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Microsatellite loci are commonly used markers in population genetic studies. In this study, we present 40 novel and polymorphic microsatellite loci elaborated for the ichneumonid parasitoid Latibulus argiolus (Rossi, 1790). Reaction condition optimisation procedures allowed 14 of these loci to be co-amplified in two PCRs and loaded in two multiplex panels onto a genetic analyser. The assay was tested on 197 individuals of L. argiolus originating from ten natural populations obtained from the host nests of paper wasps. The validated loci were polymorphic with high allele numbers ranging from eight to 27 (average 17.6 alleles per locus). Both observed and expected heterozygosity values were high, ranging between 0.75 and 0.92 for HO (mean 0.83) and from 0.70 to 0.90 for HE (mean 0.85). The optimized assay showed low genotyping error rate and negligible null allele frequency. The designed multiplex panels could be successfully applied in relatedness analyses and genetic variability studies of L. argiolus populations, which would be particularly interesting considering the coevolutionary context of this species with its social host.

Epigenetic alterations are a hallmark of aging and age‐related diseases. Computational models using DNA methylation data can create “epigenetic clocks” which are proposed to reflect “biological” aging. Thus, it is important to understand the relationship between predictive clock sites and aging biology. To do this, we examined over 450,000 methylation sites from 9,699 samples. We found ~20% of the measured genomic cytosines can be used to make many different epigenetic clocks whose age prediction performance surpasses that of telomere length. Of these predictive sites, the average methylation change over a lifetime was small (~1.5%) and these sites were under‐represented in canonical regions of epigenetic regulation. There was only a weak association between “accelerated” epigenetic aging and disease. We also compare tissue‐specific and pan‐tissue clock performance. This is critical to applying clocks both to new sample sets in basic research, as well as understanding if clinically available tissues will be feasible samples to evaluate “epigenetic aging” in unavailable tissues (e.g., brain). Despite the reproducible and accurate age predictions from DNA methylation data, these findings suggest they may have limited utility as currently designed in understanding the molecular biology of aging and may not be suitable as surrogate endpoints in studies of anti‐aging interventions. Purpose‐built clocks for specific tissues age ranges or phenotypes may perform better for their specific purpose. However, if purpose‐built clocks are necessary for meaningful predictions, then the utility of clocks and their application in the field needs to be considered in that context., Epigenetic clocks show promise as a biomarker of aging. Our study identifies components of epigenetic clocks critical to understanding their uses and where improvements are still needed. We identified a parabolic trajectory of methylation aging and the extent to which epigenetic clocks are robust to removing the most age‐predictive loci. These data shape our understanding of epigenetic age acceleration and what epigenetic clocks can tell us about the biology of aging.

Cyclin-dependent kinase inhibitor 2A (CDKN2A) gene methylation has been paramount in the development of malignant masses. The purpose of the conducted research was to evaluate the mechanism and involvement of methylation in regards to the CDKN2A gene and the specific locus region in gastric cancer (GC) with comprehensive statistical analysis utilizing statistics acquired from The Cancer Genome Atlas (TCGA) database. Gene Set Enrichment Analysis (GSEA) revealed that the level of CDKN2A gene methylation and its locus in GC tissues was increased compared to para-cancerous tissues. In multivariate analysis, low methylation of CDKN2A gene, cg03079681, cg04026675, cg07562918, and cg13601799 locus were independently linked to better OS. In addition, the methylation of CDKN2A gene, cg00718440, cg03079681, cg04026675, cg07562918, cg10848754, cg14069088 and cg14430974 locus were negative correlated with CDKN2A gene expression. Meanwhile, the methylation of cg12840719 locus was positively correlated with CDKN2A gene expression. GSEA showed that hallmark_kras_signaling_dn, hallmark_myogenesis, and hallmark_epithelial_mesenchymal_transition pathways were enriched in the CDKN2A gene hypermethylation phenotype. Taken together, the low methylation of CDKN2A gene, cg03079681, cg04026675, cg07562918, and cg13601799 locus indicated a better prognosis in GC. The methylation levels of cg14069088 were most negatively correlated with CDKN2A gene expression. Hallmark_kras_signaling_dn, Hallmark_myogenesis, and hallmark_epithelial_mesenchymal_transition pathways might be important in the regulation of CDKN2A gene hypermethylation., Graphical abstract

Myeloid cells, including antigen-presenting cells (APCs) and myeloid-derived suppressor cells (MDSCs) play opposing roles to orchestrate innate and adaptive immune responses during physiological and pathological conditions. We investigated the role of DNA methylation in regulating the transcription of inhibitory/suppressive molecules in myeloid suppressive cells (identified as CD33+HLA-DR–) in comparison to APCs. We selected a number of immune checkpoints (ICs), IC ligands, and immunosuppressive molecules that have been implicated in MDSC function, including PD-L1, TIM-3, VISTA, galectin-9, TGF-β, ARG1 and MMP9. We examined their mRNA expression levels, and investigated whether DNA methylation regulates their transcription in sorted myeloid cell subpopulations. We found that mRNA levels of PD-L1, TIM-3, TGF-β, ARG1 and MMP9 in CD33+HLA-DR– cells were higher than APCs. However, VISTA and galectin-9 mRNA levels were relatively similar in both myeloid subpopulations. CpG islands in the promoter regions of TGF-β1, TIM-3 and ARG1 were highly unmethylated in CD33+HLA-DR–cells, compared with APCs, suggesting that DNA methylation is one of the key mechanisms, which regulate their expression. However, we did not find differences in the methylation status of PD-L1 and MMP9 between CD33+HLA-DR– and APCs, suggesting that their transcription could be regulated via other genetic and epigenetic mechanisms. The promoter methylation status of VISTA was relatively similar in both myeloid subpopulations. This study provides novel insights into the epigenetic mechanisms, which control the expression of inhibitory/suppressive molecules in circulating CD33+HLA-DR– cells in a steady-state condition, possibly to maintain immune tolerance and haemostasis.

Despite recent evidence that 5-hydroxymethylcytosine (5hmC) possesses roles in gene regulation distinct from 5-methylcytosine (5mC), relatively little is known regarding the functions of 5hmC in mammalian tissues. To address this issue, we utilized an approach combining both paired bisulfite (BS) and oxidative bisulfite (oxBS) DNA treatment, to resolve genome-wide patterns of 5hmC and 5mC in normal breast tissue from disease-free women. Although less abundant than 5mC, 5hmC was differentially distributed, and consistently enriched among breast-specific enhancers and transcriptionally active chromatin. In contrast, regulatory regions associated with transcriptional inactivity, such as heterochromatin and repressed Polycomb regions, were relatively depleted of 5hmC. Gene regions containing abundant 5hmC were significantly associated with lactate oxidation, immune cell function, and prolactin signaling pathways. Furthermore, genes containing abundant 5hmC were enriched among those actively transcribed in normal breast tissue. Finally, in independent data sets, normal breast tissue 5hmC was significantly enriched among CpG loci demonstrated to have altered methylation in pre-invasive breast cancer and invasive breast tumors. Primarily, our findings identify genomic loci containing abundant 5hmC in breast tissues and provide a genome-wide map of nucleotide-level 5hmC in normal breast tissue. Additionally, these data suggest 5hmC may participate in gene regulatory programs that are dysregulated during breast-related carcinogenesis.

Background Oculocutaneous albinism (OCA) is a group of autosomal recessive disorders characterized by reduced melanin that are caused by mutations in the gene encoding tyrosinase (TYR), which is the rate-limiting enzyme in the production of the pigment melanin. Many studies or meta-analyses have suggested an association between the TYR T373K SNP and OCA1, but there is limited biochemical and genetic evidence to support this association. Methods We overexpressed TYR-WT and TYR-T373K mutants on HK293T cells and tested the changes of melanin production and tyrosinase activity. Then we generated TYR-K373T knock-in (KI) rabbits by microinjection of ssDNA and synthesized RNAs targeting C1118A using CRISPR/Cas9-HDR to observe the formation of melanin. Findings We demonstrated that the T373K mutation in TYR can reduce tyrosinase activity, leading to an absence of melanin synthesis at the cell-level. The gene-edited TYR-K373T rabbits exhibited rescued melanin production in hair follicles and irises, as inferred from the evident decrease in pigmentation in TYR-T373K rabbits, thus providing functional validation of the albinism-associated T373K SNP at the animal level. Interpretation Our study provides the first animal-level functional validation of the albinism-associated TYR K373T SNP in rabbits, and these results will facilitate gene therapy of OCA1 in pre-clinical settings in the future. Fund The National Key Research and Development Program of China Stem Cell and Translational Research, the Strategic Priority Research Program of the Chinese Academy of Sciences, the Guangdong Province Science and Technology Plan Project, and the Program for JLU Science and Technology Innovative Research Team.

Summary There is a large diversity of genetically defined resistance genes in bread wheat against the powdery mildew pathogen Blumeria graminis (B. g.) f. sp. tritici. Many confer race‐specific resistance to this pathogen, but until now only the mildew avirulence gene AvrPm3 a2/f2 that is recognized by Pm3a/f was known molecularly.We performed map‐based cloning and genome‐wide association studies to isolate a candidate for the mildew avirulence gene AvrPm2. We then used transient expression assays in Nicotiana benthamiana to demonstrate specific and strong recognition of AvrPm2 by Pm2.The virulent AvrPm2 allele arose from a conserved 12 kb deletion, while there is no protein sequence diversity in the gene pool of avirulent B. g. tritici isolates. We found one polymorphic AvrPm2 allele in B. g. triticale and one orthologue in B. g. secalis and both are recognized by Pm2. AvrPm2 belongs to a small gene family encoding structurally conserved RNase‐like effectors, including Avr a13 from B. g. hordei, the cognate Avr of the barley resistance gene Mla13.These results demonstrate the conservation of functional avirulence genes in two cereal powdery mildews specialized on different hosts, thus providing a possible explanation for successful introgression of resistance genes from rye or other grass relatives to wheat., See also the Commentary on this article by Spanu, 213: 969–971.

Summary Awns, bristle‐like structures extending from grass lemmas, provide protection against predators, contribute to photosynthesis and aid in grain dispersal. In wheat, selection of awns with minimal extension, termed awnletted, has occurred during domestication by way of loci that dominantly inhibit awn development, such as Tipped1 (B1), Tipped2 (B2), and Hooded (Hd). Here we identify and characterize the B1 gene. B1 was identified using bulked segregant RNA‐sequencing of an F2 durum wheat population and through deletion mapping of awned bread wheat mutants. Functional characterization was accomplished by gene overexpression while haplotype analyses assessed B1 polymorphisms and genetic variation.Located on chromosome 5A, B1 is a C2H2 zinc finger encoding gene with ethylene‐responsive element binding factor‐associated amphiphilic repression (EAR) motifs. Constitutive overexpression of B1 in awned wheat produced an awnletted phenotype with pleiotropic effects on plant height and fertility. Transcriptome analysis of B1 overexpression plants suggests a role as transcriptional repressor, putatively targeting pathways involved in cell proliferation. Haplotype analysis revealed a conserved B1 coding region with proximal polymorphisms and supported the contention that B1 is mainly responsible for awnletted wheats globally. B1, predominantly responsible for awn inhibition in wheat, encodes a C2H2 zinc finger protein with EAR motifs which putatively functions as a transcriptional repressor.

Human papillomavirus-related oropharyngeal squamous cell carcinoma (HPV+ OPSCC) represents a unique disease entity within head and neck cancer with rising incidence. Previous work has shown that alternative splicing events (ASEs) are prevalent in HPV+ OPSCC, but further validation is needed to understand the regulation of this process and its role in these tumours. In this study, eleven ASEs (GIT2, CTNNB1, MKNK2, MRPL33, SIPA1L3, SNHG6, SYCP2, TPRG1, ZHX2, ZNF331, and ELOVL1) were selected for validation from 109 previously published candidate ASEs to elucidate the post-transcriptional mechanisms of oncogenesis in HPV+ disease. In vitro qRT-PCR confirmed differential expression of 9 of 11 ASE candidates, and in silico analysis within the TCGA cohort confirmed 8 of 11 candidates. Six ASEs (MRPL33, SIPA1L3, SNHG6, TPRG1, ZHX2, and ELOVL1) showed significant differential expression across both methods. Further evaluation of chromatin modification revealed that ASEs strongly correlated with cancer-specific distribution of acetylated lysine 27 of histone 3 (H3K27ac). Subsequent epigenetic treatment of HPV+ HNSCC cell lines (UM-SCC-047 and UPCI-SCC-090) with JQ1 not only induced downregulation of cancer-specific ASE isoforms, but also growth inhibition in both cell lines. The UPCI-SCC-090 cell line, with greater ASE expression, also showed more significant growth inhibition after JQ1 treatment. This study confirms several novel cancer-specific ASEs in HPV+OPSCC and provides evidence for the role of chromatin modifications in regulation of alternative splicing in HPV+OPSCC. This highlights the role of epigenetic changes in the oncogenesis of HPV+OPSCC, which represents a unique, unexplored target for therapeutics that can alter the global post-transcriptional landscape.

Background: Phenylketonuria (PKU) is the most common inborn error of amino acid metabolism in Europe. The reasons underlying the high prevalence of heterozygous carriers are not clearly understood. We aimed to look for pathogenic PAH variant enrichment according to geographical areas and patients’ ethnicity using a multiethnic nationwide cohort of patients with PKU in France. We subsequently appraised the population differentiation, balancing selection and the molecular evolutionary history of the PAH locus. Methods: The French nationwide PKU study included patients who have been referred at the national level to the University Hospital of Nancy, and for whom a molecular diagnosis of phenylketonuria was made by Sanger sequencing. We performed enrichment analyses by comparing alternative allele frequencies using Fisher's exact test with Bonferroni adjustment. We estimated the amount of genetic differentiation among populations using Wright's fixation index (Fst). To estimate the molecular evolutionary history of the PAH gene, we performed phylogenetic and evolutionary analyses using whole-genome and exome-sequencing data from healthy individuals and non-PKU patients, respectively. Finally, we used exome-wide association study to decipher potential genetic loci associated with population divergence on PAH. Findings: The study included 696 patients and revealed 132 pathogenic PAH variants. Three geographical areas showed significant enrichment for a pathogenic PAH variant: North of France (p.Arg243Leu), North-West of France (p.Leu348Val), and Mediterranean coast (p.Ala403Val). One PAH variant (p.Glu280Gln) was significantly enriched among North-Africans (OR = 23·23; 95% CI: 9·75–55·38). PAH variants exhibiting a strong genetic differentiation were significantly enriched in the ‘Biopterin_H’ domain (OR = 6·45; 95% CI: 1·99–20·84), suggesting a balancing selection pressure on the biopterin function of PAH. Phylogenetic and timetree analyses were consistent with population differentiation events on European-, African-, and Asian-ancestry populations. The five PAH variants most strongly associated with a high selection pressure were phylogenetically close and were located within the biopterin domain coding region of PAH or in its vicinity. Among the non-PAH loci potentially associated with population divergence, two reached exome-wide significance: SSPO (SCO-spondin) and DBH (dopamine beta-hydroxylase), involved in neuroprotection and metabolic adaptation, respectively. Interpretation: Our data provide evidence on the combination of evolutionary and adaptive events in populations with distinct ancestries, which may explain the overdominance of some genetic variants on PAH. Funding: French National Institute of Health and Medical Research (INSERM) UMR_S 1256. Keywords: Phenylketonuria, Balancing selection, Population divergence, Overdominance, Metabolic adaptation

Storage of seeds under elevated partial pressure of oxygen mimics dry after-ripening at the genetic level, as indicated by identification of DELAY OF GERMINATION quantitative trait loci., Seed dormancy determines the timing of seed germination and may be released by dry storage, also referred to as after-ripening. Studies on dormancy-release mechanisms are often hampered by the long after-ripening requirements of seeds. After-ripening is thought to be mainly caused by oxidative processes during seed dry storage. These processes are also the main cause of seed ageing. Increasing partial oxygen pressure through the elevated partial pressure of oxygen (EPPO) system has been shown to mimic and accelerate dry seed ageing. In this study, we investigated whether the EPPO system may also release primary seed dormancy in Arabidopsis thaliana. EPPO mimics dry after-ripening at the genetic level, as quantitative trait locus (QTL) analysis after EPPO treatment identified the DELAY OF GERMINATION loci DOG1, DOG2, and DOG6 that were first described in a study using dry after-ripening to release seed dormancy. QTL analysis also showed that dormancy release by cold stratification (another common method to break seed dormancy) partly overlaps with release by after-ripening and EPPO treatment. We conclude that EPPO is an appropriate method to mimic and accelerate dormancy release and, as such, may have applications in both research and industry.

Motivation Simulation is a critical part of method development and assessment. With the increasing sophistication of multi-trait and multi-locus genetic analysis techniques, it is important that the community has flexible simulation tools to challenge and explore the properties of these methods. Results We have developed PhenotypeSimulator, a comprehensive phenotype simulation scheme that can model multiple traits with multiple underlying genetic loci as well as complex covariate and observational noise structure. This package has been designed to work with many common genetic tools both for input and output. We describe the underlying components of this simulation tool and illustrate its use on an example dataset. Availability and implementation PhenotypeSimulator is available as a well documented R/CRAN package and the code is available on github: https://github.com/HannahVMeyer/PhenotypeSimulator. Supplementary information Supplementary data are available at Bioinformatics online.

Background Headache is the most common neurological symptom and a leading cause of years lived with disability. We sought to identify the genetic variants associated with a broadly-defined headache phenotype in 223,773 subjects from the UK Biobank cohort. Methods We defined headache based on a specific question answered by the UK Biobank participants. We performed a genome-wide association study of headache as a single entity, using 74,461 cases and 149,312 controls. Results We identified 3343 SNPs which reached the genome-wide significance level of P, Highlights • This genome-wide association study identified 28 genomic loci for broadly-defined headache, among which, 14 are new. • Through tissue expression analysis, brain tissues showed significant relationships to broadly-defined headache. • Broadly-defined headache shared common genetic components with many psychological traits such as neuroticism. This genetic study using the UK Biobank resource has identified 28 genomic loci for broadly-defined headache, among which, 14 are new loci. In addition, it has provided extra evidence that the functions of brain tissues are closely related to headache. Moreover, it has suggested that headache and many psychological disorders share common genetic factors. These findings will not only contribute to the understanding of the causes of headache (and its subtypes) and its relationships with psychological disorders, it might also bring potential genetic targets for drug treatment for patients with headache and psychological disorders.

Cis-natural antisense transcripts (cis-NATs) are long noncoding RNAs transcribed from the opposite strand and overlapping coding and noncoding genes on the sense strand. cis-NATs are widely present in the human genome and can be involved in multiple mechanisms of gene regulation. Here, we describe the presence of cis-NATs in the 3′ distal region of the c-MYC locus and investigate their impact on transcriptional regulation of this key oncogene in human cancers. We found that cis-NATs are produced as consequence of the activation of cryptic transcription initiation sites in the 3′ distal region downstream of the c-MYC 3′UTR. The process is tightly regulated and leads to the formation of two main transcripts, NAT6531 and NAT6558, which differ in their ability to fold into stem-loop secondary structures. NAT6531 acts as a substrate for DICER and as a source of small RNAs capable of modulating c-MYC transcription. This complex system, based on the interplay between cis-NATs and NAT-derived small RNAs, may represent an important layer of epigenetic regulation of the expression of c-MYC and other genes in human cells.

// Yannan Jiang 1 , Xinming Qi 2 , Xinyu Liu 1 , Jun Zhang 1 , Jun Ji 1 , Zhenggang Zhu 1 , Jin Ren 2 , Yingyan Yu 1 1 Department of Surgery of Shanghai Ruijin Hospital, Shanghai Institute of Digestive Surgery, Shanghai Key Laboratory for Gastric Neoplasms, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China 2 Center for Drug Safety Evaluation and Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China Correspondence to: Yingyan Yu, email: yingyan3y@sjtu.edu.cn Keywords: Fbxw7, knockout mouse, N-Methyl-N-nitrosourea, gastric cancer, DNA damage Received: January 17, 2017 Accepted: March 22, 2017 Published: April 03, 2017 ABSTRACT Fbxw7, a subunit of the SCF E3 ubiquitin ligase, recognizes oncoprotein substrates and leads to their proteasomal degradation. Fbxw7 acts as a tumor suppressor via inducing apoptosis and growth arrest in various kinds of tumors. To clarify the initiating role in gastric carcinogenesis as well as the histologic characterization of tumor in Fbxw7 allele haploinsufficient mice, we generated Fbxw7 heterozygous knockout mice (Fbxw7 +/− ) and treated them with chemical carcinogen N-methyl-N-nitrosourea (MNU) at 5–6 weeks of age. We also treated mouse embryo fibroblasts (MEFs) from Fbxw7 +/− and Fbxw7 +/+ mice with MNU and examined cell DNA damage via comet assay. The protein expression of Fbxw7 and its substrate c-Myc from mouse tumors, as well as human tumors sampled from six patients, were detected by Western blot. As results, the tumor incidence was obviously higher in Fbxw7 +/− mice (13/20) than in Fbxw7 +/+ mice (6/20) after 35-week observation. Intestinal metaplasia ( P = 0.013) and dysplasia ( P = 0.036) were more severe in Fbxw7 +/− mice than in Fbxw7 +/+ mice. The repair potential of DNA damage was suppressed in MEFs from Fbxw7 +/− mice after MNU exposure. Increased c-Myc expression was accompanied by decreased Fbxw7 protein expression in tumor tissues from mouse and patients. In conclusion, Fbxw7 haploinsufficiency increased the risk of gastric carcinogenesis induced by MNU, which is associated with the accumulation of DNA damage as well as c-Myc oncoprotein.

// Keisuke Hitachi 1 , Kunihiro Tsuchida 1 1 Division for Therapies Against Intractable Diseases, Institute for Comprehensive Medical Science (ICMS), Fujita Health University, Toyoake, Aichi 470-1192, Japan Correspondence to: Kunihiro Tsuchida, email: tsuchida@fujita-hu.ac.jp Keywords: myostatin, miRNA, callipyge, IG-DMR ncRNA, skeletal muscle Received: July 20, 2016 Accepted: December 08, 2016 Published: December 15, 2016 ABSTRACT Myostatin, a member of the transforming growth factor-beta superfamily, is a negative regulator of skeletal muscle growth and development. Myostatin inhibition leads to increased skeletal muscle mass in mammals; hence, myostatin is considered a potential therapeutic target for skeletal muscle wasting. However, downstream molecules of myostatin in the skeletal muscle have not been fully elucidated. Here, we identified the Dlk1-Dio3 locus at the mouse chromosome 12qF1, also called as the callipyge locus in sheep, as a novel downstream target of myostatin. In skeletal muscle of myostatin knockout mice, the expression of mature miRNAs at the Dlk1-Dio3 locus was significantly increased. The increased miRNA levels are caused by the transcriptional activation of the Dlk1-Dio3 locus, because a significant increase in the primary miRNA transcript was observed in myostatin knockout mice. In addition, we found increased expression of coding and non-coding genes ( Dlk1 , Gtl2 , Rtl1/Rtl1as , and Rian ) at the Dlk1-Dio3 locus in myostatin-deficient skeletal muscle. Moreover, epigenetic changes, associated with the regulation of the Dlk1-Dio3 locus, were observed in myostatin knockout mice. Taken together, this is the first report demonstrating the role of myostatin in regulating the Dlk1-Dio3 (the callipyge ) locus in the skeletal muscle.

Vascular endothelial growth factor (VEGF)-A is differentially spliced to give two functionally different isoform families; pro-angiogenic, pro-permeability VEGF-Axxx and anti-angiogenic, anti-permeability VEGF-Axxxb. VEGF-A splicing is dysregulated in several pathologies, including cancer, diabetes, and peripheral arterial disease. The bichromatic VEGF-A splicing-sensitive fluorescent reporter harboured in a transgenic mouse is a novel approach to investigate the splicing patterns of VEGF-A in vivo. We generated a transgenic mouse harbouring a splicing-sensitive fluorescent reporter designed to mimic VEGF-A terminal exon splicing (VEGF8ab) by insertion into the ROSA26 genomic locus. dsRED expression denotes proximal splice site selection (VEGF-Axxx) and eGFP expression denotes distal splice site selection (VEGF-Axxxb). We investigated the tissue-specific expression patterns in the eye, skeletal muscle, cardiac muscle, kidney, and pancreas, and determined whether the splicing pattern could be manipulated in the same manner as endogenous VEGF-A by treatment with the SRPK1 inhibitor SPHINX 31. We confirmed expression of both dsRED and eGFP in the eye, skeletal muscle, cardiac muscle, kidney, and pancreas, with the highest expression of both fluorescent proteins observed in the exocrine pancreas. The ratio of dsRED and eGFP matched that of endogenous VEGF-Axxx and VEGF-Axxxb. Treatment of the VEGF8ab mice with SPHINX 31 increased the mRNA and protein eGFP/dsRED ratio in the exocrine pancreas, mimicking endogenous VEGF-A splicing. The VEGF-A exon 8 splicing-sensitive fluorescent reporter mouse is a novel tool to assess splicing regulation in the individual cell-types and tissues, which provides a useful screening process for potentially therapeutic splicing regulatory compounds in vivo.

The Developmental Origins of Health and Disease (DOHaD) hypothesis posits that in utero and early life conditions can disrupt normal fetal development and program susceptibility to later-life disease. Metastable epialleles are genomic loci in which CpG methylation patterning is responsive to maternal diet and conserved across time and tissues. Thus, these sites could serve as 'signatures' of gestational environment conditions. Here, we sought to determine if methylation of metastable epialleles was associated with changes in childhood body mass index (BMI) z-scores across ages one, two and ten in the Extremely Low Gestational Age Newborns (ELGAN) cohort. CpG methylation of 250 probes (corresponding to 111 genes) within metastable epiallele regions was measured in placental tissue. Linear mixed effects models were fit to evaluate the overall and sex-stratified associations between methylation and changes in BMI z-score over time. In total, 26 probes were associated (p < 0.05) with changes in BMI z-score overall, including probes within Mesoderm Specific Transcript (MEST) and Histone Deacetylase 4 (HDAC4), which have previously been associated with childhood obesity and adipogenesis. Sex-stratified analyses revealed a significant association, after adjusting for multiple comparisons (q < 0.05), within female placentas for one probe annotated to the imprinted gene PLAG1 Like Zinc Finger 1 (PLAGL1). These findings suggest epigenetic marks may be involved in programming susceptibility to obesity in utero and highlight the potential to use placental tissues in predicting growth rate trajectories among premature infants.

Marginal tests based on individual SNPs are routinely used in genetic association studies. Studies have shown that haplotype‐based methods may provide more power in disease mapping than methods based on single markers when, for example, multiple disease‐susceptibility variants occur within the same gene. A limitation of haplotype‐based methods is that the number of parameters increases exponentially with the number of SNPs, inducing a commensurate increase in the degrees of freedom and weakening the power to detect associations. To address this limitation, we introduce a hierarchical linkage disequilibrium model for disease mapping, based on a reparametrization of the multinomial haplotype distribution, where every parameter corresponds to the cumulant of each possible subset of a set of loci. This hierarchy present in the parameters enables us to employ flexible testing strategies over a range of parameter sets: from standard single SNP analyses through the full haplotype distribution tests, reducing degrees of freedom and increasing the power to detect associations. We show via extensive simulations that our approach maintains the type I error at nominal level and has increased power under many realistic scenarios, as compared to single SNP and standard haplotype‐based studies. To evaluate the performance of our proposed methodology in real data, we analyze genome‐wide data from the Wellcome Trust Case‐Control Consortium.

Introduction: Cigarette smoking has been associated with adverse health outcomes for mothers and children and is a major contributor to heart disease. Although cigarette smoking is known to affect the epigenome, few studies have been done in African American populations. In this study, we investigated the association between cigarette smoking and DNA methylation (DNAm) among African Americans from the Intergenerational Impact of Genetic and Psychological Factors on Blood Pressure Study (InterGEN), and the Genetic Epidemiology Network of Arteriopathy (GENOA). Methods: The InterGEN study aims to examine the effects of genetic and psychological factors on blood pressure among African American women and their children. Current cigarette smoking was assessed at baseline. DNAm of saliva was assessed using the 850K EPIC Illumina BeadChip for Epigenome-Wide Association analyses. A replication study was conducted among 1100 participants in the GENOA study using the same BeadChip. Results: After controlling for age, body mass index, population structure and cell composition, 26 epigenome-wide significant sites (FDR q

Increased reactive oxygen species (ROS) production and inflammation are key factors in the pathogenesis of atherosclerosis. We previously reported that NOX activator 1 (NOXA1) is the critical functional homolog of p67phox for NADPH oxidase activation in mouse vascular smooth muscle cells (VSMC). Here we investigated the effects of systemic and SMC-specific deletion of Noxa1 on VSMC phenotype during atherogenesis in mice. Neointimal hyperplasia following endovascular injury was lower in Noxa1-deficient mice versus the wild-type following endovascular injury. Noxa1 deletion in Apoe-/- or Ldlr-/- mice fed a Western diet showed 50% reduction in vascular ROS and 30% reduction in aortic atherosclerotic lesion area and aortic sinus lesion volume (P, Graphical abstract fx1, Highlights • NOXA1 is a VSMC-specific regulator of NADPH oxidase 1 activity and downstream cell signaling. • NOX1 NADPH oxidase-dependent ROS generation is required for VSMC proliferation and migration after endovascular injury. • NOXA1-dependent NOX1 activation of KLF4 in atherosclerotic lesions induces SMC phenotypic switch to macrophage-like cells. • Atherosclerotic lesion macrophage-like cells promote plaque inflammation, matrix remodeling and increase volume expansion.

Summary Heteromorphic flower development in Primula is controlled by the S locus. The S locus genes, which control anther position, pistil length and pollen size in pin and thrum flowers, have not yet been characterized. We have integrated S‐linked genes, marker sequences and mutant phenotypes to create a map of the P. vulgaris S locus region that will facilitate the identification of key S locus genes. We have generated, sequenced and annotated BAC sequences spanning the S locus, and identified its chromosomal location.We have employed a combination of classical genetics and three‐point crosses with molecular genetic analysis of recombinants to generate the map. We have characterized this region by Illumina sequencing and bioinformatic analysis, together with chromosome in situ hybridization.We present an integrated genetic and physical map across the P. vulgaris S locus flanked by phenotypic and DNA sequence markers. BAC contigs encompass a 1.5‐Mb genomic region with 1 Mb of sequence containing 82 S‐linked genes anchored to overlapping BACs. The S locus is located close to the centromere of the largest metacentric chromosome pair.These data will facilitate the identification of the genes that orchestrate heterostyly in Primula and enable evolutionary analyses of the S locus.

Motivation: The discovery of CRISPR-Cas systems almost 20 years ago rapidly changed our perception of the bacterial and archaeal immune systems. CRISPR loci consist of several repetitive DNA sequences called repeats, inter-spaced by stretches of variable length sequences called spacers. This CRISPR array is transcribed and processed into multiple mature RNA species (crRNAs). A single crRNA is integrated into an interference complex, together with CRISPR-associated (Cas) proteins, to bind and degrade invading nucleic acids. Although existing bioinformatics tools can recognize CRISPR loci by their characteristic repeat-spacer architecture, they generally output CRISPR arrays of ambiguous orientation and thus do not determine the strand from which crRNAs are processed. Knowledge of the correct orientation is crucial for many tasks, including the classification of CRISPR conservation, the detection of leader regions, the identification of target sites (protospacers) on invading genetic elements and the characterization of protospacer-adjacent motifs. Results: We present a fast and accurate tool to determine the crRNA-encoding strand at CRISPR loci by predicting the correct orientation of repeats based on an advanced machine learning approach. Both the repeat sequence and mutation information were encoded and processed by an efficient graph kernel to learn higher-order correlations. The model was trained and tested on curated data comprising >4500 CRISPRs and yielded a remarkable performance of 0.95 AUC ROC (area under the curve of the receiver operator characteristic). In addition, we show that accurate orientation information greatly improved detection of conserved repeat sequence families and structure motifs. We integrated CRISPRstrand predictions into our CRISPRmap web server of CRISPR conservation and updated the latter to version 2.0. Availability: CRISPRmap and CRISPRstrand are available at http://rna.informatik.uni-freiburg.de/CRISPRmap. Contact: backofen@informatik.uni-freiburg.de Supplementary information: Supplementary data are available at Bioinformatics online.

Directed differentiation of stem cells offers a scalable solution to the need for human cell models recapitulating islet biology and T2D pathogenesis. We profiled mRNA expression at 6 stages of an induced pluripotent stem cell (iPSC) model of endocrine pancreas development from 2 donors, and characterized the distinct transcriptomic profiles associated with each stage. Established regulators of endodermal lineage commitment, such as SOX17 (log2 fold change [FC] compared to iPSCs = 14.2, p-value = 4.9 × 10−5) and the pancreatic agenesis gene GATA6 (log2 FC = 12.1, p-value = 8.6 × 10−5), showed transcriptional variation consistent with their known developmental roles. However, these analyses highlighted many other genes with stage-specific expression patterns, some of which may be novel drivers or markers of islet development. For example, the leptin receptor gene, LEPR, was most highly expressed in published data from in vivo-matured cells compared to our endocrine pancreas-like cells (log2 FC = 5.5, p-value = 2.0 × 10−12), suggesting a role for the leptin pathway in the maturation process. Endocrine pancreas-like cells showed significant stage-selective expression of adult islet genes, including INS, ABCC8, and GLP1R, and enrichment of relevant GO-terms (e.g. “insulin secretion”; odds ratio = 4.2, p-value = 1.9 × 10−3): however, principal component analysis indicated that in vitro-differentiated cells were more immature than adult islets. Integration of the stage-specific expression information with genetic data from T2D genome-wide association studies revealed that 46 of 82 T2D-associated loci harbor genes present in at least one developmental stage, facilitating refinement of potential effector transcripts. Together, these data show that expression profiling in an iPSC islet development model can further understanding of islet biology and T2D pathogenesis.

Gene-by-gene approaches are becoming increasingly popular in bacterial genomic epidemiology and outbreak detection. However, there is a lack of open-source scalable software for schema definition and allele calling for these methodologies. The chewBBACA suite was designed to assist users in the creation and evaluation of novel whole-genome or core-genome gene-by-gene typing schemas and subsequent allele calling in bacterial strains of interest. The software can run in a laptop or in high performance clusters making it useful for both small laboratories and large reference centers. ChewBBACA is available athttps://github.com/B-UMMI/chewBBACAor as a docker image athttps://hub.docker.com/r/ummidock/chewbbaca/.DATA SUMMARYAssembled genomes used for the tutorial were downloaded from NCBI in August 2016 by selecting those submitted asStreptococcus agalactiaetaxon or sub-taxa. All the assemblies have been deposited as a zip file in FigShare (https://figshare.com/s/9cbe1d422805db54cd52), where a file with the original ftp link for each NCBI directory is also available.Code for the chewBBACA suite is available athttps://github.com/B-UMMI/chewBBACAwhile the tutorial example is found athttps://github.com/B-UMMI/chewBBACA_tutorial.I/We confirm all supporting data, code and protocols have been provided within the article or through supplementary data files. ⊠IMPACT STATEMENTThe chewBBACA software offers a computational solution for the creation, evaluation and use of whole genome (wg) and core genome (cg) multilocus sequence typing (MLST) schemas. It allows researchers to develop wg/cgMLST schemes for any bacterial species from a set of genomes of interest. The alleles identified by chewBBACA correspond to potential coding sequences, possibly offering insights into the correspondence between the genetic variability identified and phenotypic variability. The software performs allele calling in a matter of seconds to minutes per strain in a laptop but is easily scalable for the analysis of large datasets of hundreds of thousands of strains using multiprocessing options. The chewBBACA software thus provides an efficient and freely available open source solution for gene-by-gene methods. Moreover, the ability to perform these tasks locally is desirable when the submission of raw data to a central repository or web services is hindered by data protection policies or ethical or legal concerns.

Recent genome-wide association studies of glioma have led to the discovery of single nucleotide polymorphisms (SNPs) at 25 loci influencing risk. Gliomas are heterogeneous, hence to investigate the relationship between risk SNPs and glioma subtype we analysed 1659 tumours profiled for IDH mutation, TERT promoter mutation and 1p/19q co-deletion. These data allowed definition of five molecular subgroups of glioma: triple-positive (IDH mutated, 1p/19q co-deletion, TERT promoter mutated); TERT-IDH (IDH mutated, TERT promoter mutated, 1p/19q-wild-type); IDH-only (IDH mutated, 1p/19q wild-type, TERT promoter wild-type); triple-negative (IDH wild-type, 1p/19q wild-type, TERT promoter wild-type) and TERT-only (TERT promoter mutated, IDH wild-type, 1p/19q wild-type). Most glioma risk loci showed subtype specificity: (1) the 8q24.21 SNP for triple-positive glioma; (2) 5p15.33, 9p21.3, 17p13.1 and 20q13.33 SNPs for TERT-only glioma; (3) 1q44, 2q33.3, 3p14.1, 11q21, 11q23.3, 14q12, and 15q24.2 SNPs for IDH mutated glioma. To link risk SNPs to target candidate genes we analysed Hi-C and gene expression data, highlighting the potential role of IDH1 at 2q33.3, MYC at 8q24.21 and STMN3 at 20q13.33. Our observations provide further insight into the nature of susceptibility to glioma. Electronic supplementary material The online version of this article (10.1007/s00401-018-1825-z) contains supplementary material, which is available to authorized users.

// Michael T. Barrett 1 , Karen S. Anderson 2 , Elizabeth Lenkiewicz 1 , Mariacarla Andreozzi 1 , Heather E. Cunliffe 3 , Christine L. Klassen 4 , Amylou C. Dueck 5 , Ann E. McCullough 6 , Srikanth K. Reddy 7 , Ramesh K. Ramanathan 8 , Donald W. Northfelt 8 , Barbara A. Pockaj 4 1 Department of Research, Mayo Clinic in Arizona, Scottsdale, Arizona, United States of America 2 Biodesign Institute, Arizona State University, Tempe, Arizona, United States of America 3 Department of Pathology, Dunedin School of Medicine, University of Otago, Dunedin, New Zealand 4 Division of General Surgery, Section of Surgical Oncology, Mayo Clinic in Arizona, Phoenix, Arizona, United States of America 5 Section of Biostatistics, Mayo Clinic in Arizona, Scottsdale, Arizona, United States of America 6 Department of Pathology and Laboratory Medicine, Mayo Clinic in Arizona, Scottsdale, Arizona, United States of America 7 Vanderbilt University, Nashville, Tennessee, United States of America 8 Division of Hematology-Oncology, Mayo Clinic in Arizona, Scottsdale, Arizona, United States of America Correspondence to: Michael T. Barrett, e-mail: Barrett.michael@mayo.edu Keywords: 9p24.1 amplicon, flow sorting, triple negative breast cancer, JAK2, PD-L1 Received: May 05, 2015 Accepted: June 22, 2015 Published: July 03, 2015 ABSTRACT We used DNA content flow cytometry followed by oligonucleotide array based comparative genomic hybridization to survey the genomes of 326 tumors, including 41 untreated surgically resected triple negative breast cancers (TNBC). A high level (log 2 ratio ≥1) 9p24 amplicon was found in TNBC (12/41), glioblastomas (2/44), and colon carcinomas (2/68). The shortest region of overlap for the amplicon targets 9p24.1 and includes the loci for PD-L1 , PD-L2 , and JAK2 (PDJ amplicon). In contrast this amplicon was absent in ER+ (0/8) and HER2+ (0/15) breast tumors, and in pancreatic ductal adenocarcinomas (0/150). The PDJ amplicon in TNBCs was correlated with clinical outcomes in group comparisons by two-sample t -tests for continuous variables and chi-squared tests for categorical variables. TNBC patients with the PDJ amplicon had a worse outcome with worse disease-free and overall survival. Quantitative RT-PCR confirmed that the PDJ amplicon in TNBC is associated with elevated expression of JAK2 and of the PD-1 ligands. These initial findings demonstrate that the PDJ amplicon is enriched in TNBC, targets signaling pathways that activate the PD-1 mediated immune checkpoint, and identifies patients with a poor prognosis.

Motivation MicroRNAs are a class of ∼21–22 nt small RNAs which are excised from a stable hairpin-like secondary structure. They have important gene regulatory functions and are involved in many pathways including developmental timing, organogenesis and development in eukaryotes. There are several computational tools for miRNA detection from next-generation sequencing datasets. However, many of these tools suffer from high false positive and false negative rates. Here we present a novel miRNA prediction algorithm, miRCat2. miRCat2 incorporates a new entropy-based approach to detect miRNA loci, which is designed to cope with the high sequencing depth of current next-generation sequencing datasets. It has a user-friendly interface and produces graphical representations of the hairpin structure and plots depicting the alignment of sequences on the secondary structure. Results We test miRCat2 on a number of animal and plant datasets and present a comparative analysis with miRCat, miRDeep2, miRPlant and miReap. We also use mutants in the miRNA biogenesis pathway to evaluate the predictions of these tools. Results indicate that miRCat2 has an improved accuracy compared with other methods tested. Moreover, miRCat2 predicts several new miRNAs that are differentially expressed in wild-type versus mutants in the miRNA biogenesis pathway. Availability and Implementation miRCat2 is part of the UEA small RNA Workbench and is freely available from http://srna-workbench.cmp.uea.ac.uk/. Contact v.moulton@uea.ac.uk or s.moxon@uea.ac.uk Supplementary information Supplementary data are available at Bioinformatics online.

The timing of de novo DNA methylation in male germ cells during human testicular development is yet unsolved. Apart from that, the stability of established imprinting patterns in vitro is controversially discussed. This study aimed at determining the timing of DNA de novo methylation and at assessing the stability of the methylation status in vitro. We employed the marmoset monkey (Callithrix jacchus) as it is considered the best non-human primate model for human testicular development. We selected neonatal, pre-pubertal, pubertal, and adult animals (n = 3, each) and assessed germ cell global DNA methylation levels by 5-methyl cytosine staining, and Alu elements and gene-specific methylation (H19, LIT1, SNRPN, MEST, OCT4, MAGE-A4, and DDX-4) by pyrosequencing. De novo methylation is progressively established during postnatal primate development and continues until adulthood, a process that is different in most other species. Importantly, once established, methylation patterns remained stable, as demonstrated using in vitro cultures. Thus, the marmoset monkey is a unique model for the study of postnatal DNA methylation mechanisms in germ cells and for the identification of epimutations and their causes.

Highlight Identification of three quantitative trait loci associated with root bark percentage in apple rootstocks is predictive of rootstock-induced dwarfing of the scion., Rootstock-induced dwarfing of apple scions revolutionized global apple production during the twentieth century, leading to the development of modern intensive orchards. A high root bark percentage (the percentage of the whole root area constituted by root cortex) has previously been associated with rootstock-induced dwarfing in apple. In this study, the root bark percentage was measured in a full-sib family of ungrafted apple rootstocks and found to be under the control of three loci. Two quantitative trait loci (QTLs) for root bark percentage were found to co-localize to the same genomic regions on chromosome 5 and chromosome 11 previously identified as controlling dwarfing, Dw1 and Dw2, respectively. A third QTL was identified on chromosome 13 in a region that has not been previously associated with dwarfing. The development of closely linked sequence-tagged site markers improved the resolution of allelic classes, thereby allowing the detection of dominance and epistatic interactions between loci, with high root bark percentage only occurring in specific allelic combinations. In addition, we report a significant negative correlation between root bark percentage and stem diameter (an indicator of tree vigour), measured on a clonally propagated grafted subset of the mapping population. The demonstrated link between root bark percentage and rootstock-induced dwarfing of the scion leads us to propose a three-locus model that is able to explain levels of dwarfing from the dwarf ‘M.27’ to the semi-invigorating rootstock ‘M.116’. Moreover, we suggest that the QTL on chromosome 13 (Rb3) might be analogous to a third dwarfing QTL, Dw3, which has not previously been identified.

The transcription factor MYB has a key role in hematopoietic progenitor cells (HPCs) lineage choice, by enhancing erythropoiesis at the expense of megakaryopoiesis. We previously demonstrated that MYB controls erythroid versus megakaryocyte lineage decision by transactivating KLF1 and LMO2 expression. To further unravel the molecular mechanisms through which MYB affects lineage fate decision, we performed the integrative analysis of miRNA and mRNA changes in MYB-silenced human primary CD34+ HPCs. Among the miRNAs with the highest number of predicted targets, we focused our studies on hsa-miR-486-3p by demonstrating that MYB controls miR-486-3p expression through the transactivation of its host gene, ankyrin-1 (ANK1) and that miR-486-3p affects HPCs commitment. Indeed, overexpression and knockdown experiments demonstrated that miR-486-3p supports the erythropoiesis while restraining the megakaryopoiesis. Of note, miR-486-3p also favors granulocyte differentiation while repressing the macrophage differentiation. To shed some light on the molecular mechanisms through which miR-486-3p affects HPCs lineage commitment, we profiled the gene expression changes upon miR-486-3p overexpression in CD34+ cells. Among the genes downregulated in miR-486-3p-overexpressing HPCs and computationally predicted to be miR-486-3p targets, we identified MAF as a miR-486-3p target by 3'UTR luciferase reporter assay. Noteworthy, MAF overexpression was able to partially reverse the effects of miR-486-3p overexpression on erythroid versus megakaryocyte lineage choice. Moreover, the MYB/MAF co-silencing constrained the skewing of erythroid versus megakaryocyte lineage commitment in MYB-silenced CD34+ cells, by restraining the expansion of megakaryocyte lineage while partially rescuing the impairment of erythropoiesis. Therefore, our data collectively demonstrate that MYB favors erythropoiesis and restrains megakaryopoiesis through the transactivation of miR-486-3p expression and the subsequent downregulation of MAF. As a whole, our study uncovers the MYB/miR-486-3p/MAF axis as a new mechanism underlying the MYB-driven control of erythroid versus megakaryocyte lineage fate decision.