Browsing by Subject "DNA methylation"

Our primary goal is to better understand complex diseases using statistically disciplined approaches. As multi-modal data is streaming out of consortium projects like Genotype-Tissue Expression (GTEx) project, which aims at collecting samples from various tissue sites in order to understand tissue-specific gene regulation, new approaches are needed that can efficiently model groups of data with minimal loss of power. For example, GTEx project delivers RNA-Seq, Microarray gene expression and genotype data (SNP Arrays) from a vast number of tissues in a given individual subject. In order to analyze this type of multi-level (hierarchical) multi-modal data, we proposed a series of efficient-score based tests or score tests and leveraged groups of tissues or gene isoforms in order map genomic biomarkers. We model group-specific variability as a random effect within a mixed effects model framework. In one instance, we proposed a score-test based approach to map expression quantitative trait loci (eQTL) across multiple-tissues. In order to do that we jointly model all the tissues and make use of all the information available to maximize the power of eQTL mapping and investigate an overall shift in the gene expression combined with tissue-specific effects due to genetic variants. In the second instance, we showed the flexibility of our model framework by expanding it to include tissue-specific epigenetic data (DNA methylation) and map eQTL by leveraging both tissues and methylation. Finally, we also showed that our methods are applicable on different data type such as whole transcriptome expression data, which is designed to analyze genomic events such alternative gene splicing. In order to accomplish this, we proposed two different models that exploit gene expression data of all available gene-isoforms within a gene to map biomarkers of interest (either genes or gene-sets) in paired early-stage breast tumor samples before and after treatment with external beam radiation. Our efficient score-based approaches have very distinct advantages. They have a computational edge over existing methods because they do not need parameter estimation under the alternative hypothesis. As a result, model parameters only have to be estimated once per genome, significantly decreasing computation time. Also, the efficient score is the locally most powerful test and is guaranteed a theoretical optimality over all other approaches in a neighborhood of the null hypothesis. This theoretical performance is born out in extensive simulation studies which show that our approaches consistently outperform existing methods both in statistical power and computational speed. We applied our methods to publicly available datasets. It is important to note that all of our methods also accommodate the analysis of next-generation sequencing data.

In many species, early life experiences have striking effects on health, reproduction, and survival in adulthood. Thus, early life conditions shape a range of evolutionarily relevant traits, and in doing so alter the genotype-phenotype relationship and the phenotypic distribution on which selection acts. Because of the key role early life effects play in generating variation in fitness-related traits, understanding their evolution and mechanistic basis is crucial. To gain traction on these topics, my dissertation draws on ecological, demographic, and genomic data from a long-term study population of wild baboons in Amboseli, Kenya to address three major themes: (i) the adaptive significance of early life effects, (ii) the molecular mechanisms that connect early life experiences with later life traits, and (iii) the development of laboratory tools for understanding the role of one particular mechanism—DNA methylation—in translating environmental inputs into phenotypic variation. In chapter one, I empirically test two competing explanations for how early life effects evolve, providing novel insight into the evolution of developmental plasticity in long-lived species. In chapter two, I address the degree to which ecological effects on fitness-related traits are potentially mediated by changes in DNA methylation. Finally, in chapter three, I develop a high-throughput assay to improve our knowledge of the phenotypic relevance of changes in the epigenome. Together, this work provides some of the first empirical data on the genes and mechanisms involved in sensing and responding to environmental variation in wild mammals, and more generally addresses several critical gaps in our understanding of how early experiences affect evolutionarily relevant traits.

Changes in gene regulation are thought to play an important role in primate evolution and divergence. In support of this hypothesis, comparative evidence clearly demonstrate that gene expression patterns differ between closely related species and tend to evolve under selective constraint. However, we know little about the evolutionary forces that shape gene regulation across primates, particularly outside of humans and the other great apes. To address this gap, my dissertation draws on population and functional genomic variation between baboon species and within an admixed wild baboon population to address two themes: (i) how is gene regulatory divergence related to genetic divergence? and (ii) to what extent has natural selection shaped regulatory variation? Using interspecific comparative approaches, I show that changes in DNA methylation accumulate with increasing sequence divergence. While most changes in methylation can be explained by genetic drift, a subset are likely to have evolved under positive selection. Then, using genomic data from admixed baboons, I show that interspecific changes in DNA methylation are linked to genetic effects on DNA methylation (i.e., methylation quantitative trait loci, meQTL) and differences in allele frequency between baboon species. I also show that changes in DNA methylation are associated with changes in gene expression. Finally, I identify genomic evidence for selection against admixture in baboons, especially near genes that are differentially expressed between species. Together, my work highlights the close relationship between genetic and gene regulatory divergence in baboons. It also emphasizes the importance of natural selection in shaping genetic and regulatory variation throughout primate evolution, including in a living model for admixture in our own lineage.

Lichens are symbioses between a fungus and a photosynthesizing partner such as a green alga or a cyanobacterium. Unlike mycorrhizal or rhizobial symbioses, the lichen symbiosis is not well understood either morphologically or molecularly. The lichen symbiosis has been somewhat neglected for several reasons. Lichens grow very slowly in nature (less than 1 cm a year), it is difficult to grow the fungus and the alga separately and, moreover, it remains difficult to resynthesize the mature symbiosis in the laboratory. It is not yet possible to delete genes, nor has any transformation method been established to introduce genes into the genomes of either the fungus or the alga. However, the lack of genetic tools for these organisms has been partially compensated for by the sequencing of the genomes of the lichenizing fungus Cladonia grayi and its green algal partner Asterochloris sp. This work uses the model lichen system Cladonia grayi and the associated genomes to explore one evolutionary and one developmental question concerning the lichen symbiosis.

Chapter One uses data from the genomes to assess whether there was evidence of horizontal gene transfer between the lichen symbionts in the evolution of this very intimate association; that is, whether genes of algal origin could be found in the fungal genome or vise versa. An initial homology search of the two genomes demonstrated that the fungus had, in addition to ammonium transporter/ammonia permease genes that were clearly fungal in origin, ammonium transporter/ammonia permease genes which appeared to be of plant origin. Using cultures of various lichenizing fungi, plant-like ammonium transporter/ammonia permease genes were identified by degenerate PCR in ten additional species of lichen in three classes of lichenizing fungi including the Lecanoromycetes, the Eurotiomycetes, and the Dothidiomycetes. Using the sequences of these transporter genes as well as data from publically available genome sequences of diverse organisms, I constructed a phylogy of 513 ammonium transporter/ammonia permease sequences from 191 genomes representing all main lineages of life to infer the evolutionary history of this family of proteins. In this phylogeny I detected several horizontal gene transfer events, including the aforementioned one which was demonstrated to be not a transfer from plants to fungi or vise versa, but a gene gain from a group of phylognetically unrelated hyperthermophilic chemoautolithotrophic prokaryotes during the early evolution of land plants (Embryophyta), and an independent gain of this same gene in the filamentous ascomycetes (Pezizomycotina), which was subsequently lost in most lineages but retained in even distantly related lichenized fungi. Also demonstrated was the loss of the native fungal ammonium transporter and the subsequent replacement of this gene with a bacterial ammonium transporter during the early evolution of the fungi. Several additional recent horizontal gene transfers into lineages of eukaryotes were demonstrated as well. The phylogenetic analysis suggests that what has heretofore been conceived of as a protein family with two clades (AMT/MEP and Rh) is instead a protein family with three clades (AMT, MEP, and Rh). I show that the AMT/MEP/Rh family illustrates two contrasting modes of gene transmission: AMT family as defined here exhibits standard parent-to-offspring inheritance, whereas the MEP family as defined here is characterized by several ancient independent horizontal gene transfers (HGTs) into eukaryotes. The clades as depicted in this phylogenetic study appear to correspond to functionally different groups, with ammonium transporters and ammonia permeases forming two distinct and possibly monophyletic groups.

In Chapter Two I address a follow-up question: in key lichenizing lineages for which ammonium transporter/ammonia permease (AMTP) genes were not found in Chapter One, were the genes lost? The only definitive infomation which can demonstrate absence of a gene from a genome is a full genome sequence. To this end, the genomes of eight additional lichenizing fungi in the key clades including the Caliciales (sensu Gaya 2011), the Peltigerales, the Ostropomycetidae, the Acarosporomycetidae, the Verrucariales, the Arthoniomycetidae and the Lichinales were sequenced using the Ilumina HiSeq technology and assembled with the short reads assembly software Velvet. These genomes were searched for ammonium transporter/ammonia permease sequences as well as 20 test genes to assess the completeness of each assembly. The genes recovered were included in a refined phylogenetic analysis. The hypothesis that lichens symbiotic with a nitrogen-fixing cyanobacteria as a primary photobiont or living in high nitrogen environments lose the plant-like ammonium transporters was upheld, but did not account for additional losses of ammonium transporters/ammonia permeases in the Acarosporomyetidae and Arthoniomycetes. In addition, the four AMTP genes from Cladonia grayi were shown to be functional by expression of the lichen genes in a strain of Saccharomyces cerevisiae in which all three native ammonium transporters were deleted, and assaying for growth on limiting ammonia as a sole nitrogen source.

In Chapter Three I use genome data to address a developmental aspect of the lichen symbiosis. The finding that DNA in three genera of lichenizing fungi is methylated in symbiotic tissues and not methylated in aposymbiotic tissues or in the free-living fungus (Armaleo & Miao 1999a) suggested that epigenetic silencing may play a key role in the development of the symbiosis. Epigenetic silencing involves several steps that are conserved in many eukaryotes, including methylation of histone H3 at lysine 9 (H3K9) in nucleosomes within the silenced region, subsequent binding of heterochromatin-binding protein (HP1) over the region, and the recruitment of DNA methyltransferases to methylate the DNA, all of which causes the underlying chromatin to adopt a closed conformation, inhibiting the transcriptional machinery from binding. In this chapter I both identify the genes encoding the silencing machinery and determine the targets of the silencing machinery. I use degenerate PCR and genome sequencing to identify the genes encoding the H3K9 histone methyltransferase, the heterochromatin binding protein, and the DNA methyltransferases. I use whole genome bisulfite sequencing of DNA from the symbiotic structures of Cladonia grayi including podetia, squamules and soredia as well as DNA from cultures of the free-living fungus and free-living alga to determine which regions of the genome are methylated in the symbiotic and aposymbiotic states. In particular I examine regions of the genomes which appear to be differentially methylated in the symbiotic versus the aposymbiotic state. I show that DNA methylation is uncommon in the genome of the fungus in the symbiotic and aposymbiotic states, and that the genome of the alga is methylated in the symbiotic and aposymbiotic states.

DNA methylation is one of the most studied and important epigenetic modifications in cells, playing a role in DNA transcription, splicing, and imprinting. Recently, advanced genome-wide DNA methylation profiling technologies have been developed, making it possible to conduct methylome-wide association studies. One of the problems with large scale DNA methylation studies is that the current technologies are either targeting only a limited number of CpG sites in the genome or whole genome sequencing is expensive and time consuming for most laboratories. Computational prediction of CpG site-specific methylation levels is the cost-saving and time-saving alternative.

In this work, we found striking patterns of DNA methylation across the genome. We show that correlation among CpG sites decays rapidly within several hundreds base pairs in contrast to the LD structure of genotypes which holds for up to several KB. Using genomic features including, neighbor CpG site methylation and genomic distance, genomic context such as CpG island regions, and genomic regulatory elements, we built random forest classifiers to predict CpG site methylation levels. Our approach achieves 92% prediction accuracy at single CpG sites in different genome-wide methylation datasets. We achieves the highest accuracy as 98% for prediction within CpG island regions. What's more, our method identifies genomic features that interact with DNA methylation, which improves our understanding of mechanisms involved in DNA methylation modification and regulation.

While phenotypic plasticity has been widely documented, the relative contribution of parent versus offspring environment to determining progeny phenotypes and the persistence of parental effects throughout progeny development under different environments remain unknown. In predictable and/or seasonal environments, parental effects are predicted to be favored during early life stages of offspring, while offspring environment is predicted to increase in relative importance as the accuracy of offspring perception increases over the course of development. Furthermore, as plastic responses to the environment are both transmissible across generations and dynamic across development, epigenetic mechanisms are likely involved in the regulation of parental effects and phenotypic plasticity. The neighboring community is an important environmental factor for plants since sessile plants cannot escape competition within their current generation. Whether interactions with neighbors are positive or negative likely depends on the environment, as competition is hypothesized to increase in favorable environments and the neighboring community of plants may change over a seasonal progression. For these reasons, neighbor environment is an interesting and ecologically important environmental variable that can be used to investigate how parental and progeny environment regulate progeny phenotypes throughout development. To test these predictions, this dissertation evaluated how progeny phenotypes responded to the combination of parental and offspring environments, quantified how parental and progeny methylation regulate offspring phenotypes, and examined their effects on plasticity. To address which generation’s environment and DNA methylation affect phenotypes in offspring, I manipulated simulated and real neighbor environments and DNA methylation within and across generations in Arabidopsis thaliana, a winter annual native to Eurasia widely introduced across North America. In Chapter 1, using chemical demethylation, I tested whether parental and progeny DNA methylation influences germination, and whether parental DNA regulates germination response to past and present simulated canopy. I found that germination of offspring is regulated by parental DNA methylation and is responsive to parent, not seed, environment for most genotypes. Furthermore, I confirmed using mutant lines that all contexts of DNA methylation were involved in the transmission of parental effects, but they may operate through different pathways controlling germination. In Chapter 2, I quantified how parental versus progeny methylation regulate progeny phenotypic responses to parental and progeny canopy shade. I found that both parent and offspring canopy affect offspring traits across development, but parental environment had stronger effects at the seedling stage. Both parent and offspring DNA methylation affected offspring response to canopy, but parental DNA methylation only affected traits at the seedling stage. Trait correlations were significantly altered by chemical demethylation of parents and offspring, indicating that DNA methylation of both generations are important in regulating development and integrating phenotypic response to canopy. Finally, in Chapter 3, I tested whether parent or offspring DNA methylation affected response to the heterospecific neighbor, Stellaria media, under simulated seasonal conditions. I found that growing with competitors decreased fitness for all genotypes, but genotypes varied in the effect of neighbors on morphology and fitness. Both parent and offspring DNA methylation had direct effects on growth and fitness in all genotypes, but genotypes varied in how DNA methylation influenced response to neighbors. In one genotype, plastic response to neighbors was unaffected by chemical demethylation treatments, indicating that neighbor-induced plasticity is not always mediated via DNA methylation. Together, these results indicated that offspring phenotypes are shaped by both parent and offspring environment, and that parental environment and parental DNA methylation are especially important in regulating offspring traits early in life. The genetic variation observed in the expression of phenotypic plasticity via parental and progeny DNA methylation suggests that the epigenetic regulation of progeny phenotypes has a genetic basis and may evolve.

We have a rudimentary understanding of the consequences of preconception exposure to cannabis. As the most commonly used illicit psychoactive drug, cannabis prevalence is rapidly increasing across the United States (US), and consumers are increasingly perceiving it as safe. Recreational cannabis use is especially common among American men, rendering the paternal preconception environment potentially vulnerable to deleterious effects. Parental cannabis use has been associated with adverse developmental outcomes in offspring, but little is known about how such phenotypes are transmitted. Gametic epigenetic changes – the collection of molecular modifications made to DNA and histone proteins that play a role in regulating gene activity – provide one potential explanation. In a pilot study, our group recently demonstrated that cannabis use in humans, and exposure to delta-9-tetrahydrocannabinol (THC, the main psychoactive component of cannabis) in rats, is associated with significantly altered levels of DNA methylation in sperm. Epidemiological studies have further associated prenatal cannabis use with an increased risk of numerous teratologies including neurodevelopmental disorders and cardiovascular defects. While these studies illuminate the risks associated with cannabis and the prenatal environment, little attention has been paid to the effects of paternal preconception exposures alone on such congenital anomalies. There remains an urgent need to investigate the effects of cannabis on the sperm epigenome as use increases across the globe. Further, it is critical to investigate these effects on genes that are uniquely positioned to contribute to early development. The hypothesis of this research was that cannabis exposure is associated with heritable, but reversible, changes in DNA methylation in sperm at genes important for early life development. Broadly, the main objectives of this thesis research were to generate meaningful data to contribute to the gaps in knowledge of how cannabis can impact sperm DNA methylation. The results of this thesis research are described beginning in chapter 2. An initial pilot study from our group used reduced representation bisulfite sequencing (RRBS) to analyze methylation changes in sperm between cannabis users and controls. One gene identified as being significantly differentially methylated was an autism candidate gene, Discs-Large Associated Protein 2 (DLGAP2). Quantitative bisulfite pyrosequencing confirmed that an intronic region of DLGAP2 was significantly hypomethylated in the sperm of cannabis exposed men compared to controls. Use of human fetal brain tissues demonstrated that there is a significant, sex-specific, inverse relationship between DNA methylation and gene expression at this locus. A paternal rat model of THC exposure was used to determine whether these effects at Dlgap2 were heritable. Bisulfite pyrosequencing identified significant changes in rat sperm DNA methylation at Dlgap2, as well as significant losses of methylation at the same locus in F1 nucleus accumbens (NAc) tissues. Having demonstrated that autism candidate gene DLGAP2 shows functional changes in DNA methylation, the next question addressed was whether different routes of THC exposure, and exposure to different drugs, could similarly impact DNA methylation at a select group of neuroactive genes. Sperm DNA from rats exposed to THC or vehicle control via oral gavage underwent RRBS. Bisulfite pyrosequencing of sperm DNA from rats exposed to injected THC or vehicle control was performed to examine methylation at regions identified by RRBS. Sperm DNA from rats exposed to nicotine or vehicle control underwent pyrosequencing at the same regions. Lastly, two publicly available datasets were investigated to determine significant overlap between a known list of autism candidate genes and a list of genes with bivalent chromatin, a unique epigenetic feature. In the sperm of rats injected with THC and those nicotine-exposed, significant differential methylation at five of seven neurodevelopmentally active genes that were initially identified as significantly altered by oral gavage was identified. It was further discovered that autism candidate genes are significantly enriched for genes containing bivalent chromatin. Enrichment of both autism candidate genes and genes possessing bivalent chromatin was identified in the human RRBS dataset of genes significantly differentially methylated in sperm of cannabis users. These studies demonstrated THC and nicotine exposure in rats can impact DNA methylation in sperm at neuroactive genes. Further, this work provides initial evidence that genes with bivalent chromatin may be particularly vulnerable to DNA methylation changes resulting from environmental exposures. The fourth chapter of this thesis research employed a novel in vitro model of human spermatogenesis to identify the impact of exposure to a cannabis smoke extract (CSE) on DNA methylation at two groups of genes important for early life development. Human embryonic stem cells (hECS) exposed to CSE or vehicle control were differentiated into a mixed population of spermatogonial stem-like cells (SSCs), primary spermatocyte-like cells, secondary spermatocyte-like cells, and round haploid spermatid-like cells over a ten-day period. Following differentiation, flow cytometry was performed to isolate SSC-like cells and haploid spermatid-like cells for DNA methylation analyses. Methylation was first analyzed at a group of imprinted genes. Significant effects of exposure were identified in SSC-like cells at Sarcoglycan Epsilon (SGCE) and in haploid spermatid-like cells at Paternally Expressed 3 (PEG3) and Growth Factor Receptor-Bound Protein 10 (GRB10). Next, methylation was assessed at a group of genes randomly chosen genes from the Simons Foundation Autism Research Initiative (SFARI) autism candidate gene list, half of which possessed bivalent chromatin at the specific CpG sites analyzed and half of which did not. Significant methylation changes were identified in SSC-like cells at genes from the SFARI list possessing bivalent chromatin, but not at genes from the SFARI list without this epigenetic modification. These results support the hypothesis that bivalent chromatin may make genes more vulnerable to environmental exposures. Chapter five of this thesis research addressed the potential heritability of the impacts of exposure to CSE. Changes in F0 sperm DNA methylation were initially identified via whole genome bisulfite sequencing (WGBS) and methylation changes were validated at select genes via bisulfite pyrosequencing. Methylation changes validated in F0 sperm at the gene 2-Phosphoxylose Phosphatase 1 (Pxylp1) were similarly present in F1 sperm, while changes validated in F0 sperm at Gamma-Aminobutyric Acid Type A Receptor Subunit Beta2 (Gabrb2) and Metastasis Suppressor 1-Like Protein (Mtss1l) were similarly present in F1 hippocampal, and NAc and hippocampal tissues, respectively. Further, for Mtss1l a significant, sex-specific relationship between DNA methylation and gene expression in offspring NAc was demonstrated. Phenotypically, rats born to CSE-exposed fathers exhibited significant cardiomegaly relative to those whose fathers were CSE-naïve. Finally, chapter six of this thesis research addressed whether or not the effects of cannabis on human sperm DNA methylation were reversible. Men were recruited to participate in the study as cannabis users and non-user controls. Semen samples were collected at baseline, and then again following an 11-week cannabis-abstinence period. WGBS was performed on all sperm samples. There were no significant differences between users and controls based on demographic information or measured semen parameters. WGBS quantified DNA methylation changes in sperm. Importantly, a reduction in the magnitude of methylation difference between users and controls after the abstinence period relative to the methylation difference present before abstinence was observed. However, select genes retained their altered methylation patterns after abstinence, suggesting not all cannabis-induced effects were ameliorated. Bioinformatic analysis of genes associated with significantly differentially methylated CpG sites revealed terms associated with nervous system development, cardiovascular system development, and embryonic development. Together, this suggests that the abstinence period is at least partially effective at resolving the methylation changes observed following cannabis use at genes important for early development. This research adds to the emergent literature that cannabis is able to impact DNA methylation in sperm at genes important for early life development. It demonstrates in rodents the ability of this exposure to induce heritable epigenetic and phenotypic effects in offspring. Further, it provides the first evidence that abstinence from cannabis use might help resolve the methylation changes that arise in sperm following this exposure. Future work should assess the ability of this exposure to impact offspring methylation and neurodevelopmental outcomes in children and should define how long abstinence from cannabis use must last to produce the most robust amelioration effects.

Maternal smoking is a deleterious and preventable risk to fetal health. Maternal tobacco smoke (TS) exposure in humans has been linked to impaired fetal growth, preterm birth, sudden infant death syndrome, and neurobehavioral disorders including cognitive dysfunction, attention-deficit hyperactivity disorder (ADHD) and autism spectrum disorder (ASD). In the United States, nearly 10% of pregnant women smoke despite ongoing public health efforts to reduce the incidence of smoking. Of additional concern is the steady rise of electronic nicotine delivery systems (ENDS) use among pregnant women over the past decade. Further, ENDS are often used in conjunction with tobacco cigarettes, compounding exposure effects. In animal models, both maternal TS and nicotine exposure lead to adverse neurodevelopmental outcomes, including increased anxiety and ADHD-like behavior, that are transmitted to subsequent generations. A likely explanation for this phenomenon lies in early developmental epigenetic programming. Epigenetic markers that are established early in development, like DNA methylation, can persist throughout somatic cell division and gametogenesis. During early development, zygotic DNA methylation is reprogrammed following a wave of global demethylation, and only re-established during the peri-implantation period. Environmental perturbations during these critical phases of reprogramming have been associated with persistent, and even transgenerationally-inherited effects, underscoring the importance of examining these associations in the context of human health and disease.

The broader goals of this dissertation were to identify alterations in DNA methylation patterning in the brain as a result of maternal TS exposure, and assess their neurodevelopmental significance. In an effort to better understand the effects of nicotine alone, especially given the increasing usage of ENDS during pregnancy, we chose to examine developmental nicotine exposure in addition to TS exposure. Finally, we evaluated the translational significance of these alterations by examining correlations in humans developmentally exposed to TS. Using a rat model for gestational nicotine exposure, DNA methylation levels were measured in the brains of neonatal and adult rats to determine the persistence of exposure effects. Specific brain regions, including the rat preoptic area (POA), hippocampus and cortex were targeted for evaluation based on their neurobehavioral significance. A rat gestational exposure model for tobacco smoke extract (TSE) was further employed to determine potential overlap with methylomic regions affected by developmental nicotine exposure. Finally, to derive translational implications, DNA methylation was examined on both epigenome-wide and targeted levels in human cord blood from newborns exposed to maternal TS.

In neonatal rats developmentally exposed to nicotine, DNA methylation was reduced in regions implicated in masculinization of the preoptic area (POA), a region of the brain that requires epigenetic reprogramming events to sexually differentiate. Subsequent behavioral analyses in adulthood revealed that these alterations may have contributed to the developmental masculinization of the POA in nicotine-exposed females. In adults males developmentally exposed to nicotine, alterations to DNA methylation were observed in the hippocampus and cortex, two brain regions that are also associated with ADHD- and ASD-like behaviors, respectively. Further, a comparison of differentially-methylated regions (DMRs) between the brains of animals exposed to developmental nicotine and TSE revealed significant overlap, indicating that nicotine is largely driving the developmental alterations to DNA methylation observed in TSE-exposed animals. Examination of DNA methylation alterations in human infant cord blood as a result of maternal TS exposure indicated significant overlap with those revealed in rats, supporting common impacts on developmental epigenetic reprogramming across species. Moreover, nearly half of these common regions were implicated in neurodevelopmental disorders, namely ASD and ADHD. Alterations to DNA methylation at human metastable epialleles, or regions for which DNA methylation is stochastically established during early development, were observed in the cord blood of infants exposed to TS in utero, supporting the ability of TS-exposure to alter vulnerable regions of the epigenome during early developmental reprogramming.