Epigenetics and Early Life Stress : Experimental Brood Size Affects DNA Methylation in Great Tits (Parus major)

Early developmental conditions are known to have life-long effects on an individual's behavior, physiology and fitness. In altricial birds, a majority of these conditions, such as the number of siblings and the amount of food provisioned, are controlled by the parents. This opens up the potential for parents to adjust the behavior and physiology of their offspring according to local post-natal circumstances. However, the mechanisms underlying such intergenerational regulation remain largely unknown. A mechanism often proposed to possibly explain how parental effects mediate consistent phenotypic change is DNA methylation. To investigate whether early life effects on offspring phenotypes are mediated by DNA methylation, we cross-fostered great tit (Parus major) nestlings and manipulated their brood size in a natural study population. We assessed genome-wide DNA methylation levels of CpG sites in erythrocyte DNA, using Reduced Representation Bisulfite Sequencing (RRBS). By comparing DNA methylation levels between biological siblings raised in enlarged and reduced broods and between biological siblings of control broods, we assessed which CpG sites were differentially methylated due to brood size. We found 32 differentially methylated sites (DMS) between siblings from enlarged and reduced broods, a larger number than in the comparison between siblings from control broods. A considerable number of these DMS were located in or near genes involved in development, growth, metabolism, behavior and cognition. Since the biological functions of these genes line up with previously found effects of brood size and food availability, it is likely that the nestlings in the enlarged broods suffered from nutritional stress. We therefore conclude that early life stress might directly affect epigenetic regulation of genes related to early life conditions. Future studies should link such experimentally induced DNA methylation changes to expression of phenotypic traits and assess whether these effects affect parental fitness to determine if such changes are also adaptive.Peer reviewe

Performance of methods to detect genetic variants from bisulphite sequencing data in a non-model species

The profiling of epigenetic marks like DNA methylation has become a central aspect of studies in evolution and ecology. Bisulphite sequencing is commonly used for assessing genome-wide DNA methylation at single nucleotide resolution but these data can also provide information on genetic variants like single nucleotide polymorphisms (SNPs). However, bisulphite conversion causes unmethylated cytosines to appear as thymines, complicating the alignment and subsequent SNP calling. Several tools have been developed to overcome this challenge, but there is no independent evaluation of such tools for non-model species, which often lack genomic references. Here, we used whole-genome bisulphite sequencing (WGBS) data from four female great tits (Parus major) to evaluate the performance of seven tools for SNP calling from bisulphite sequencing data. We used SNPs from whole-genome resequencing data of the same samples as baseline SNPs to assess common performance metrics like sensitivity, precision, and the number of true positive, false positive, and false negative SNPs for the full range of variant and genotype quality values. We found clear differences between the tools in either optimizing precision (Bis-SNP), sensitivity (biscuit), or a compromise between both (all other tools). Overall, the choice of SNP caller strongly depends on which performance parameter should be maximized and whether ascertainment bias should be minimized to optimize downstream analysis, highlighting the need for studies that assess such differences.Peer reviewe

An ecologist's guide for studying DNA methylation variation in wild vertebrates

The field of molecular biology is advancing fast with new powerful technologies, sequencing methods and analysis software being developed constantly. Commonly used tools originally developed for research on humans and model species are now regularly used in ecological and evolutionary research. There is also a growing interest in the causes and consequences of epigenetic variation in natural populations. Studying ecological epigenetics is currently challenging, especially for vertebrate systems, because of the required technical expertise, complications with analyses and interpretation, and limitations in acquiring sufficiently high sample sizes. Importantly, neglecting the limitations of the experimental setup, technology and analyses may affect the reliability and reproducibility, and the extent to which unbiased conclusions can be drawn from these studies. Here, we provide a practical guide for researchers aiming to study DNA methylation variation in wild vertebrates. We review the technical aspects of epigenetic research, concentrating on DNA methylation using bisulfite sequencing, discuss the limitations and possible pitfalls, and how to overcome them through rigid and reproducible data analysis. This review provides a solid foundation for the proper design of epigenetic studies, a clear roadmap on the best practices for correct data analysis and a realistic view on the limitations for studying ecological epigenetics in vertebrates. This review will help researchers studying the ecological and evolutionary implications of epigenetic variation in wild populations

The Potato Nucleotide-Binding Leucine-Rich Repeat (NLR) Immune Receptor Rx1 is a Pathogen Dependent DNA-Deforming Protein

Plant NLR proteins enable cells to respond to pathogen attack. Several NLRs act in the nucleus, however, conserved nuclear targets that support their role in immunity are unknown. Previously we noted a structural homology between the NB domain of NLRs and DNA replication origin-binding Cdc6/Orc1 proteins. Here we show that the NB-ARC domain of the Rx1 NLR of potato binds nucleic acids. Rx1 induces ATP-dependent bending and melting of DNA in vitro dependent upon a functional P-loop. In situ full-length Rx1 binds nuclear DNA following activation by its cognate pathogen-derived effector protein, the coat protein of potato virus X. In line with its obligatory nucleocytoplasmic distribution, DNA-binding was only observed when Rx1 was allowed to freely translocate between both compartments and was activated in the cytoplasm. Immune activation induced by an unrelated NLR-effector pair did not trigger a Rx1-DNA interaction. DNA-binding is therefore not merely a consequence of immune activation. These data establish a role for DNA distortion in Rx1 immune signalling and defines DNA as a molecular target of an activated NLR

An ecologist’s guide for studying DNA methylation variation in wild vertebrates

The field of molecular biology is advancing fast with new powerful technologies, sequencing methods and analysis software being developed constantly. Commonly used tools originally developed for research on humans and model species are now regularly used in ecological and evolutionary research. There is also a growing interest in the causes and consequences of epigenetic variation in natural populations. Studying ecological epigenetics is currently challenging, especially for vertebrate systems, because of the required technical expertise, complications with analyses and interpretation, and limitations in acquiring sufficiently high sample sizes. Importantly, neglecting the limitations of the experimental setup, technology and analyses may affect the reliability and reproducibility, and the extent to which unbiased conclusions can be drawn from these studies. Here, we provide a practical guide for researchers aiming to study DNA methylation variation in wild vertebrates. We review the technical aspects of epigenetic research, concentrating on DNA methylation using bisulfite sequencing, discuss the limitations and possible pitfalls and how to overcome them through rigid and reproducible data analysis. This review provides a solid foundation for the proper design of epigenetic studies, a clear roadmap on the best practices for correct data analysis and a realistic view on the limitations for studying ecological epigenetics in vertebrates. This review will help researchers studying the ecological and evolutionary implications of epigenetic variation in wild populations.peerReviewe

An ecologist's guide for studying DNA methylation variation in wild vertebrates

The field of molecular biology is advancing fast with new powerful technologies, sequencing methods and analysis software being developed constantly. Commonly used tools originally developed for research on humans and model species are now regularly used in ecological and evolutionary research. There is also a growing interest in the causes and consequences of epigenetic variation in natural populations. Studying ecological epigenetics is currently challenging, especially for vertebrate systems, because of the required technical expertise, complications with analyses and interpretation, and limitations in acquiring sufficiently high sample sizes. Importantly, neglecting the limitations of the experimental setup, technology and analyses may affect the reliability and reproducibility, and the extent to which unbiased conclusions can be drawn from these studies. Here, we provide a practical guide for researchers aiming to study DNA methylation variation in wild vertebrates. We review the technical aspects of epigenetic research, concentrating on DNA methylation using bisulfite sequencing, discuss the limitations and possible pitfalls, and how to overcome them through rigid and reproducible data analysis. This review provides a solid foundation for the proper design of epigenetic studies, a clear roadmap on the best practices for correct data analysis and a realistic view on the limitations for studying ecological epigenetics in vertebrates. This review will help researchers studying the ecological and evolutionary implications of epigenetic variation in wild populations

Epigenetics of animal personality : DNA methylation cannot explain the heritability of exploratory behavior in a songbird

The search for the hereditary mechanisms underlying quantitative traits traditionally focused on the identification of underlying genomic polymorphisms such as single-nucleotide polymorphisms. It has now become clear that epigenetic mechanisms, such as DNA methylation, can consistently alter gene expression over multiple generations. It is unclear, however, if and how DNA methylation can stably be transferred from one generation to the next and can thereby be a component of the heritable variation of a trait. In this study, we explore whether DNA methylation responds to phenotypic selection using whole-genome and genome-wide bisulfite approaches. We assessed differential erythrocyte DNA methylation patterns between extreme personality types in the Great Tit (Parus major). For this, we used individuals from a four-generation artificial bi-directional selection experiment and siblings from eight F2 intercross families. We find no differentially methylated sites when comparing the selected personality lines, providing no evidence for the so-called epialleles associated with exploratory behavior. Using a pair-wise sibling design in the F2 intercrosses, we show that the genome-wide DNA methylation profiles of individuals are mainly explained by family structure, indicating that the majority of variation in DNA methylation in CpG sites between individuals can be explained by genetic differences. Although we found some candidates explaining behavioral differences between F2 siblings, we could not confirm this with a whole-genome approach, thereby confirming the absence of epialleles in these F2 intercrosses. We conclude that while epigenetic variation may underlie phenotypic variation in behavioral traits, we were not able to find evidence that DNA methylation can explain heritable variation in personality traits in Great Tits.</p

Developmental stress does not induce genome‐wide methylation changes in wild great tit ( ) nestlings

Sepers B, Mateman AC, Gawehns F, Verhoeven KJF, van Oers K. Developmental stress does not induce genome‐wide methylation changes in wild great tit ( ) nestlings. Molecular Ecology. 2023;32(14):3960-3974.**Abstract** The environment experienced during early life is a crucial factor in the life of many organisms. This early life environment has been shown to have profound effects on morphology, physiology and fitness. However, the molecular mechanisms that mediate these effects are largely unknown, even though they are essential for our understanding of the processes that induce phenotypic variation in natural populations. DNA methylation is an epigenetic mechanism that has been suggested to explain such environmentally induced phenotypic changes early in life. To investigate whether DNA methylation changes are associated with experimentally induced early developmental effects, we cross‐fostered great tit (Parus major) nestlings and manipulated their brood sizes in a natural study population. We assessed experimental brood size effects on pre‐fledging biometry and behaviour. We linked this to genome‐wide DNA methylation levels of CpG sites in erythrocyte DNA, using 122 individuals and an improved epiGBS2 laboratory protocol. Brood enlargement caused developmental stress and negatively affected nestling condition, predominantly during the second half of the breeding season, when conditions are harsher. Brood enlargement, however, affected nestling DNA methylation in only one CpG site and only if the hatch date was taken into account. In conclusion, this study shows that nutritional stress in enlarged broods does not associate with direct effects on genome‐wide DNA methylation. Future studies should assess whether genome‐wide DNA methylation variation may arise later in life as a consequence of phenotypic changes during early development

Performance of methods to detect genetic variants from bisulphite sequencing data in a non-model species

The profiling of epigenetic marks like DNA methylation has become a central aspect of studies in evolution and ecology. Bisulphite sequencing is commonly used for assessing genome-wide DNA methylation at single nucleotide resolution but these data can also provide information on genetic variants like single nucleotide polymorphisms (SNPs). However, bisulphite conversion causes unmethylated cytosines to appear as thymines, complicating the alignment and subsequent SNP calling. Several tools have been developed to overcome this challenge, but there is no independent evaluation of such tools for non-model species, which often lack genomic references. Here, we used whole-genome bisulphite sequencing (WGBS) data from four female great tits (Parus major) to evaluate the performance of seven tools for SNP calling from bisulphite sequencing data. We used SNPs from whole-genome resequencing data of the same samples as baseline SNPs to assess common performance metrics like sensitivity, precision, and the number of true positive, false positive, and false negative SNPs for the full range of variant and genotype quality values. We found clear differences between the tools in either optimizing precision (Bis-SNP), sensitivity (biscuit), or a compromise between both (all other tools). Overall, the choice of SNP caller strongly depends on which performance parameter should be maximized and whether ascertainment bias should be minimized to optimize downstream analysis, highlighting the need for studies that assess such differences