Characterizing short-term evolution of DNA methylation in A. thaliana using next-generation sequencing

DNA sequence mutations are the principal source of natural variation. Over the last few decades, however, an increasing number of studies have suggested that also epigenetic components can be at the basis of differences in phenotypic traits. These epigenetic marks allow a flexible modulation of gene activity without changes in the DNA sequence. One of the most prominent epigenetic modifications is DNA methylation, which consists of cytosines that carry an additional methyl group. Such chemical marks can be inherited across cell divisions and generations, and there are many durable methylation differences between individuals, so-called epimutations. These can originate from mainly three different sources: most epimutations are coupled to genetic mutations, yet they can also arise spontaneously, or they can be induced by environmental stimuli. The latter case enables rapid adaptation to changing environments, which in the short term is usually not possible via genetic mutations. A current debate revolves around the question whether adaptive environmentally induced epimutations can be heritable, which would contradict the random mutagenesis assumption of Darwinian evolutionary theory. However, the experimental setup of most studies that have examined epigenetic variation did not allow the clear separation of different sources of variable methylation. These studies typically did not inspect genome-wide genetic variation, or did not monitor environmentally induced changes for more than one or two generations. Thus it has remained largely unresolved how frequently methylation differences arise spontaneously on the whole-genome level, and how strongly and durably environmental conditions impact the methylation landscape. This work addresses these questions in the model plant Arabidopsis thaliana. I present whole-genome DNA methylation analyses at base-pair resolution of two different populations, originating from unique experimental settings that largely eliminate specific sources of epimutations. Investigation of genetically quasi identical lines propagated for thirty generations in uniform greenhouse conditions – thus largely without genetic and environmental influences – revealed that spontaneously occurring epimutations emerged frequently, but seemed to be largely short-lived. Plants with minimal genetic divergence that had grown in diverse natural sites over a previously uncharted time period of over one hundred years exhibited a methylation pattern that was largely stable on the whole-genome level and that was in many aspects intriguingly similar to that of the greenhouse-grown lines. Thus, environmentally induced epimutations seem to be only minor contributors to heritable methylation differences, which challenges published claims of broad-scale inheritance of adaptive epigenetic variation. This thesis also provides technical and methodological advances of next-generation sequencing (NGS) data analysis. To gauge the genome-wide genetic influence on epimutations, this work provides an iterative workflow that maximizes the detection of a wide range of DNA sequence variants using short NGS reads by integrating several different genetic variation detection approaches. Finally, while previous epigenetic studies in plants, due to rather simplistic statistical testing, largely revealed a biased picture of differential methylation in the genome, this work introduces a comprehensive DNA methylation pipeline for NGS data that includes a novel approach to obtain more sensitive and more unbiased calls of differentially methylated regions. Together, this work presents advanced computational methods to profile genome-wide genetic and methylation variation, and inspects the rate and spectrum of naturally occurring methylation changes, thus contributing to elucidating the role of epimutations in evolution

Long-term balancing selection drives evolution of immunity genes in Capsella.

Genetic drift is expected to remove polymorphism from populations over long periods of time, with the rate of polymorphism loss being accelerated when species experience strong reductions in population size. Adaptive forces that maintain genetic variation in populations, or balancing selection, might counteract this process. To understand the extent to which natural selection can drive the retention of genetic diversity, we document genomic variability after two parallel species-wide bottlenecks in the genus Capsella. We find that ancestral variation preferentially persists at immunity related loci, and that the same collection of alleles has been maintained in different lineages that have been separated for several million years. By reconstructing the evolution of the disease-related locus MLO2b, we find that divergence between ancient haplotypes can be obscured by referenced based re-sequencing methods, and that trans-specific alleles can encode substantially diverged protein sequences. Our data point to long-term balancing selection as an important factor shaping the genetics of immune systems in plants and as the predominant driver of genomic variability after a population bottleneck

Arabidopsis BTB/POZ protein-dependent PENETRATION3 trafficking and disease susceptibility

The outermost cell layer of plant roots (epidermis) constantly encounters environmental challenges. The epidermal outer plasma membrane domain harbours the PENETRATION3 (PEN3)/ABCG36/PDR8 ATP-binding cassette transporter that confers non-host resistance to several pathogens. Here, we show that the Arabidopsis ENDOPLASMIC RETICULUM-ARRESTED PEN3 (EAP3) BTB/POZ-domain protein specifically mediates PEN3 exit from the endoplasmic reticulum and confers resistance to a root-penetrating fungus, providing prime evidence for BTB/POZ- domain protein-dependent membrane trafficking underlying disease resistance.The PENETRATION3 (PEN3/ABCG36/PDR8) ATP-binding cassette transporter of Arabidopsis thaliana is a crucial component of preinvasive defence against some fungal and bacterial non-host pathogens entering by direct penetration1,2,3,4. In above-ground organs, PEN3 is recruited to sites of pathogen attack at the cell surface3,4. In seedling roots, PEN3 polarly localizes to the epidermal outer membrane domain in the absence of pathogens5,6. Root epidermal cells display four major polar plasma membrane domains: the outer domain facing the environment, the inner domain oriented towards the cortical cell layer, the shootward-oriented, apical, and the root tip-oriented, basal, domain6. Proteins in the outer domain that function in regulating the transport of inorganic compounds include, for example, the NIP5;1 boric acid uptake channel7. Factors required for PEN3 and NIP5;1 trafficking from the trans-Golgi network to the outer domain have been identified8,9,10, and exocyst complex components promote polar tethering of several outer domain proteins9,11. However, factors that specifically mediate trafficking of polar outer membrane cargos involved in responses to root-penetrating pathogens remain to be discovered.In a genetic screen for mislocalization of PEN3 fused to green-fluorescent protein (PEN3- GFP) in the root epidermis of seedlings9, we recovered one recessive mutant in which PEN3-GFP localized to a cytoplasmic structure resembling the endoplasmic reticulum (ER) (Fig. 1a–d). This er-arrested pen3-1 (eap3-1) mutation indistinguishably affected localization of PEN3-GFP from that of PEN3-mCherry (Supplementary Fig. 1a,b), which colocalized with the ER-intrinsic chaperone BIP in the eap3-1 mutant (Supplementary Fig. 1c,d), corroborating an ER arrest of PEN3

Simultaneous alignment of short reads against multiple genomes

New software for the alignment of short-read sequence data to multiple genomes allows identification of polymorphisms that cannot be identified by alignment to a single reference genome

Improved Electrical, Thermal, and Thermoelectric Properties Through Sample‐to‐Sample Fluctuations in Near‐Percolation Threshold Composite Materials

Effective medium theories (EMT) are powerful tools to calculate sample averaged thermoelectric material properties of composite materials. However, averaging over the heterogeneous spatial distribution of the phases can lead to incorrect estimates of the thermoelectric transport properties and the figure of merit ZT in compositions close to the percolation threshold. This is particularly true when the phases’ electronic properties are rather distinct leading to pronounced percolation effects. The authors propose an alternative model to calculate the thermoelectric properties of multi-phased materials that are based on an expanded nodal analysis of random resistor networks (RRN). This method conserves the information about the morphology of the individual phases, allowing the study of the current paths through the phases and the influence of heterogeneous charge transport and cluster formation on the effective material properties of the composite. The authors show that in composites with strongly differing phases close to the percolation threshold the thermoelectric properties and the ZT value are always dominated exclusively by one phase or the other and never by an average of both. For these compositions, the individual samples display properties vastly different from EMT predictions and can be exploited for an increased thermoelectric performance

Paralemmin-1 is expressed in lymphatic endothelial cells and modulates cell migration, cell maturation and tumor lymphangiogenesis

The lymphatic system, the network of lymphatic vessels and lymphoid organs, maintains the body fluid balance and ensures the immunological surveillance of the body. In the adult organism, the de novo formation of lymphatic vessels is mainly observed in pathological conditions. In contrast to the molecular mechanisms governing the generation of the lymphatic vasculature during embryogenesis, the processes underlying pathological lymphangiogenesis are less well understood. A genome-wide screen comparing the transcriptome of tumor-derived lymphatic endothelial cells with that of blood vessel endothelial cells identified paralemmin-1 as a protein prominently expressed in lymphatic endothelial cells. Paralemmin-1 is a lipid-anchored membrane protein that in fibroblasts and neurons plays a role in the regulation of cell shape, plasma membrane dynamics and cell motility. Here, we show that paralemmin-1 is expressed in tumor-derived lymphatic endothelial cells as well as in lymphatic endothelial cells of normal, non-tumorigenic tissue. Paralemmin-1 represses cell migration and delays the formation of tube-like structures of lymphatic endothelial cells in vitro by modulating cell-substrate adhesion, filopodia formation and plasma membrane blebbing. While constitutive genetic ablation of paralemmin-1 expression in mice has no effect on the development and physiological function of the lymphatic system, the loss of paralemmin-1 impaired tumor-associated lymphangiogenesis. Together, these results newly identify paralemmin-1 as a protein highly expressed in lymphatic endothelial cells. Similar to its function in neurons, it may link the cytoskeleton to the plasma membrane and thereby modulate lymphatic endothelial cell adhesion, migration and lymphangiogenesi

Targeting Metabolic Symbiosis to Overcome Resistance to Anti-angiogenic Therapy

Despite the approval of several anti-angiogenic therapies, clinical results remain unsatisfactory, and transient benefits are followed by rapid tumor recurrence. Here, we demonstrate potent anti-angiogenic efficacy of the multi-kinase inhibitors nintedanib and sunitinib in a mouse model of breast cancer. However, after an initial regression, tumors resume growth in the absence of active tumor angiogenesis. Gene expression profiling of tumor cells reveals metabolic reprogramming toward anaerobic glycolysis. Indeed, combinatorial treatment with a glycolysis inhibitor (3PO) efficiently inhibits tumor growth. Moreover, tumors establish metabolic symbiosis, illustrated by the differential expression of MCT1 and MCT4, monocarboxylate transporters active in lactate exchange in glycolytic tumors. Accordingly, genetic ablation of MCT4 expression overcomes adaptive resistance against anti-angiogenic therapy. Hence, targeting metabolic symbiosis may be an attractive avenue to avoid resistance development to anti-angiogenic therapy in patients

The impact of the COVID-19 pandemic on administrative eating disorder prevalence in the outpatient sector and on severity of anorexia nervosa.

The COVID-19 pandemic appears to have had a considerable impact on the mental health of children and adolescents, particularly regarding eating disorders. However, it remains unclear whether the pandemic affected only the frequency or also the severity of eating disorders. We examined potential pandemic-related changes in the administrative prevalence of eating disorders in the outpatient sector compared with other mental disorders using German statutory health insurance data for the age group 10 to 16 years. We also examined disorder severity of anorexia nervosa using data from the multicenter German Registry of Children and Adolescents with Anorexia Nervosa in the same age group. Our results showed a marked increase in the administrative prevalence of eating disorders (based on documented diagnoses) in the outpatient sector among girls but not among boys. A similar pattern was found for internalizing disorders, whereas the administrative prevalences of externalizing disorders decreased. Regarding the severity of anorexia nervosa among inpatients, we found no pandemic-related changes in body mass index standard deviation score at admission, body weight loss before admission, psychiatric comorbidities and psychopharmacological medication. Given the administrative prevalence increase in the outpatient sector, the lack of impact of the pandemic on the inpatient sector may also be partly due to a shift in healthcare utilization towards outpatient services during the pandemic. Thus, the higher number of children and adolescents requiring specialized and timely outpatient care may be a major concern under pandemic conditions

Genome expansion of Arabis alpina linked with retrotransposition and reduced symmetric DNA methylation

This document is the Accepted Manuscript version, made available in accordance to Springer Nature Terms of reuse of archived manuscripts.Despite evolutionary conserved mechanisms to silence transposable element activity, there are drastic differences in the abundance of transposable elements even among closely related plant species. We conducted a de novo assembly for the 375 .Mb genome of the perennial model plant, Arabis alpina. Analysing this genome revealed long-lasting and recent transposable element activity predominately driven by Gypsy long terminal repeat retrotransposons, which extended the low-recombining pericentromeres and transformed large formerly euchromatic regions into repeat-rich pericentromeric regions. This reduced capacity for long terminal repeat retrotransposon silencing and removal in A. alpina co-occurs with unexpectedly low levels of DNA methylation. Most remarkably, the striking reduction of symmetrical CG and CHG methylation suggests weakened DNA methylation maintenance in A. alpina compared with Arabidopsis thaliana. Phylogenetic analyses indicate a highly dynamic evolution of some components of methylation maintenance machinery that might be related to the unique methylation in A. alpina.Peer reviewe