Parental micronutrient deficiency distorts liver DNA methylation and expression of lipid genes associated with a fatty-liver-like phenotype in offspring

Micronutrient status of parents can affect long term health of their progeny. Around 2 billion humans are affected by chronic micronutrient deficiency. In this study we use zebrafish as a model system to examine morphological, molecular and epigenetic changes in mature offspring of parents that experienced a one-carbon (1-C) micronutrient deficiency. Zebrafish were fed a diet sufficient, or marginally deficient in 1-C nutrients (folate, vitamin B12, vitamin B6, methionine, choline), and then mated. Offspring livers underwent histological examination, RNA sequencing and genome-wide DNA methylation analysis. Parental 1-C micronutrient deficiency resulted in increased lipid inclusion and we identified 686 differentially expressed genes in offspring liver, the majority of which were downregulated. Downregulated genes were enriched for functional categories related to sterol, steroid and lipid biosynthesis, as well as mitochondrial protein synthesis. Differential DNA methylation was found at 2869 CpG sites, enriched in promoter regions and permutation analyses confirmed the association with parental feed. Our data indicate that parental 1-C nutrient status can persist as locus specific DNA methylation marks in descendants and suggest an effect on lipid utilization and mitochondrial protein translation in F1 livers. This points toward parental micronutrients status as an important factor for offspring health and welfare.publishedVersio

Paternal chronic colitis causes epigenetic inheritance of susceptibility to colitis.

Inflammatory bowel disease (IBD) arises by unknown environmental triggers in genetically susceptible individuals. Epigenetic regulation of gene expression may integrate internal and external influences and may thereby modulate disease susceptibility. Epigenetic modification may also affect the germ-line and in certain contexts can be inherited to offspring. This study investigates epigenetic alterations consequent to experimental murine colitis induced by dextran sodium sulphate (DSS), and their paternal transmission to offspring. Genome-wide methylome- and transcriptome-profiling of intestinal epithelial cells (IECs) and sperm cells of males of the F0 generation, which received either DSS and consequently developed colitis (F0(DSS)), or non-supplemented tap water (F0(Ctrl)) and hence remained healthy, and of their F1 offspring was performed using reduced representation bisulfite sequencing (RRBS) and RNA-sequencing (RNA-Seq), respectively. Offspring of F0(DSS) males exhibited aberrant methylation and expression patterns of multiple genes, including Igf1r and Nr4a2, which are involved in energy metabolism. Importantly, DSS colitis in F0(DSS) mice was associated with decreased body weight at baseline of their F1 offspring, and these F1 mice exhibited increased susceptibility to DSS-induced colitis compared to offspring from F0(Ctrl) males. This study hence demonstrates epigenetic transmissibility of metabolic and inflammatory traits resulting from experimental colitis.This study was carried out as part of the Research Training Group “Genes, Environment and Inflammation”, supported by the Deutsche Forschungsgemeinschaft (RTG 1743/1) of which A.F. is the spokesperson, the European Research Council under the European Community’s Seventh Framework Programme (FP7/2007–2013)/ERC Grant agreement no. 260961 (A.K.), the Austrian Science Fund and Ministry of Science P21530-B18 and START Y446-B18 (A.K.), the Wellcome Trust (investigator award 106260/Z/14/Z) to A.K., the Cambridge Biomedical Research Centre (A.K.), a fellowship from the European Crohn’s and Colitis Organisation (M.T. and T.E.A.) and a DOC fellowship from the Austrian Academy of Sciences (J.K.).This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/srep3164

Genomic and phenotypic insights from an atlas of genetic effects on DNA methylation

Characterizing genetic influences on DNA methylation (DNAm) provides an opportunity to understand mechanisms underpinning gene regulation and disease. In the present study, we describe results of DNAm quantitative trait locus (mQTL) analyses on 32,851 participants, identifying genetic variants associated with DNAm at 420,509 DNAm sites in blood. We present a database of >270,000 independent mQTLs, of which 8.5% comprise long-range (trans) associations. Identified mQTL associations explain 15–17% of the additive genetic variance of DNAm. We show that the genetic architecture of DNAm levels is highly polygenic. Using shared genetic control between distal DNAm sites, we constructed networks, identifying 405 discrete genomic communities enriched for genomic annotations and complex traits. Shared genetic variants are associated with both DNAm levels and complex diseases, but only in a minority of cases do these associations reflect causal relationships from DNAm to trait or vice versa, indicating a more complex genotype–phenotype map than previously anticipated

Genomic and phenotypic insights from an atlas of genetic effects on DNA methylation.

Characterizing genetic influences on DNA methylation (DNAm) provides an opportunity to understand mechanisms underpinning gene regulation and disease. In the present study, we describe results of DNAm quantitative trait locus (mQTL) analyses on 32,851 participants, identifying genetic variants associated with DNAm at 420,509 DNAm sites in blood. We present a database of >270,000 independent mQTLs, of which 8.5% comprise long-range (trans) associations. Identified mQTL associations explain 15–17% of the additive genetic variance of DNAm. We show that the genetic architecture of DNAm levels is highly polygenic. Using shared genetic control between distal DNAm sites, we constructed networks, identifying 405 discrete genomic communities enriched for genomic annotations and complex traits. Shared genetic variants are associated with both DNAm levels and complex diseases, but only in a minority of cases do these associations reflect causal relationships from DNAm to trait or vice versa, indicating a more complex genotype–phenotype map than previously anticipated.C.L.R., G.D.S., G.S., J.L.M., K.B., M. Suderman, T.G.R. and T.R.G. are supported by the UK Medical Research Council (MRC) Integrative Epidemiology Unit at the University of Bristol (MC_UU_00011/1, MC_UU_00011/4, MC_UU_00011/5). C.L.R. receives support from a Cancer Research UK Programme grant (no. C18281/A191169). G.H. is funded by the Wellcome Trust and the Royal Society (208806/Z/17/Z). E.H. and J.M. were supported by MRC project grants (nos. MR/K013807/1 and MR/R005176/1 to J.M.) and an MRC Clinical Infrastructure award (no. MR/M008924/1 to J.M.). B.T.H. is supported by the Netherlands CardioVascular Research Initiative (the Dutch Heart Foundation, Dutch Federation of University Medical Centres, the Netherlands Organisation for Health Research and Development, and the Royal Netherlands Academy of Sciences) for the GENIUS project ‘Generating the best evidence-based pharmaceutical targets for atherosclerosis’ (CVON2011-19, CVON2017-20). J.T.B. was supported by the Economic and Social Research Council (grant no. ES/N000404/1). The present study was also supported by JPI HDHL-funded DIMENSION project (administered by the BBSRC UK, grant no. BB/S020845/1 to J.T.B., and by ZonMW the Netherlands, grant no. 529051021 to B.T.H). A.D.B. has been supported by a Wellcome Trust PhD Training Fellowship for Clinicians and the Edinburgh Clinical Academic Track programme (204979/Z/16/Z). J. Klughammer was supported by a DOC fellowship of the Austrian Academy of Sciences. Cohort-specific acknowledgements and funding are presented in the Supplementary Note

Microevolution of Neisseria lactamica during nasopharyngeal colonisation induced by controlled human infection.

Neisseria lactamica is a harmless coloniser of the infant respiratory tract, and has a mutually-excluding relationship with the pathogen Neisseria meningitidis. Here we report controlled human infection with genomically-defined N. lactamica and subsequent bacterial microevolution during 26 weeks of colonisation. We find that most mutations that occur during nasopharyngeal carriage are transient indels within repetitive tracts of putative phase-variable loci associated with host-microbe interactions (pgl and lgt) and iron acquisition (fetA promotor and hpuA). Recurrent polymorphisms occurred in genes associated with energy metabolism (nuoN, rssA) and the CRISPR-associated cas1. A gene encoding a large hypothetical protein was often mutated in 27% of the subjects. In volunteers who were naturally co-colonised with meningococci, recombination altered allelic identity in N. lactamica to resemble meningococcal alleles, including loci associated with metabolism, outer membrane proteins and immune response activators. Our results suggest that phase variable genes are often mutated during carriage-associated microevolution

Epigenomic and transcriptional determination of cellular identity

Einer der faszinierendsten Aspekte multizellulären Lebens ist die Ausbildung morphologisch und funktionell unterschiedlicher Zelltypen auf Basis derselben genomischen DNA-Sequenz. Molekularbiologische Forschungen haben das Zusammenspiel zwischen Genom, Epigenom und Transkriptom als zentral für die Regulation der zellulären Identität identifiziert. Technologische Fortschritte in der Hochdurchsatz-Sequenzierung bieten die Werkzeuge, diese Mechanismen im Detail zu untersuchen. Trotz des wissenschaftlichen Fortschritts sind viele fundamentale Fragen bisher unbeantwortet geblieben. Zum Beispiel haben Untersuchungen in Mensch und Maus gezeigt, dass die DNA-Methylierung in Säugetieren wichtig für die Festlegung zellulärer Identität ist, was über alle Vertebraten hinweg konserviert zu seinen scheint. Invertebraten jedoch zeigen grundlegend andere DNA-Methylierungsmuster, und in manchen Spezies ist überhaupt keine DNA-Methylierung messbar. Dies wirft die Frage auf, wie und warum DNA-Methylierung in Vertebraten seine zelluläre Identität definierende Funktion erlangt hat. Solche evolutionären Fragen zu beantworten könnte zur Entdeckung neuer Funktionen der DNA-Methylierung führen. Um die Forschung in dieser Richtung voranzubringen haben wir eine computerbasierte Analysemethode (RefFreeDMA) entwickelt. Diese Methode ermöglicht differentielle DNA-Methylierungsanalysen auch ohne Referenzgenome, womit die Untersuchung von DNA-Methylierung in praktisch jeder Spezies möglich wird. Wir haben unseren Ansatz erfolgreich in drei Spezies (Mensch, Rind und Karpfen) validiert und planen diese Analysemethode zur Untersuchung gewebespezifischer DNA Methylierung in vielen weiteren Spezies zu verwenden. Im Kontext humaner Erkrankungen ist die klinische Relevanz von Veränderungen der zellulären Identität gut etabliert. Fehlentwicklungen zellulärer Identität wurden als fundamentaler Faktor in der Entstehung von Krebs erkannt. Mit Fokus auf dem Glioblastom, dem häufigsten und bösartigsten Tumor des adulten zentralen Nervensystems, haben wir in der Tumorprogression involvierte Prozesse untersucht, indem wir DNA-Methylierungsprofile von primären und rezidivierten Tumoren in 112 Patienten vergleichen haben. Diese Untersuchung hat ergeben, dass sowohl primäre als auch rezidivierte Tumore eine erhebliche Heterogenität in ihrer Subtyp-Zusammensetzung zeigen, dass sich die Glioblastom-Subtypen durch distinkte Epigenom-regulatorische Signaturen unterscheiden lassen und dass im Laufe der Tumorprogression eine Reduktion der DNA-Methylierung in Genen der Wnt-Signalkaskade auftritt. Unsere Ergebnisse beschreiben die Dynamik der DNA-Methylierung in der Progression von Glioblastomen und etablieren die Machbarkeit von DNA-Methylierungs-Studien, die auf im klinischen Routinebetrieb gesammelten Proben basieren. Im Bereich der regenerativen Medizin hat sich die Forschung intensiv mit der Möglichkeit beschäftigt, verlorengegangene Betazellen in Diabetes-Patienten durch die Umwandlung von anderen pankreatischen Inselzellen zu ersetzen. Um diese Bemühungen zu unterstützen, haben wir Transkriptome von 64 gesunden, humanen, einzelnen, pankreatischen Inselzellen produziert und ausgewertet. Wir konnten vier endokrine und zwei exokrine pankreatische Typen von Inselzellen identifizieren, was das Erstellen von zelltypspezifischen Expressionsprofilen ermöglichte. In einer darauf aufbauenden Studie, die das Transdifferenzierungspotential von Wirkstoffen kleiner molekularer Größe untersuchte, dienten diese Expressionsprofile dann als Referenz, um charakteristische Veränderungen in den Expressionsprofilen von mit Artemisininen behandelten Alphazellen festzustellen. Zusammen genommen trägt die hier präsentierte Arbeit biologisch und medizinisch relevante Ergebnisse zum Verständnis der zellulären Identität bei. Sie betont und fördert außerdem das Potential neuartiger Hochdurchsatz-Sequenzierungstechnologien für die biomedizinische Forschung.One of the most fascinating aspects of multicellular life is how a shared genomic sequence supports morphologically and functionally diverse cell types. Understanding the regulatory processes underlying cellular identity is therefore important to understanding multicellular life. With the advances of modern molecular biology, the interplay between genome, epigenome, and transcriptome has emerged as the leading determinant of cellular identity. Technological advances in high throughput sequencing have further provided the necessary tools to study these molecular determinants of cellular identity in detail. Despite substantial scientific progress, many fundamental questions remain unanswered. For example, studies conducted mainly in human and mouse have shown that DNA methylation is crucially involved in the determination of cellular identity, which appears to be conserved across vertebrates. Invertebrates, however, display fundamentally different DNA methylation patterns and in some species, DNA methylation cannot be detected at all. This raises the question of how and why DNA methylation acquired its defining role for cellular identity in vertebrates, what the role of DNA methylation in invertebrates might be, and how some higher organisms can exist without detectable levels of DNA methylation. Answering these evolutionary questions might identify yet unknown mechanisms involved in the determination of cellular identity or new functions of DNA methylation that may be relevant to human physiology. To advance research in this direction, we have developed a computational framework called RefFreeDMA that allows differential DNA methylation analysis without the need of a reference genome, enabling the assessment of DNA methylation in virtually any species. We successfully validated our approach in three species (human, cow, and carp) and plan to apply it next in the assessment of tissue specific DNA methylation across many more vertebrate and invertebrate species. In the context of human diseases, the clinical relevance of changes in cellular identity is widely accepted. Aberration of cellular identity has been recognized as a fundamental factor in cancerogenesis. Focusing on glioblastoma, the most common malignant tumour of the adult central nervous system, we assessed the processes involved in tumour progression by comparing the DNA methylation profiles of primary and recurring tumours in 112 patients. We found that primary and recurring tumours display considerable variability in their subtype compositions and identified subtype specific epigenome regulatory patterns and a loss of DNA methylation in Wnt signalling genes during progression. Our results chart the dynamics of DNA methylation in the progression of glioblastoma and establish the feasibility of conducting a DNA methylation study on samples collected in a routine clinical setting. Finally, the field of regenerative medicine has been actively researching the possibility to replenish lost pancreatic beta cell mass in diabetic patients by reprogramming the identity of other, more abundant cell types to beta cells. To support these efforts, we generated and analysed transcriptomes of 64 healthy human pancreatic islet cells by single-cell sequencing. We were able to identify four endocrine and two exocrine human pancreatic islet cell types, which allowed the generation of accurate, cell type specific expression profiles. In a subsequent study assessing the trans-differentiating potential of small molecule drugs, these expression profiles then served as reference to identify characteristic changes in the expression profiles of alpha cells upon treatment with Artemisinins. Taken together, the work presented in this thesis contributes biologically and medically relevant results to the understanding of cellular identity. Moreover, it emphasizes and promotes the promising potential of recent high throughput sequencing technology for the advancement of this field.submitted by Johanna KlughammerZusammenfassung in deutscher SpracheAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität Wien, Dissertation, 2017OeBB(VLID)223348

Divergent Expression Regulation of Gonad Development Genes in Medaka Shows Incomplete Conservation of the Downstream Regulatory Network of Vertebrate Sex Determination

Genetic control of male or female gonad development displays between different groups of organisms a remarkable diversity of “master sex-determining genes” at the top of the genetic hierarchies, whereas downstream components surprisingly appear to be evolutionarily more conserved. Without much further studies, conservation of sequence has been equalized to conservation of function. We have used the medaka fish to investigate the generality of this paradigm. In medaka, the master male sex-determining gene is dmrt1bY, a highly conserved downstream regulator of sex determination in vertebrates. To understand its function in orchestrating the complex gene regulatory network, we have identified targets genes and regulated pathways of Dmrt1bY. Monitoring gene expression and interactions by transgenic fluorescent reporter fish lines, in vivo tissue-chromatin immunoprecipitation and in vitro gene regulation assays revealed concordance but also major discrepancies between mammals and medaka, notably amongst spatial, temporal expression patterns and regulations of the canonical Hedgehog and R-spondin/Wnt/Follistatin signaling pathways. Examination of Foxl2 protein distribution in the medaka ovary defined a new subpopulation of theca cells, where ovarian-type aromatase transcriptional regulation appears to be independent of Foxl2. In summary, these data show that the regulation of the downstream regulatory network of sex determination is less conserved than previously thought

Cytoplasmic flows in starfish oocytes are fully determined by cortical contractions.

Cytoplasmic flows are an ubiquitous feature of biological systems, in particular in large cells, such as oocytes and eggs in early animal development. Here we show that cytoplasmic flows in starfish oocytes, which can be imaged well with transmission light microscopy, are fully determined by the cortical dynamics during surface contraction waves. We first show that the dynamics of the oocyte surface is highly symmetric around the animal-vegetal axis. We then mathematically solve the Stokes equation for flows inside a deforming sphere using the measured surface displacements as boundary conditions. Our theoretical predictions agree very well with the intracellular flows quantified by particle image velocimetry, proving that during this stage the starfish cytoplasm behaves as a simple Newtonian fluid on the micrometer scale. We calculate the pressure field inside the oocyte and find that its gradient is too small as to explain polar body extrusion, in contrast to earlier suggestions. Myosin II inhibition by blebbistatin confirms this conclusion, because it diminishes cell shape changes and hydrodynamic flow, but does not abolish polar body formation

SM-Omics: An automated platform for high-throughput spatial multi-omics

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