Clinical features characterizing Angelman syndrome, previously shown to be caused by disruption of UBE3A, were recently also described in neurologically disabled patients with mutations in SLC9A6, which encodes the Na(+)/H(+) exchanger NHE6. In the present work we have focused on NHE6Delta255-256, the protein product of a specific 6-bp patient deletion in SLC9A6. To resolve the molecular mechanism causing the cellular dysfunction associated with this mutant, we have characterized its intracellular behaviour in comparison to wild type NHE6. Our study demonstrates that NHE6Delta255-256 is much less stable than the wild type protein. Whereas wild type NHE6 is transported to the plasma membrane and early endosomes and remains stable, NHE6Delta255-256 is degraded via two independent pathways mediated by proteasomes and lysosomes, respectively. Depletion of NHE6 had no detectable effect on endosomal pH, but co-depletion of NHE6 and the closely related NHE9 caused enhanced acidification of early endosomes. Our results suggest that NHE6 participates in regulation of endosomal pH and provides a cellular basis for understanding the loss of NHE6 function leading to a neurological phenotype resembling Angelman syndrome.

The current epidemic of obesity and associated diseases calls for swift actions to better understand the mechanisms by which genetics and environmental factors affect metabolic health in humans. Monozygotic (MZ) twin pairs showing discordance for obesity suggest that epigenetic influences represent one such mechanism. We studied genome-wide leukocyte DNA methylation variation in 30 clinically healthy young adult MZ twin pairs discordant for body mass index (BMI; average within-pair BMI difference: 5.4 ± 2.0 kg/m(2)).

The cholesterol-lowering drug atorvastatin is among the most prescribed drug in the world. Alternative splicing in a number of genes has been reported to be associated with variable statin response. RNA-seq has proven to be a powerful technique for genome-wide splice variant analysis. In the present study, we sought to investigate atorvastatin responsive splice variants in HepG2 cells using RNA-seq analysis to identify novel candidate genes implicated in cholesterol homeostasis and in the statin response. HepG2 cells were treated with 10 µM atorvastatin for 24 hours. RNA-seq and exon array analyses were performed. The validation of selected genes was performed using Taqman gene expression assays. RNA-seq analysis identified 121 genes and 98 specific splice variants, of which four were minor splice variants to be differentially expressed, 11 were genes with potential changes in their splicing patterns (SYCP3, ZNF195, ZNF674, MYD88, WHSC1, KIF16B, ZNF92, AGER, FCHO1, SLC6A12 and AKAP9), and one was a gene (RAP1GAP) with differential promoter usage. The IL21R transcript was detected to be differentially expressed via RNA-seq and RT-qPCR, but not in the exon array. In conclusion, several novel candidate genes that are affected by atorvastatin treatment were identified in this study. Further studies are needed to determine the biological significance of the atorvastatin responsive splice variants that have been uniquely identified using RNA-seq.

Overweight and obesity lead to changes in adipose tissue such as inflammation and reduced insulin sensitivity. The aim of this study was to assess how altered energy balance by reduced food intake or enhanced physical activity affect these processes. We studied sedentary subjects with overweight/obesity in two intervention studies, each lasting 12 weeks affecting energy balance either by energy restriction (~20% reduced intake of energy from food) in one group, or by enhanced energy expenditure due to physical exercise (combined endurance- and strength-training) in the other group. We monitored mRNA expression by microarray and mRNA sequencing from adipose tissue biopsies. We also measured several plasma parameters as well as fat distribution with magnetic resonance imaging and spectroscopy. Comparison of microarray and mRNA sequencing showed strong correlations, which were also confirmed using RT-PCR In the energy restricted subjects (body weight reduced by 5% during a 12 weeks intervention), there were clear signs of enhanced lipolysis as monitored by mRNA in adipose tissue as well as plasma concentration of free-fatty acids. This increase was strongly related to increased expression of markers for M1-like macrophages in adipose tissue. In the exercising subjects (glucose infusion rate increased by 29% during a 12-week intervention), there was a marked reduction in the expression of markers of M2-like macrophages and T cells, suggesting that physical exercise was especially important for reducing inflammation in adipose tissue with insignificant reduction in total body weight. Our data indicate that energy restriction and physical exercise affect energy-related pathways as well as inflammatory processes in different ways, probably related to macrophages in adipose tissue.

Four patients from three Norwegian families presented with a common skin phenotype of warts, molluscum contagiosum, and dermatitis since early childhood, and various other immunological features. Warts are a common manifestation of human papilloma virus (HPV), but when they are overwhelming, disseminated and/or persistent, and presenting together with other immunological features, a primary immunodeficiency disease (PIDD) may be suspected.

Hematopoietic stem cell renewal and differentiation are regulated through epigenetic processes. The conversion of 5-methylcytosine into 5-hydroxymethylcytosine (5hmC) by ten-eleven-translocation enzymes provides new insights into the epigenetic regulation of gene expression during development. Here, we studied the potential gene regulatory role of 5hmC during human hematopoiesis.

Expression of the mannose receptor (MRC1/CD206) identifies macrophage subtypes, such as alternatively activated macrophages (AAMs) and M2-polarized tumor-associated macrophages (TAMs), which are endowed with tissue-remodeling, proangiogenic, and protumoral activity. However, the significance of MRC1 expression for TAM's protumoral activity is unclear. Here, we describe and characterize miR-511-3p, an intronic microRNA (miRNA) encoded by both mouse and human MRC1 genes. By using sensitive miRNA reporter vectors, we demonstrate robust expression and bioactivity of miR-511-3p in MRC1(+) AAMs and TAMs. Unexpectedly, enforced expression of miR-511-3p tuned down the protumoral gene signature of MRC1(+) TAMs and inhibited tumor growth. Our findings suggest that transcriptional activation of Mrc1 in TAMs evokes a genetic program orchestrated by miR-511-3p, which limits rather than enhances their protumoral functions. Besides uncovering a role for MRC1 as gatekeeper of TAM's protumoral genetic programs, these observations suggest that endogenous miRNAs may operate to establish thresholds for inflammatory cell activation in tumors.

Down syndrome (DS) is characterized by extensive phenotypic variability, with most traits occurring in only a fraction of affected individuals. Substantial gene-expression variation is present among unaffected individuals, and this variation has a strong genetic component. Since DS is caused by genomic-dosage imbalance, we hypothesize that gene-expression variation of human chromosome 21 (HSA21) genes in individuals with DS has an impact on the phenotypic variability among affected individuals. We studied gene-expression variation in 14 lymphoblastoid and 17 fibroblast cell lines from individuals with DS and an equal number of controls. Gene expression was assayed using quantitative real-time polymerase chain reaction on 100 and 106 HSA21 genes and 23 and 26 non-HSA21 genes in lymphoblastoid and fibroblast cell lines, respectively. Surprisingly, only 39% and 62% of HSA21 genes in lymphoblastoid and fibroblast cells, respectively, showed a statistically significant difference between DS and normal samples, although the average up-regulation of HSA21 genes was close to the expected 1.5-fold in both cell types. Gene-expression variation in DS and normal samples was evaluated using the Kolmogorov-Smirnov test. According to the degree of overlap in expression levels, we classified all genes into 3 groups: (A) nonoverlapping, (B) partially overlapping, and (C) extensively overlapping expression distributions between normal and DS samples. We hypothesize that, in each cell type, group A genes are the most dosage sensitive and are most likely involved in the constant DS traits, group B genes might be involved in variable DS traits, and group C genes are not dosage sensitive and are least likely to participate in DS pathological phenotypes. This study provides the first extensive data set on HSA21 gene-expression variation in DS and underscores its role in modulating the outcome of gene-dosage imbalance.

Transcriptomic profiling of the immune response induced by vaccine adjuvants is of critical importance for the rational design of vaccination strategies. In this study, transcriptomics was employed to profile the effect of the vaccine adjuvant used for priming on the immune response following re-exposure to the vaccine antigen alone. Mice were primed with the chimeric vaccine antigen H56 of Mycobacterium tuberculosis administered alone or with the CAF01 adjuvant and boosted with the antigen alone. mRNA sequencing was performed on blood samples collected 1, 2, and 7 days after priming and after boosting. Gene expression analysis at day 2 after priming showed that the CAF01 adjuvanted vaccine induced a stronger upregulation of the innate immunity modules compared with the unadjuvanted formulation. The immunostimulant effect of the CAF01 adjuvant, used in the primary immunization, was clearly seen after a booster immunization with a low dose of antigen alone. One day after boost, we observed a strong upregulation of multiple genes in blood of mice primed with H56 + CAF01 compared with mice primed with the H56 alone. In particular, blood transcription modules related to innate immune response, such as monocyte and neutrophil recruitment, activation of antigen-presenting cells, and interferon response were activated. Seven days after boost, differential expression of innate response genes faded while a moderate differential expression of T cell activation modules was appreciable. Indeed, immunological analysis showed a higher frequency of H56-specific CD4+ T cells and germinal center B cells in draining lymph nodes, a strong H56-specific humoral response and a higher frequency of antibody-secreting cells in spleen of mice primed with H56 + CAF01. Taken together, these data indicate that the adjuvant used for priming strongly reprograms the immune response that, upon boosting, results in a stronger recall innate response essential for shaping the downstream adaptive response.

Prenatal symptoms of depression (PND) and anxiety affect up to every third pregnancy. Children of mothers with mental health problems are at higher risk of developmental problems, possibly through epigenetic mechanisms together with other factors such as genetic and environmental. We investigated DNA methylation in cord blood in relation to PND, taking into consideration a history of depression, co-morbidity with anxiety and selective serotonin reuptake inhibitors (SSRI) use, and stratified by sex of the child. Mothers (N = 373) prospectively filled out web-based questionnaires regarding mood symptoms and SSRI use throughout pregnancy. Cord blood was collected at birth and DNA methylation was measured using Illumina MethylationEPIC array at 850 000 CpG sites throughout the genome. Differentially methylated regions were identified using Kruskal-Wallis test, and Benjamini-Hochberg adjusted p-values < 0.05 were considered significant.

Assisted reproductive technology (ART) may affect fetal development through epigenetic mechanisms as the timing of ART procedures coincides with the extensive epigenetic remodeling occurring between fertilization and embryo implantation. However, it is unknown to what extent ART procedures alter the fetal epigenome. Underlying parental characteristics and subfertility may also play a role. Here we identify differences in cord blood DNA methylation, measured using the Illumina EPIC platform, between 962 ART conceived and 983 naturally conceived singleton newborns. We show that ART conceived newborns display widespread differences in DNA methylation, and overall less methylation across the genome. There were 607 genome-wide differentially methylated CpGs. We find differences in 176 known genes, including genes related to growth, neurodevelopment, and other health outcomes that have been associated with ART. Both fresh and frozen embryo transfer show DNA methylation differences. Associations persist after controlling for parents' DNA methylation, and are not explained by parental subfertility.

Children of mothers with prenatal depressive symptoms (PND) have a higher risk of behavioral problems; fetal programming through DNA methylation is a possible underlying mechanism. This study investigated DNA methylation in cord blood to identify possible "at birth" signatures that may indicate susceptibility to behavioral problems at 18 months of age. Cord blood was collected from 256 children of mothers who had self-reported on symptoms of depression during pregnancy and the behavior of their child at 18 months of age. Whole genome DNA methylation was assessed using Illumina MethylationEPIC assay. The mother and child pairs were categorized into four groups, based on both self-reported depressive symptoms, PND or Healthy control (HC), and scores from the Child Behavior checklist (high or low for internalizing, externalizing, and total scores). Adjustments were made for batch effects, cell-type, and clinical covariates. Differentially methylated sites were identified using Kruskal-Wallis test, and Benjamini-Hochberg adjusted p values < 0.05 were considered significant. The analysis was also stratified by sex of the child. Among boys, we observed higher and correlated DNA methylation of one CpG-site in the promoter region of TPP1 in the HC group, with high externalizing scores compared to HC with low externalizing scores. Boys in the PND group showed lower DNA methylation in NUDT15 among those with high, compared to low, internalizing scores; the DNA methylation levels of CpGs in this gene were positively correlated with the CBCL scores. Hence, the differentially methylated CpG sites could be of interest for resilience, regardless of maternal mental health during pregnancy. The findings are in a relatively healthy study cohort, thus limiting the possibility of detecting strong effects associated with behavioral difficulties. This is the first investigation of cord blood DNA methylation signs of fetal programming of PND on child behavior at 18 months of age and thus calls for independent replications.

Virus-host interactions are regulated by complex coevolutionary dynamics. In Streptococcus pneumoniae, phase-variable type I restriction-modification (R-M) systems are part of the core genome. We hypothesized that the ability of the R-M systems to switch between six target DNA specificities also has a key role in preventing the spread of bacteriophages. Using the streptococcal temperate bacteriophage SpSL1, we show that the variants of both the SpnIII and SpnIV R-M systems are able to restrict invading bacteriophage with an efficiency approximately proportional to the number of target sites in the bacteriophage genome. In addition to restriction of lytic replication, SpnIII also led to abortive infection in the majority of host cells. During lytic infection, transcriptional analysis found evidence of phage-host interaction through the strong upregulation of the nrdR nucleotide biosynthesis regulon. During lysogeny, the phage had less of an effect on host gene regulation. This research demonstrates a novel combined bacteriophage restriction and abortive infection mechanism, highlighting the importance that the phase-variable type I R-M systems have in the multifunctional defense against bacteriophage infection in the respiratory pathogen S. pneumoniaeIMPORTANCE With antimicrobial drug resistance becoming an increasing burden on human health, much attention has been focused on the potential use of bacteriophages and their enzymes as therapeutics. However, the investigations into the physiology of the complex interactions of bacteriophages with their hosts have attracted far less attention, in comparison. This work describes the molecular characterization of the infectious cycle of a bacteriophage in the important human pathogen Streptococcus pneumoniae and explores the intricate relationship between phase-variable host defense mechanisms and the virus. This is the first report showing how a phase-variable type I restriction-modification system is involved in bacteriophage restriction while it also provides an additional level of infection control through abortive infection.

The hepatokine fetuin-A can together with free fatty acids (FFAs) enhance adipose tissue (AT) inflammation and insulin resistance via toll-like receptor 4 (TLR4). Although some of the health benefits of exercise can be explained by altered release of myokines from the skeletal muscle, it is not well documented if some of the beneficial effects of exercise can be explained by altered secretion of hepatokines. The aim of this study was to examine the effect of interaction between fetuin-A and FFAs on insulin sensitivity after physical exercise. In this study, 26 sedentary men who underwent 12 weeks of combined endurance and strength exercise were included. Insulin sensitivity was measured using euglycemic-hyperinsulinemic clamp, and AT insulin resistance was indicated by the product of fasting plasma concentration of FFAs and insulin. Blood samples and biopsies from skeletal muscle and subcutaneous AT were collected. Several phenotypic markers were measured, and mRNA sequencing was performed on the biopsies. AT macrophages were analyzed based on mRNA markers. The intervention improved hepatic parameters, reduced plasma fetuin-A concentration (~11%, P < 0.01), slightly changed FFAs concentration, and improved glucose infusion rate (GIR) (~33%, P < 0.01) across all participants. The change in circulating fetuin-A and FFAs interacted to predict some of the change in GIR (β = -42.16, P = 0.030), AT insulin resistance (β = 0.579, P = 0.003), gene expression related to TLR-signaling in AT and AT macrophage mRNA (β = 94.10, P = 0.034) after exercise. We observed no interaction effects between FFAs concentrations and leptin and adiponectin on insulin sensitivity, or any interaction effects between Fetuin-A and FFAs concentrations on skeletal muscle TLR-signaling. The relationship between FFAs levels and insulin sensitivity seemed to be specific for fetuin-A and the AT Some of the beneficial effects of exercise on insulin sensitivity may be explained by changes in circulating fetuin-A and FFAs, promoting less TLR4 signaling in AT perhaps by modulating AT macrophages.

We summarize current knowledge regarding regulatory functions of long noncoding RNAs (lncRNAs) in yeast, with emphasis on lncRNAs identified recently in yeast colonies and biofilms. Potential regulatory functions of these lncRNAs in differentiated cells of domesticated colonies adapted to plentiful conditions versus yeast colony biofilms are discussed. We show that specific cell types differ in their complements of lncRNA, that this complement changes over time in differentiating upper cells, and that these lncRNAs target diverse functional categories of genes in different cell subpopulations and specific colony types.

Monozygotic (MZ) twins do not show complete concordance for many complex diseases; for example, discordance rates for autoimmune diseases are 20%-80%. MZ discordance indicates a role for epigenetic or environmental factors in disease. We used MZ twins discordant for psoriasis to search for genome-wide differences in DNA methylation and gene expression in CD4(+) and CD8(+) cells using Illumina's HumanMethylation27 and HT-12 expression assays, respectively. Analysis of these data revealed no differentially methylated or expressed genes between co-twins when analyzed separately, although we observed a substantial amount of small differences. However, combined analysis of DNA methylation and gene expression identified genes where differences in DNA methylation between unaffected and affected twins were correlated with differences in gene expression. Several of the top-ranked genes according to significance of the correlation in CD4(+) cells are known to be associated with psoriasis. Further, gene ontology (GO) analysis revealed enrichment of biological processes associated with the immune response and clustering of genes in a biological pathway comprising cytokines and chemokines. These data suggest that DNA methylation is involved in an epigenetic dysregulation of biological pathways involved in the pathogenesis of psoriasis. This is the first study based on data from MZ twins discordant for psoriasis to detect epigenetic alterations that potentially contribute to development of the disease.

Dosage compensation in male Drosophila relies on the X chromosome-specific recruitment of a chromatin-modifying machinery, the dosage compensation complex (DCC). The principles that assure selective targeting of the DCC are unknown. According to a prevalent model, X chromosome targeting is initiated by recruitment of the DCC core components, MSL1 and MSL2, to a limited number of so-called "high-affinity sites" (HAS). Only very few such sites are known at the DNA sequence level, which has precluded the definition of DCC targeting principles. Combining RNA interference against DCC subunits, limited crosslinking, and chromatin immunoprecipitation coupled to probing high-resolution DNA microarrays, we identified a set of 131 HAS for MSL1 and MSL2 and confirmed their properties by various means. The HAS sites are distributed all over the X chromosome and are functionally important, since the extent of dosage compensation of a given gene and its proximity to a HAS are positively correlated. The sites are mainly located on non-coding parts of genes and predominantly map to regions that are devoid of nucleosomes. In contrast, the bulk of DCC binding is in coding regions and is marked by histone H3K36 methylation. Within the HAS, repetitive DNA sequences mainly based on GA and CA dinucleotides are enriched. Interestingly, DCC subcomplexes bind a small number of autosomal locations with similar features.

The exploration of quantitative variation in human populations has become one of the major priorities for medical genetics. The successful identification of variants that contribute to complex traits is highly dependent on reliable assays and genetic maps. We have performed a genome-wide quantitative trait analysis of 630 genes in 60 unrelated Utah residents with ancestry from Northern and Western Europe using the publicly available phase I data of the International HapMap project. The genes are located in regions of the human genome with elevated functional annotation and disease interest including the ENCODE regions spanning 1% of the genome, Chromosome 21 and Chromosome 20q12-13.2. We apply three different methods of multiple test correction, including Bonferroni, false discovery rate, and permutations. For the 374 expressed genes, we find many regions with statistically significant association of single nucleotide polymorphisms (SNPs) with expression variation in lymphoblastoid cell lines after correcting for multiple tests. Based on our analyses, the signal proximal (cis-) to the genes of interest is more abundant and more stable than distal and trans across statistical methodologies. Our results suggest that regulatory polymorphism is widespread in the human genome and show that the 5-kb (phase I) HapMap has sufficient density to enable linkage disequilibrium mapping in humans. Such studies will significantly enhance our ability to annotate the non-coding part of the genome and interpret functional variation. In addition, we demonstrate that the HapMap cell lines themselves may serve as a useful resource for quantitative measurements at the cellular level.

The dosage compensation complex (DCC) in Drosophila melanogaster is responsible for up-regulating transcription from the single male X chromosome to equal the transcription from the two X chromosomes in females. Visualization of the DCC, a large ribonucleoprotein complex, on male larval polytene chromosomes reveals that the complex binds selectively to many interbands on the X chromosome. The targeting of the DCC is thought to be in part determined by DNA sequences that are enriched on the X. So far, lack of knowledge about DCC binding sites has prevented the identification of sequence determinants. Only three binding sites have been identified to date, but analysis of their DNA sequence did not allow the prediction of further binding sites. We have used chromatin immunoprecipitation to identify a number of new DCC binding fragments and characterized them in vivo by visualizing DCC binding to autosomal insertions of these fragments, and we have demonstrated that they possess a wide range of potential to recruit the DCC. By varying the in vivo concentration of the DCC, we provide evidence that this range of recruitment potential is due to differences in affinity of the complex to these sites. We were also able to establish that DCC binding to ectopic high-affinity sites can allow nearby low-affinity sites to recruit the complex. Using the sequences of the newly identified and previously characterized binding fragments, we have uncovered a number of short sequence motifs, which in combination may contribute to DCC recruitment. Our findings suggest that the DCC is recruited to the X via a number of binding sites of decreasing affinities, and that the presence of high- and moderate-affinity sites on the X may ensure that lower-affinity sites are occupied in a context-dependent manner. Our bioinformatics analysis suggests that DCC binding sites may be composed of variable combinations of degenerate motifs.

Pre-eclampsia (PE) and gestational diabetes mellitus (GDM) are common complications of pregnancy, but the mechanisms underlying these disorders remain unclear. The aim was to identify the extent of altered gene expression in term placentas from pregnant women with late-onset PE and GDM compared to controls. RNAseq identified few significantly differentially regulated genes in placental biopsies between PE, GDM, or uncomplicated pregnancy (n = 10 each group). Five genes were altered in placentas from PE including 4 non-coding genes and Angiopoietin 2 (ANGPT2). No genes were significantly regulated by GDM. In contrast, many genes were significantly regulated by fetal, maternal and delivery-specific variables, particularly spinal and epidural anesthesia. We selected ANGPT2 and Chemokine (C-X-C motif) ligand 14 (CXCL14) to test with qPCR in a larger set of placentas (n = 475) and found no differences between the groups. However, regression analysis revealed a stronger association between placental ANGPT2 and CXCL14 mRNA expression and fetal, maternal and delivery-specific variables than diagnostic group. To conclude, the gene expression in term placentas are highly affected by fetal, maternal and delivery specific variables. Few regulated genes were found in late-onset PE and GDM placentas, which may suggest that these conditions could be more affected by maternal factors.