Measuring stress-induced DNA methylation in apomictic Dandelions
Gurp, Thomas P. van - \ 2017
Wageningen University. Promotor(en): W.H. van der Putten, co-promotor(en): K.J.F. Verhoeven; A. Biere. - Wageningen : Wageningen University - ISBN 9789463436045 - 176
taraxacum officinale - epigenetics - dna methylation - inheritance - apomixis - environmental factors - taraxacum officinale - epigenetica - dna-methylering - overerving - apomixis - milieufactoren
The success or continuous existence of species requires continuous adaptation to changes in the environment to survive and contribute offspring to the next generation. Selection acts on the phenotype, which is in turn determined by the complex interplay of genetic, epigenetic and environmental variation. (Natural) selection leads to ‘survival of the fittest’ or best-adapted individuals to their local environment, ultimately determining which individuals contribute offspring to the next generation. Understanding the mechanisms by which epigenetic and genetic variation can arise and get passed on through generations determines our understanding of inheritance and evolution. Hitherto, the mechanistic understanding of genetics has shaped the scientific view of inheritance and evolution, leading to the gene-centered paradigm of Neo-Darwinism. However, recent studies indicate that besides genetic (DNA sequence) variation, epigenetic variation can also be transmitted between generations. Further studies on the properties and transgenerational dynamics of epigenetic variation are needed to enhance our understanding of heritability and evolution.
Epigenetic variation has distinct properties and different transgenerational dynamics compared to genetic variation. Epigenetic variation helps to regulate gene expression and determines the different cell types and function in eukaryotes. The main function of DNA methylation, an important part of the epigenetic code, is to prevent the spread of selfish genetic elements in the genome and to establish the different cellular profiles observed in multicellular organisms. One differentiating feature of epigenetic variation compared to genetic variation is that (specific) epigenetic variation can arise under the influence of stress. This can enable a trans-generational stress-response of organisms which can have a positive influence on the phenotype and (natural) selection on either the (enhanced level of) transgenerational phenotypic plasticity or the epigenetic variation itself, potentially influencing natural selection and ultimately evolution. Where genetic variation can be characterized as hard-inheritance, the inheritance of epigenetic variation is often referred to as ‘soft-inheritance’ due to the lower transgenerational stability and resetting that occurs in the intergenerational transfer of epigenetic variation. Epigenetic variation is also often dependent on, or a downstream consequence, of genetic variation, suggesting that it is (in part) determined by genetic variation.
Mechanistic studies in model species have contributed greatly to the understanding of the molecular mechanisms that control the dynamics of different epigenetic marks present in multicellular organisms. In plants, studies in the model plant Arabidopsis thaliana have resulted in deciphering the most important molecular mechanisms and actors, giving an ever-increasing insight into the dynamics of epigenetic regulation of cells and organisms. A key feature of model systems is the ability to ‘switch’ off certain genes or molecular pathways, for instance via the experimental use of mutants, enabling the study of their role in the heritability of epigenetic marks. DNA methylation is a well-studied epigenetic mark, which has shown high stability even in transgenerational experiments.
From the perspective of studying epigenetic variation, plants are particularly interesting for several reasons, most importantly: 1) The separation between soma and germline, the Weismann barrier, is less strict in plants compared to other eukaryotes, as in higher plants
germline cells are formed during floral development from somatic cells (which can occur throughout the life of the plant), whereas in most eukaryotes germline cell development is restricted to a defined point (early) in the organismal development. 2) The sessile nature of plants makes an adaptive plastic response to changing environments an important feature, a plant cannot just walk away when the going gets tough. 3) The transgenerational stability of DNA methylation is higher in plants compared to other eukaryotes such as mammals, in which epigenetic information is erased during germline reprogramming. These factors combined suggest that the potential importance of epigenetic variation in plants might be high.
In this thesis, I focus on studying DNA methylation in apomictic Dandelions, applying Next Generation Sequencing (NGS) approaches to the study of this non-model plant species. Apomictic dandelions produce seeds that are genetically identical to the ‘mother’ plant, which makes it easier to study the influence of epigenetic variation without confounding effects of genetic variation. Working with Next Generation Sequencing data is still relatively new and therefore not always optimized for specific types of analysis. I discovered a distinct error pattern in RNAseq data that indicated an artificial source of variation that could be traced back to the way the RNAseq libraries were constructed. The first publication of this thesis contains a technical analysis of such artefacts present in RNAseq data, suggesting that these errors are related to random hexamer mispriming during library construction (Chapter 2).
The main goal of my work is to better understand the role of epigenetic variation in adaptation and plasticity of plants. This role remains poorly understood. This is in part due to the lack of high-resolution techniques that allow for the detailed study of epigenetic marks such as DNA methylation in non-model organisms. Existing techniques for measuring DNA methylation such as methylation sensitive AFLPs offer only information on DNA methylation variation in an anonymous and limited fashion. The plummeting costs of sequencing techniques have enabled large-scale genotyping efforts (focusing on genetic variation only) for a wide variety of non-model organisms. Here, I extended this popular genotyping by sequencing technique, to allow for sequencing-based epigenotyping or epiGBS (chapter 3), which allows for measuring DNA methylation and genetic variation in hundreds of samples simultaneously. I have extensively validated the approach, providing evidence that with the right design, the accuracy of the DNA methylation measurements with epiGBS are as high as those with the gold standard Whole Genome Bisulfite Sequencing.
An important aim of my PhD research was to investigate the stability of (stress induced) DNA methylation variation in apomictic dandelions and the potential of phenotypic variation underpinned by DNA methylation variation to be subjected to selection. I therefore studied the transgenerational stability of both stress induced and natural DNA methylation variation in different genotypes of apomictic dandelions in a six-generation experiment, comparing DNA methylation patterns between generations and tracking changes in them (chapter 4) using epiGBS. I found clear but limited evidence for environmental induction of heritable DNA methylation changes after application of Jasmonic Acid. Furthermore, I found a significant negative relation between the similarity of DNA methylation patterns and intergenerational distance, indicating epigenetic divergence over generations. I conclude that DNA methylation in both CG and CHG (where H can be any nucleotide except for G) sequence context are heritable and that environmental perturbation can result in heritable DNA methylation changes which are however not widespread.
A prerequisite for epigenetic variation to contribute to adaptation is that epigenetic variants that affect the phenotype are heritable. To test whether an epigenetics-based selection response is possible, at least over the time course of a few generations, I selected early flowering for two subsequent generations in three genotypes of apomictic dandelions. This selection effort included lines that received a stress pre-treatment with either Jasmonic Acid or 5-azacytidine, to determine if stress-induced DNA methylation variation would increase the capacity to respond to selection. The selection experiment on flowering time (chapter 5) resulted in a shift in flowering time for all treatments in a young apomict, suggesting that natural and heritable epigenetic variation can underpin quantitative traits such as flowering time. I also found evidence for treatment induced (epi)genetic variation leading to a stronger selection response in one out of 3 genotypes. This suggests that stress- induced heritable epigenetic variation can lead to a selection response. Further study is however required to rule out genetic variants and to study the long-term stability of the variation selected upon.
Finally, in the General Discussion I summarize the findings, putting them in context with recently published studies. I reflect on the state of the field of ecological epigenetics and in what sense the epiGBS technique that I developed and other emerging techniques can contribute to a better understanding of the role of epigenetic variation in ecology and evolution. I reflect on the place of epiGBS compared to other techniques. I point out that with the growing evidence of the inadequacy and misinterpretation of MS-AFLP results a systematic review of such results by replicating the studies employing sequencing based techniques such as epiGBS instead of MS-AFLP is in order.
Epigenetic inheritance in apomictic dandelions : stress-induced and heritable modifications in DNA methylation and small RNA
Preite, V. - \ 2016
Wageningen University. Promotor(en): Wim van der Putten, co-promotor(en): K.J.F. Verhoeven. - Wageningen : Wageningen University - ISBN 9789462578715 - 152
taraxacum officinale - epigenetics - inheritance - apomixis - dna methylation - rna - heritability - stress - taraxacum officinale - epigenetica - overerving - apomixis - dna-methylering - rna - heritability - stress
Epigenetic variation, such as changes in DNA methylations, regulatory small RNAs (sRNAs) and chromatin modifications can be induced by environmental stress. There is increasing information that such induced epigenetic modifications can be transmitted to offspring, potentially mediating adaptive transgenerational responses to environmental changes. However, it is unclear if this phenomenon is common and relevant for adaptation under natural conditions. My thesis study aimed to examine epigenetic inheritance in common and widespread apomictic dandelions (Taraxacum officinale Wig.). Due to their asexual reproduction mode by producing clonal seeds offspring from seeds are genetically uniform and thus suitable to investigate epigenetic effects that are not confounded with genetic variation.
I exposed apomictic dandelion lineages to drought and salicylic acid (SA) stress, which induces plant defense responses following pathogen attack, and found effects on patterns of DNA methylation up to two stress-free offspring generations after exposure. However, a heritable stress signal was not present in all tests and was stress- and lineage-dependent. Drought stress triggered a weak and lineage-dependent signal that was lost again in the second offspring generation. SA treatment revealed a stress-related increased rate of DNA methylation changes in the two offspring generations, but no stress signal was found in the stressed generation itself. I also observed changes in small RNA production due the drought and SA stress experienced two generations ago. These transgenerational sRNA effects showed association with gene functions related to grandparental drought and SA stress, which suggests functional relevance of the transgenerational effects.
I used a reciprocal transplantation field experiment to investigate whether exposing dandelions to natural field stresses also triggers DNA methylation changes. The experiment revealed evidence of adaptive divergence between the populations, suggesting that non-native habitats are experienced as more stressful. However, under these field conditions no induction-based DNA methylation changes were found that persisted into offspring.
By using AFLP and MS-AFLP screening of natural apomictic dandelion populations across a north-south transect in Europe I examined if natural, heritable DNA methylation variation reflects underlying genetic variation, or if it shows patterns that are not predictable from underlying genetics. I found that a large part of heritable DNA methylation differentiation along the north-south transect was correlated with genetic differentiation. However, a fraction of differentiation in heritable DNA methylation was independent from genetic variation. This suggests a potential of epigenetics to play an evolutionary role independently, at least to some extent, from underlying genetics. Overall, I found indications of epigenetic inheritance in apomictic dandelions. Whether epigenetic variation would result in adaptive phenotypic variation in nature and whether it would persist long enough to play a relevant role in adaptation remains unclear and requires further study.
Adventitious root formation in Arabidopsis : underlying mechanisms and applications
Massoumi, Mehdi - \ 2016
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Geert-Jan de Klerk; Frans Krens. - Wageningen : Wageningen University - ISBN 9789462578524 - 191
arabidopsis thaliana - adventitious roots - formation - plant development - quantitative traits - etiolation - auxins - explants - molecular biology - gene expression - dna methylation - rooting - ontogeny - plant breeding - arabidopsis thaliana - adventiefwortels - formatie - plantenontwikkeling - kwantitatieve kenmerken - etiolering - auxinen - explantaten - moleculaire biologie - genexpressie - dna-methylering - beworteling - ontogenie - plantenveredeling
Adventitious root (AR) formation is indispensable in vegetative propagation and is widely used. A better understanding of the underlying mechanisms is needed to improve rooting treatments. We first established a system to study rooting in Arabidopsis, the model organism in plant biology but only occasionally used to study adventitious rooting. Inhibition of polar auxin transport reduced AR formation. The role of auxin transporter proteins (several PIN-proteins) was found to be tissue-specific. Maturation (the transition from juvenile to adult) negatively influenced AR formation. Maturation was associated with increased DNA methylation and decreased miR156 level. 5-Azacytidine, a drug that reduces DNA methylation, increased rooting. We also examined the effect of two donor plant pre-treatments, etiolation and flooding, on rooting. Both increased AR formation.
Persistent organic pollutants : aberrant DNA methylation underlying potential health effects
Dungen, M.W. van den - \ 2016
Wageningen University. Promotor(en): Tinka Murk; Ellen Kampman, co-promotor(en): Wilma Steegenga; Dieuwertje van Gils-Kok. - Wageningen : Wageningen University - ISBN 9789462577893 - 207
persistent organic pollutants - dna methylation - molecular genetics - epigenetics - health hazards - toxic substances - endocrine disruptors - eels - fish consumption - toxicology - persistente organische verontreinigende stoffen - dna-methylering - moleculaire genetica - epigenetica - gezondheidsgevaren - toxische stoffen - hormoonverstoorders - palingen - visconsumptie - toxicologie
Wild caught fish, especially marine fish, can contain high levels of persistent organic pollutants (POPs). In the Netherlands, especially eel from the main rivers have high POP levels. This led to a ban in 2011 on eel fishing due to health concerns. Many of the marine POPs have been related to adverse health effects such as endocrine disruption, neurodevelopmental problems, immune suppression and cancer. Although some mechanisms of action of POPs are clear, like dioxins binding to the aryl hydrocarbon receptor and OH-PCBs binding to thyroid transport proteins, not all adverse health effects can be explained by these mechanisms of action. Epigenetic phenomena, such as DNA methylation, have been proposed as a possible molecular mechanism underlying adverse health effects. DNA methylation is a heritable modification, which refers to the addition of a methyl group to cytosine in a CpG dinucleotide. Observational studies have indeed shown that POPs can affect global DNA methylation, although results are inconsistent. Some animal studies as well as in vitro experiments suggest that POPs can affect gene-specific DNA methylation, however, the biological significance and relevance for humans is not clear. Therefore, this thesis aimed to 1) study the accumulation of POPs in men consuming eel from high-polluted areas 2) elucidate whether seafood-related POPs can induce aberrant DNA methylation and 3) to determine whether DNA methylation is related to functional endpoints and gene expression in vitro.
For this purpose eight POPs that are abundantly present in seafood were chosen, namely 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorobiphenyl (PCB) 126 and 153, perfluorooctanesulfonic acid (PFOS), hexabromocyclododecane (HBCD), 2,2′,4,4′- tetrabromodiphenyl ether (BDE-47), tributyltin (TBT), and methylmercury (MeHg). Chapter 2 describes the in vitro effects of these POPs and mixtures thereof in H295R adrenocortical carcinoma cells. Relative responses for 13 steroid hormones and 7 genes involved in the steroidogenic pathway, and CYP1A1, were analysed. PFOS induced the most pronounced effects on steroid hormone levels by significantly affecting 9 out of 13 hormone levels measured, with the largest increases found for 17β-estradiol, corticosterone, and cortisol. Furthermore, TCDD, both PCBs, and TBT significantly altered steroidogenesis. Increased steroid hormone levels were accompanied by related increased gene expression levels. The differently expressed genes were MC2R, CYP11B1, CYP11B2, and CYP19A1 and changes in gene expression levels were more sensitive than changes in hormone levels. The POP mixtures tested showed mostly additive effects, especially for DHEA and 17β-estradiol levels. This study shows that some seafood POPs are capable of altering steroidogenesis in H295R cells at concentrations that mixtures might reach in human blood, suggesting that adverse health effects cannot be excluded. DNA methylation was not measured in this study due to the short exposure time, which was expected not to be sufficient for long-term epigenetic marks. Therefore, in chapters 3A and 3B a differentiation experiment was performed enabling long-term exposure to POPs. Human mesenchymal stem cells (hMSCs) were differentiated into mature adipocytes over a time-course of 10 days. The transcriptional regulatory cascade involved in adipocyte differentiation has been extensively studied, however the mechanisms driving the transcription are poorly understood. In chapter 3A we therefore first explored the involvement of DNA methylation in transcriptional regulation during adipocyte differentiation. Genome-wide changes in DNA methylation were measured as well as the expression of adipogenic genes. The majority of these genes showed significant expression changes during the differentiation process. There were, however, only a couple of these differentially expressed genes that were differentially methylated. Genome-wide DNA methylation changes were most often located in intergenic regions, and underrepresented close to the transcription start site. This suggested that changes in DNA methylation are not the underlying mechanism regulating gene expression during adipocyte differentiation. Nevertheless, we explored DNA methylation differences after continuous exposure to POPs to investigate whether this could be an underlying mechanism by which POPs affect adipocyte differentiation. TCDD and PFOS decreased lipid accumulation, while TBT increased lipid accumulation. TCDD and TBT induced opposite gene expression profiles, whereas after PFOS exposure gene expression remained relatively stable. Genome-wide DNA methylation analysis showed that all three POPs affected DNA methylation patterns in adipogenic and other genes, but without concomitant gene expression changes. Differential methylation was again predominantly detected in intergenic regions, where the biological relevance of alterations in DNA methylation is unclear. This study demonstrated that POPs, at environmentally relevant levels, are able to induce differential DNA methylation in differentiating adipocytes. However, the biological relevance of this aberrant DNA methylation remains unclear.
The in vitro results showed a proof of principle that POPs could be capable of altering DNA methylation. To this date, no human studies were performed investigating the relationship between POP levels and genome-wide DNA methylation. In order to investigate this, we first measured POP levels in eel consumers from the high-polluted areas (areas with a ban on eel fishing) and compared these levels to men consuming eel from low-polluted areas or aquaculture (chapter 4). We aimed to investigate the accumulation of these POPs and determine whether the predictions made in an earlier risk assessment were valid. This was indeed the case as levels of dioxins and dioxin-like compounds were on average 2.5 times higher in men consuming eel from high-polluted areas. Furthermore, PCBs with their hydroxylated metabolites, and perfluoroalkyl substances (PFASs) were, up to ten times, higher in these consumers. Especially the high levels of dioxins and dioxin-like compounds as well as the OH-PCBs are expected to be of health concern. We continued this research in chapter 5 by associating all the measured POPs to clinical parameters related to e.g. thyroid hormones and liver enzymes, but found no relationship. Subsequently, we investigated the association between dioxins and dioxin-like compounds, the sum of seven indicator PCBs, and PFOS with genome-wide DNA methylation. We detected a number of differentially methylated regions (DMRs) related to genes involved in carcinogenesis (e.g. BRCA1, MAGEE2, HOXA5), the immune system (e.g. RNF39, HLA-DQB1), in retinol homeostasis (DHRS4L2), or in metabolism (CYP1A1). In contrast to the in vitro data, most significant effects were detected in CpG islands and were annotated close to the promoter region. This suggests that the differential methylation might be related to differential expression and possibly induce adverse health effects. The hypermethylation of some of these gene related to cancer could be an explanation of the carcinogenic effects that are observed with POP exposure.
Based on the results of this thesis we can conclude that the consumption of eel from high-polluted areas lead to accumulation of POPs above safe levels and that POP levels are associated with gene-specific DNA methylation in vitro as well as in environmentally exposed men. More research, however, is needed to fully elucidate the biological implications of this aberrant DNA methylation. A first step can be to measure histone modifications, as these two epigenetic marks together are likely better in predicting gene expression. The second step can be to investigate the potential health effects related to these epigenetic marks and to determine whether there is a causal relationship. Although at this point there is a lack of knowledge with regard to health effects caused by DNA methylation, the consumption of eel from these high-polluted areas is ill- advised, because adverse health effects cannot be excluded based on our results and can even be expected based on literature.
Opportunities of New Plant Breeding Techniques
Schaart, Jan ; Riemens, M.M. ; Wiel, C.C.M. van de; Lotz, L.A.P. ; Smulders, M.J.M. - \ 2015
Wageningen : Wageningen UR - 24
plantenveredeling - plantenveredelingsmethoden - resistentieveredeling - cisgenese - intragenic recombination - mutagenese - dna-methylering - bloei - plant breeding - plant breeding methods - resistance breeding - cisgenesis - intragenic recombination - mutagenesis - dna methylation - flowering
This brochure gives an overview of new plant breeding techniques. This overview is based on a more technical review of the scientific literature, published in a separate report. The overview presents the opportunities and limitations of these techniques from the point of view of potential applications in plant breeding with promising results for improving agricultural sustainability.
Epigenetics, an update
Nap, J.P.H. ; Geurts van Kessel, A. - \ 2011
Wageningen : Plant Research International (Rapport / Plant Research International 397)
epigenetica - genen - dna - rna - genexpressie - dna-modificatie - dna-methylering - epigenetics - genes - dna - rna - gene expression - dna modification - dna methylation
Chromatin organisation during Arabidopsis root development
Lorvellec, M. - \ 2007
Wageningen University. Promotor(en): Ton Bisseling, co-promotor(en): Hans de Jong; Olga Kulikova. - [S.l.] : S.n. - ISBN 9789085046219 - 121
arabidopsis - chromatine - wortels - plantenontwikkeling - genen - dna - wortelmeristemen - dna-methylering - arabidopsis - chromatin - roots - plant development - genes - dna - root meristems - dna methylation
The genetic information is stored in a highly compact manner in every nucleus. About 150 bp of DNA is packed around a histone octamer constituting a nucleosome. Nucleosomes are linked together by histone H1 and further compaction of this "beads on a string" form higher-order chromatin structures. DNA staining reveals two cytologically different chromatin states: weakly stained euchromatin and brightly stained heterochromatin. Euchromatin is gene-rich and decondensed during interphase, whereas heterochromatin is rich in repetitive sequences, low in gene density, and remains mostly condensed throughout the cell cycle. Euchromatin and heterochromatin differ also by their epigenetic modifications. Epigenetic modifications of chromatin are for example methylated cytosine and acetylation or methylation of histones tails. Acetylation of histones is in general a mark of euchromatin, whereas DNA methylation and histone methylation are marks of heterochromatin. To access the chromatin to perform processes such as DNA replication or to modify the expression of a gene, chromatin remodelling is necessary and performed by chromatin modifiers such as Heterochromatin Protein 1.In this thesis, we studied how chromatin is organised through development of the root ofArabidopsis. This model plant has a simple organized root meristem. Further the distribution of eu- and heterochromatin in interphase nuclei is rather simple. This allowsus to follow the chromatin organisation of a cell through development from stem cell into a fully differentiated cell.DNA methylation is one of the most abundant epigenetic modifications and varies through development. It is involved in the defence of the genome against transposable elements and retroviruses, in the control of genomic imprinting and in the regulation of gene expression.In Arabidopsis, we showed that Quiescent Center (QC) cells and stem cells are highly methylated contrarily to stem cells in animals. When cells divide their DNA methylation level decreases to increase again when cells differentiate. DRM1 and DRM2, de novo DNA methyltransferases, and HDA1, a histone deacetyltransferase, appear to be involved in establishing the hypermethylated DNA state in nuclei of QC and stem cells.Heterochromatin Protein 1 in animals is a chromatin modifier first discovered as a protein involved in heterochromatin formation. Nowadays it is thought to be a bridging protein, connecting histones through its chromodomain and non-histone chromosomal proteins through its chromoshadow domain. The homologue of Heterochromatin Protein 1 in Arabidopsis is Like Heterochromatin Protein 1 (LHP1). LHP1 was shown to be located in the euchromatic part of interphase nuclei like the animal isoform HP1γ and to form foci in differentiated cells. We showed that these foci are most likely chromatin complexes bound to the DNA and that LHP1 binds probably trimethylated lysine 9 and/or trimethylated lysine 27 of histone H3.HP1 in animal was shown to bind to trimethylated lysine 9 of histone H3 (H3K9m3) and to interact with the H3K9 trimethyltransferaseSU(VAR)3-9. In Arabidopsis, we tried to identify among the family ofSU(VAR)3-9 homologues, the SUVH proteins, which is responsible for trimethylating H3K9 and might interact with LHP1. We showed that SUVH3, SUVH7 and SUVH9 are tissue specifically expressed and their encoded proteins are located in the euchromatic regions where they most likely form chromatin complexes. SUVH3 and SUVH9 form foci depending on the developmental stage of the cell. SUVH9 might be a candidate for trimethylating histone H3 lysine 9 however neither SUVH3,-7 or -9 are likely to interact with LHP1.