pore expansion and thus the balance of hormone and transmitter release during insulin granule exocytosis.

Mutations in voltage-gated sodium channels from pyrethroid resistant salmon lice (Lepeophtheirus salmonis).

BACKGROUND: Parasitic salmon lice (Lepeophtheirus salmonis) cause high economic losses in Atlantic salmon farming. Pyrethroids, which block arthropod voltage-gated sodium channels (Nav 1), are used for salmon delousing. However, pyrethroid resistance is common in L. salmonis. The present study characterized Nav 1 homologues in L. salmonis in order to identify channel mutations associated to resistance, called kdr (knockdown) mutations. RESULTS: Genome scans identified three L. salmonis Nav 1 homologues, LsNav 1.1, LsNav 1.2 and LsNav 1.3. Arthropod kdr mutations map to specific Nav 1 regions within domains DI-III, namely segments S5 and S6 and the linker helix connecting S4 and S5. The above channel regions were amplified by RT-PCR and sequenced in deltamethrin-susceptible and deltamethrin-resistant L. salmonis. While LsNav 1.1 and LsNav 1.2 lacked nucleotide polymorphisms showing association to resistance, LsNav 1.3 showed a non-synonymous mutation in S5 of DII occurring in deltamethrin-resistant parasites. The mutation is homologous to a previously described kdr mutation (I936V, numbering according to Musca domestica Vssc1) and was present in two pyrethroid-resistant L. salmonis strains (allele frequencies of 0.800 and 0.357), but absent in two pyrethroid-susceptible strains. CONCLUSIONS: The present study indicates that a kdr-mutation in LsNaV 1.3 may contribute to deltamethrin resistance in L. salmonis. © 2018 Society of Chemical Industry.

Deletion of exchange proteins directly activated by cAMP (Epac) causes defects in hippocampal signaling in female mice.

Previous studies demonstrate essential roles for the exchange proteins directly activated by cAMP 1 and 2 (Epac1 and Epac2; here collectively referred to as Epac) in the brain. In the hippocampus, Epac contributes to the control of neuronal growth and differentiation and has been implicated in memory and learning as well as in anxiety and depression. In the present study we address the hypothesis that Epac affects hippocampal cellular responses to acute restraint stress. Stress causes activation of the hypothalamus-pituitary-adrenal (HPA)-axis, and glucocorticoid receptor (GR) signaling is essential for proper feedback regulation of the stress response, both in the brain and along the HPA axis. In the hippocampus, GR expression is regulated by cAMP and the brain enriched micro RNA miR-124. Epac has been associated with miR-124 expression in hippocampal neurons, but not in regulation of GR. We report that hippocampal expression of Epac1 and Epac2 increased in response to acute stress in female wild type mice. In female mice genetically deleted for Epac, nuclear translocation of GR in response to restraint stress was significantly delayed, and moreover, miR-124 expression was decreased in these mice. Male mice lacking Epac also showed abnormalities in miR-124 expression, but the phenotype was less profound than in females. Serum corticosterone levels were slightly altered immediately after stress in both male and female mice deleted for Epac. The presented data indicate that Epac1 and Epac2 are involved in controlling cellular responses to acute stress in the mouse hippocampus and provide novel insights into the underlying transcriptional and signaling networks. Interestingly, we observe sex specific differences when Epac is deleted. As the incidence and prevalence of stress-related diseases are higher in women than in men, the Epac knockout models might serve as genetic tools to further elucidate the cellular mechanisms underlying differences between male and female with regard to regulation of stress.

Deltamethrin resistance in the salmon louse, Lepeophtheirus salmonis (Krøyer): Maternal inheritance and reduced apoptosis.

Resistance towards deltamethrin (DMT) in the crustacean ectoparasite Lepeophtheirus salmonis (Caligidae) is a problem on fish farms lining the North Atlantic Ocean. Two Norwegian strains with different susceptibility towards DMT were crossed in the parental generation (P0), females from a sensitive strain were crossed with males from a resistant strain and vice versa. Individual susceptibility towards DMT was assessed in the second filial generation (F2). DMT resistance was only found in F2 descendants when the P0 females were from the resistant strain, pointing to maternal inheritance. Since maternal inheritance might be linked to the mitochondrial (mt) genome, the nucleotide sequences and the gene expressions of mt-genes were analysed. Twenty non-synonymous single nucleotide polymorphisms (SNPs) were identified in mt-transcripts from resistant F2 parasites, including SNPs in two cytochrome C oxidase subunits (COX1 and COX3) and two subunits of the NADH dehydrogenase complex (ND1 and ND5) previously linked to DMT resistance in the salmon louse. Differential expression analysis between the sensitive and resistant strain revealed strain effect in seven out of twelve mt-genes. The current study also show that DNA fragmentation (indicating apoptosis) was affected by DMT exposure in skeletal muscle tissue and that resistant parasites undergo less apoptosis than sensitive parasites.

Epac1-deficient mice have bleeding phenotype and thrombocytes with decreased GPIbß expression.

Epac1 (Exchange protein directly activated by cAMP 1) limits fluid loss from the circulation by tightening the endothelial barrier. We show here that Epac1-/- mice, but not Epac2-/- mice, have prolonged bleeding time, suggesting that Epac1 may limit fluid loss also by restraining bleeding. The Epac1-/- mice had deficient in vitro secondary hemostasis. Quantitative comprehensive proteomics analysis revealed that Epac1-/- mouse platelets (thrombocytes) had unbalanced expression of key components of the glycoprotein Ib-IX-V (GPIb-IX-V) complex, with decrease of GP1bß and no change of GP1bα. This complex is critical for platelet adhesion under arterial shear conditions. Furthermore, Epac1-/- mice have reduced levels of plasma coagulation factors and fibrinogen, increased size of circulating platelets, increased megakaryocytes (the GP1bß level was decreased also in Epac1-/- bone marrow) and higher abundance of reticulated platelets. Viscoelastic measurement of clotting function revealed Epac1-/- mice with a dysfunction in the clotting process, which corresponds to reduced plasma levels of coagulation factors like factor XIII and fibrinogen. We propose that the observed platelet phenotype is due to deficient Epac1 activity during megakaryopoiesis and thrombopoiesis, and that the defects in blood clotting for Epac1-/- is connected to secondary hemostasis.

A selection study on a laboratory-designed population of salmon lic (Lepeophtheirus salmonis) using organophosphate and pyrethroid pesticides.

Resistance towards antiparasitic agents in the salmon louse (Lepeophtheirus salmonis) is a widespread problem along the Norwegian coast, reducing treatments efficacies and slowing down the envisioned expansion of Norwegian salmon production. The present study was conducted in order to assess the efficacies of two of the most widely used anti-parasitic substances-azamethiphos and deltamethrin-as well as assessing the benefit of having a resistant genotype compared to being fully sensitive when exposed to one of these substances. Atlantic salmon were exposed to a mix of salmon lice copepodids from a fully sensitive, a double resistant and a multi-resistant strain. Once the lice reached pre-adult stages, one group was exposed to 100 µg/L azamethiphos for 60 minutes, the other to 2 µg/L deltamethrin for 30 minutes, and the last was kept in a seawater control. Detached lice were collected at a series of time points following exposure, and all lice (immobilized and surviving) were analysed for both pyrethroid (sensitive "S" and resistant "R") and azamethiphos (fully sensitive "SS", heterozygous resistant "RS" and fully resistant "RR") resistance markers. We found that the efficacies of deltamethrin on parasites with genotype S and R were 70.3 and 13.2%, respectively. The overall efficacy of the deltamethrin treatment was 32.3%. The efficacies of azamethiphos on parasites with genotype SS, RS and RR were 100, 80 and 19.1%, respectively. The overall efficacy of the azamethiphos treatment was 80.4%. Survival analyses revealed that the median survival time in deltamethrin-sensitive and-resistant parasites were 16.8 and >172 hours, respectively. The differences were even more pronounced in the azamethiphos-treated group, where SS, RS and RR parasites survived for 0.26, 6.6 and >172 hours, respectively. The substantial differences in survival between sensitive and resistant lice following treatment demonstrate the ability of medicinal treatments to drive genetic selection towards a much more resistant salmon lice population within a very short time span if there is no influx of sensitive genotypes.

Role of EPAC in cAMP-Mediated Actions in Adrenocortical Cells.

Adrenocorticotropic hormone regulates adrenal steroidogenesis mainly via the intracellular signaling molecule cAMP. The effects of cAMP are principally relayed by activating protein kinase A (PKA) and the more recently discovered exchange proteins directly activated by cAMP 1 and 2 (EPAC1 and EPAC2). While the intracellular roles of PKA have been extensively studied in steroidogenic tissues, those of EPACs are only emerging. EPAC1 and EPAC2 are encoded by the genes RAPGEF3 and RAPGEF4, respectively. Whereas EPAC1 is ubiquitously expressed, the expression of EPAC2 is more restricted, and typically found in endocrine tissues. Alternative promoter usage of RAPGEF4 gives rise to three different isoforms of EPAC2 that vary in their N-termini (EPAC2A, EPAC2B, and EPAC2C) and that exhibit distinct expression patterns. EPAC2A is expressed in the brain and pancreas, EPAC2B in steroidogenic cells of the adrenal gland and testis, and EPAC2C has until now only been found in the liver. In this review, we discuss current knowledge on EPAC expression and function with focus on the known roles of EPAC in adrenal gland physiology.

The effect of dietary lipid composition on the intestinal uptake and tissue distribution of benzo[a]pyrene and phenanthrene in Atlantic salmon (Salmo salar).

Uptake of polycyclic aromatic hydrocarbons (PAHs) across the intestine is suggested to occur in association with dietary lipids. Partial replacement of fish ingredients by vegetable ingredients in aquafeeds has led to increased levels of PAHs in marine farmed fish. We therefore investigated, intestinal uptake, tissue distribution and PAH metabolism after a single dose of (14)C-benzo[a]pyrene (BaP) or (14)C-phenanthrene (PHE) given to Atlantic salmon (Salmo salar) acclimatized to a fish oil or vegetable oil based diet. Both BaP and PHE were absorbed along the intestine. Fish oil based feed increased BaP concentration in the pyloric caeca and that of PHE in the proximal intestine. In contrast, vegetable oil increased BaP concentrations in the distal intestine. Extraction of whole body autoradiograms removed PHE-associated radiolabeling almost completely from the intestinal mucosa, but not BaP-associated radiolabeling, indicating the presence of BaP metabolites bound to cellular macromolecules. This observation correlates with the increased cyp1a expression in the proximal intestine, distal intestine and liver in the BaP exposed group. Furthermore, BaP-induced cyp1a expression was higher in the distal intestine of salmon fed fish oil compared to the vegetable oil fed group. PHE had no significant effect on cyp1a expression in any of these tissues. We conclude that dietary lipid composition affects intestinal PAH uptake. Fish oil based feed increased intestinal PAH concentrations probably due to an enhanced solubility in micelles composed of fish oil fatty acids. Increased BaP accumulation in the distal intestine of vegetable oil fed fish seems to be associated with a reduced Cyp1a-mediated BaP metabolism.

Identification and Molecular Characterization of Two Acetylcholinesterases from the Salmon Louse, Lepeophtheirus salmonis.

Acetylcholinesterase (AChE) is an important enzyme in cholinergic synapses. Most arthropods have two genes (ace1 and ace2), but only one encodes the predominant synaptic AChE, the main target for organophosphates. Resistance towards organophosphates is widespread in the marine arthropod Lepeophtheirus salmonis. To understand this trait, it is essential to characterize the gene(s) coding for AChE(s). The full length cDNA sequences encoding two AChEs in L. salmonis were molecularly characterized in this study. The two ace genes were highly similar (83.5% similarity at protein level). Alignment to the L. salmonis genome revealed that both genes were located close to each other (separated by just 26.4 kbp on the L. salmonis genome), resulting from a recent gene duplication. Both proteins had all the typical features of functional AChE and clustered together with AChE-type 1 proteins in other species, an observation that has not been described in other arthropods. We therefore concluded the presence of two versions of ace1 gene in L. salmonis, named ace1a and ace1b. Ace1a was predominantly expressed in different developmental stages compared to ace1b and was possibly active in the cephalothorax, indicating that ace1a is more likely to play the major role in cholinergic synaptic transmission. The study is essential to understand the role of AChEs in resistance against organophosphates in L. salmonis.

Peripheral Blood Cells from Patients with Autoimmune Addison's Disease Poorly Respond to Interferons In Vitro, Despite Elevated Serum Levels of Interferon-Inducible Chemokines.

Autoimmune Addison's disease (AAD) is a disorder caused by an immunological attack on the adrenal cortex. The interferon (IFN)-inducible chemokine CXCL10 is elevated in serum of AAD patients, suggesting a peripheral IFN signature. However, CXCL10 can also be induced in adrenocortical cells stimulated with IFNs, cytokines, or microbial components. We therefore investigated whether peripheral blood mononuclear cells (PBMCs) from AAD patients display an enhanced propensity to produce CXCL10 and the related chemokine CXCL9, after stimulation with type I or II IFNs or the IFN inducer poly (I:C). Although serum levels of CXCL10 and CXCL9 were significantly elevated in patients compared with controls, IFN stimulated patient PBMC produced significantly less CXCL10/CXCL9 than control PBMC. Low CXCL10 production was not significantly associated with medication, disease duration, or comorbidities, but the low production of poly (I:C)-induced CXCL10 among patients was associated with an AAD risk allele in the phosphatase nonreceptor type 22 (PTPN22) gene. PBMC levels of total STAT1 and -2, and IFN-induced phosphorylated STAT1 and -2, were not significantly different between patients and controls. We conclude that PBMC from patients with AAD are deficient in their response to IFNs, and that the adrenal cortex itself may be responsible for the increased serum levels of CXCL10.

Mechanism behind Resistance against the Organophosphate Azamethiphos in Salmon Lice (Lepeophtheirus salmonis).

Acetylcholinesterase (AChE) is the primary target for organophosphates (OP). Several mutations have been reported in AChE to be associated with the reduced sensitivity against OP in various arthropods. However, to the best of our knowledge, no such reports are available for Lepeophtheirus salmonis. Hence, in the present study, we aimed to determine the association of AChE(s) gene(s) with resistance against OP. We screened the AChE genes (L. salmonis ace1a and ace1b) in two salmon lice populations: one sensitive (n=5) and the other resistant (n=5) for azamethiphos, a commonly used OP in salmon farming. The screening led to the identification of a missense mutation Phe362Tyr in L. salmonis ace1a, (corresponding to Phe331 in Torpedo californica AChE) in all the samples of the resistant population. We confirmed the potential role of the mutation, with reduced sensitivity against azamethiphos in L. salmonis, by screening for Phe362Tyr in 2 sensitive and 5 resistant strains. The significantly higher frequency of the mutant allele (362Tyr) in the resistant strains clearly indicated the possible association of Phe362Tyr mutation in L. salmonis ace1a with resistance towards azamethiphos. The 3D modelling, short term survival experiments and enzymatic assays further supported the imperative role of Phe362Tyr in reduced sensitivity of L. salmonis for azamethiphos. Based on all these observations, the present study, for the first time, presents the mechanism of resistance in L. salmonis against azamethiphos. In addition, we developed a rapid diagnostic tool for the high throughput screening of Phe362Tyr mutation using High Resolution Melt analysis.

Drug resistance in sea lice: a threat to salmonid aquaculture.

Sea lice are copepod ectoparasites with vast reproductive potential and affect a wide variety of fish species. The number of parasites causing morbidity is proportional to fish size. Natural low host density restricts massive parasite dispersal. However, expanded salmon farming has shifted the conditions in favor of the parasite. Salmon farms are often situated near wild salmonid migrating routes, with smolts being particularly vulnerable to sea lice infestation. In order to protect both farmed and wild salmonids passing or residing in the proximity of the farms, several measures are taken. Medicinal treatment of farmed fish has been the most predictable and efficacious, leading to extensive use of the available compounds. This has resulted in drug-resistant parasites occurring on farmed and possibly wild salmonids.

Regulation of CDKN2B expression by interaction of Arnt with Miz-1--a basis for functional integration between the HIF and Myc gene regulatory pathways.

BACKGROUND: Hypoxia- and Myc-dependent transcriptional regulatory pathways are frequently deregulated in cancer cells. These pathways converge in many cellular responses, but the underlying molecular mechanisms are unclear. METHODS: The ability of Miz-1 and Arnt to interact was identified in a yeast two-hybrid screen. The mode of interaction and the functional consequences of complex formation were analyzed by diverse molecular biology methods, in vitro. Statistical analyses were performed by Student's t-test and ANOVA. RESULTS: In the present study we demonstrate that the aryl hydrocarbon receptor nuclear translocator (Arnt), which is central in hypoxia-induced signaling, forms a complex with Miz-1, an important transcriptional regulator in Myc-mediated transcriptional repression. Overexpression of Arnt induced reporter gene activity driven by the proximal promoter of the cyclin-dependent kinase inhibitor 2B gene (CDKN2B), which is an established target for the Myc/Miz-1 complex. In contrast, mutated forms of Arnt, that were unable to interact with Miz-1, had reduced capability to activate transcription. Moreover, repression of Arnt reduced endogenous CDKN2B expression, and chromatin immunoprecipitation demonstrated that Arnt interacts with the CDKN2B promoter. The transcriptional activity of Arnt was counteracted by Myc, but not by a mutated variant of Myc that is unable to interact with Miz-1, suggesting mutually exclusive interaction of Arnt and Myc with Miz-1. Our results also establish CDKN2B as a hypoxia regulated gene, as endogenous CDKN2B mRNA and protein levels were reduced by hypoxic treatment of U2OS cells. CONCLUSIONS: Our data reveal a novel mode of regulation by protein-protein interaction that directly ties together, at the transcriptional level, the Myc- and hypoxia-dependent signaling pathways and expands our understanding of the roles of hypoxia and cell cycle alterations during tumorigenesis.

Altered DNA methylation profile in Norwegian patients with Autoimmune Addison's Disease.

Autoimmune Addison's Disease (AAD) is an endocrine and immunological disease of uncertain pathogenesis resulting from the immune system's destruction of the hormone producing cells of the adrenal cortex. The underlying molecular mechanisms are largely unknown, but it is commonly accepted that a combination of genetic susceptibility and environmental impact is critical. In the present study, we identified multiple hypomethylated gene promoter regions in patients with isolated AAD using DNA isolated from CD4+ T cells. The identified differentially methylated regions were distributed evenly across the 10.5-kb-promoter regions covered by the array, and a substantial number localized to promoters of genes involved in immune regulation and autoimmunity. This study reveals a hypomethylated status in CD4+ T cells from AAD patients and indicates differential methylation of promoters of key genes involved in immune responses.

Computational characterization of modes of transcriptional regulation of nuclear receptor genes.

BACKGROUND: Nuclear receptors are a large structural class of transcription factors that act with their co-regulators and repressors to maintain a variety of biological and physiological processes such as metabolism, development and reproduction. They are activated through the binding of small ligands, which can be replaced by drug molecules, making nuclear receptors promising drug targets. Transcriptional regulation of the genes that encode them is central to gaining a deeper understanding of the diversity of their biochemical and biophysical roles and their role in disease and therapy. Even though they share evolutionary history, nuclear receptor genes have fundamentally different expression patterns, ranging from ubiquitously expressed to tissue-specific and spatiotemporally complex. However, current understanding of regulation in nuclear receptor gene family is still nascent. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigate the relationship between long-range regulation of nuclear receptor family and their known functionality. Towards this goal, we identify the nuclear receptor genes that are potential targets based on counts of highly conserved non-coding elements. We validate our results using publicly available expression (RNA-seq) and histone modification (ChIP-seq) data from the ENCODE project. We find that nuclear receptor genes involved in developmental roles show strong evidence of long-range mechanism of transcription regulation with distinct cis-regulatory content they feature clusters of highly conserved non-coding elements distributed in regions spanning several Megabases, long and multiple CpG islands, bivalent promoter marks and statistically significant higher enrichment of enhancer mark around their gene loci. On the other hand nuclear receptor genes that are involved in tissue-specific roles lack these features, having simple transcriptional controls and a greater variety of mechanisms for producing paralogs. We further examine the combinatorial patterns of histone maps associated with dynamic functional elements in order to explore the regulatory landscape of the gene family. The results show that our proposed classification capturing long-range regulation is strongly indicative of the functional roles of the nuclear receptors compared to existing classifications. CONCLUSIONS/SIGNIFICANCE: We present a new classification for nuclear receptor gene family capturing whether a nuclear receptor is a possible target of long-range regulation or not. We compare our classification to existing structural (mechanism of action) and homology-based classifications. Our results show that understanding long-range regulation of nuclear receptors can provide key insight into their functional roles as well as evolutionary history; and this strongly merits further study.

DNA methylation of alternative promoters directs tissue specific expression of Epac2 isoforms.

Epac 1 and Epac 2 (Epac1/2; exchange factors directly activated by cAMP) are multidomain proteins that mediate cellular responses upon activation by the signaling molecule cAMP. Epac1 is ubiquitously expressed, whereas Epac2 exhibits a restricted expression pattern. The gene encoding Epac2 gives rise to at least three protein isoforms (Epac2A, Epac2B and Epac2C) that exhibit confined tissue and cell specific expression profiles. Here, we describe alternative promoter usage for the different isoforms of Epac2, and demonstrate that the activity of these promoters depend on the DNA methylation status. Bisulfite sequencing demonstrated that the level of methylation of the promoters in different tissues correlates with Epac2 isoform expression. The presented data indicate that the tissue-specific expression of the Epac2 isoforms is epigenetically regulated, and identify tissue-specific differentially methylated promoter regions within the Epac2 locus that are essential for its transcriptional control.

Epigenetic regulation of the gene encoding steroidogenic factor-1.

The nuclear receptor steroidogenic factor 1 (SF-1) is expressed in a precise time and cell-specific pattern in the endocrine system. Three intronic enhancers and one upstream enhancer, which are required for controlling the restricted expression of SF-1, have been identified in the mouse gene encoding SF-1. In recent years, efforts from several laboratories have established that expression of SF-1 is controlled by DNA methylation. CpG-sites are found in the basal promoter as well as in the intronic enhancers, and the methylation status of these genomic regions nearly perfectly correlates with their transcriptional activity such that they are hypomethylated in tissues where they are active, and generally hypermethylated in tissues where they are not active. This review summarizes the present knowledge of how tissue differentially methylated regions control the transcriptional activity of the SF-1 gene, and how irregularities in the methylation pattern can contribute to disease development.