Epigenome-wide association study of physical activity and physiological parameters in discordant monozygotic twins.

An epigenome-wide association study (EWAS) was performed on buccal cells from monozygotic-twins (MZ) reared together as children, but who live apart as adults. Cohorts of twin pairs were used to investigate associations between neighborhood walkability and objectively measured physical activity (PA) levels. Due to dramatic cellular epigenetic sex differences, male and female MZ twin pairs were analyzed separately to identify differential DNA methylation regions (DMRs). A priori comparisons were made on MZ twin pairs discordant on body mass index (BMI), PA levels, and neighborhood walkability. In addition to direct comparative analysis to identify specific DMRs, a weighted genome coexpression network analysis (WGCNA) was performed to identify DNA methylation sites associated with the physiological traits of interest. The pairs discordant in PA levels had epigenetic alterations that correlated with reduced metabolic parameters (i.e., BMI and waist circumference). The DNA methylation sites are associated with over fifty genes previously found to be specific to vigorous PA, metabolic risk factors, and sex. Combined observations demonstrate that behavioral factors, such as physical activity, appear to promote systemic epigenetic alterations that impact metabolic risk factors. The epigenetic DNA methylation sites and associated genes identified provide insight into PA impacts on metabolic parameters and the etiology of obesity.

Ancestral plastics exposure induces transgenerational disease-specific sperm epigenome-wide association biomarkers.

Plastic-derived compounds are one of the most frequent daily worldwide exposures. Previously a mixture of plastic-derived toxicants composed of bisphenol A, bis(2-ethylhexyl) phthalate, and dibutyl phthalate at low-dose exposures of a gestating female rats was found to promote the epigenetic transgenerational inheritance of disease to the offspring (F1 generation), grand-offspring (F2 generation), and great-grand-offspring (F3 generation). Epigenetic analysis of the male sperm was found to result in differential DNA methylation regions (DMRs) in the transgenerational F3 generation male sperm. The current study is distinct and was designed to use an epigenome-wide association study to identify potential sperm DNA methylation biomarkers for specific transgenerational diseases. Observations indicate disease-specific DMRs called epimutations in the transgenerational F3 generation great-grand-offspring of rats ancestrally exposed to plastics. The epigenetic DMR biomarkers were identified for testis disease, kidney disease, and multiple (≥2) diseases. These disease sperm epimutation biomarkers were found to be predominantly disease-specific. The genomic locations and features of these DMRs were identified. Interestingly, the disease-specific DMR-associated genes were previously shown to be linked with each of the specific diseases. Therefore, the germline has ancestrally derived epimutations that potentially transmit transgenerational disease susceptibilities. Epigenetic biomarkers for specific diseases could be used as diagnostics to facilitate clinical management of disease and preventative medicine.

Transgenerational disease specific epigenetic sperm biomarkers after ancestral exposure to dioxin.

Dioxin was historically one of the most common industrial contaminants with several major industry accidents, as well as governmental actions involving military service, having exposed large numbers of the worldwide population over the past century. Previous rat studies have demonstrated the ability of dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)) exposure to promote the epigenetic transgenerational inheritance of disease susceptibility in subsequent generations. The types of disease previously observed include puberty abnormalities, testis, ovary, kidney, prostate and obesity pathologies. The current study was designed to use an epigenome-wide association study (EWAS) to identify potential sperm DNA methylation biomarkers for specific transgenerational diseases. Therefore, the transgenerational F3 generation dioxin lineage male rats with and without a specific disease were compared to identify differential DNA methylation regions (DMRs) as biomarkers for disease. The genomic features of the disease-specific DMRs were characterized. Observations demonstrate that disease-specific epimutation DMRs exist for the transgenerational dioxin lineage rats that can potentially be used as epigenetic biomarkers for testis, kidney, prostate and obesity diseases. These disease-specific DMRs were associated with genes that have previously been shown to be linked with the specific diseases. This EWAS for transgenerational disease identified potential epigenetic biomarkers and provides the proof of concept of the potential to develop similar biomarkers for humans to diagnose disease susceptibilities and facilitate preventative medicine.

Epigenome-wide association study for atrazine induced transgenerational DNA methylation and histone retention sperm epigenetic biomarkers for disease.

Atrazine is a common agricultural herbicide previously shown to promote epigenetic transgenerational inheritance of disease to subsequent generations. The current study was designed as an epigenome-wide association study (EWAS) to identify transgenerational sperm disease associated differential DNA methylation regions (DMRs) and differential histone retention regions (DHRs). Gestating female F0 generation rats were transiently exposed to atrazine during the period of embryonic gonadal sex determination, and then subsequent F1, F2, and F3 generations obtained in the absence of any continued exposure. The transgenerational F3 generation males were assessed for disease and sperm collected for epigenetic analysis. Pathology was observed in pubertal onset and for testis disease, prostate disease, kidney disease, lean pathology, and multiple disease. For these pathologies, sufficient numbers of individual males with only a single specific disease were identified. The sperm DNA and chromatin were isolated from adult one-year animals with the specific diseases and analyzed for DMRs with methylated DNA immunoprecipitation (MeDIP) sequencing and DHRs with histone chromatin immunoprecipitation (ChIP) sequencing. Transgenerational F3 generation males with or without disease were compared to identify the disease specific epimutation biomarkers. All pathologies were found to have disease specific DMRs and DHRs which were found to predominantly be distinct for each disease. No common DMRs or DHRs were found among all the pathologies. Epimutation gene associations were identified and found to correlate to previously known disease linked genes. This is one of the first observations of potential sperm disease biomarkers for histone retention sites. Although further studies with expanded animal numbers are required, the current study provides evidence the EWAS analysis is effective for the identification of potential pathology epimutation biomarkers for disease susceptibility.

Epigenome-wide association study for pesticide (Permethrin and DEET) induced DNA methylation epimutation biomarkers for specific transgenerational disease.

BACKGROUND: Permethrin and N,N-diethyl-meta-toluamide (DEET) are the pesticides and insect repellent most commonly used by humans. These pesticides have been shown to promote the epigenetic transgenerational inheritance of disease in rats. The current study was designed as an epigenome-wide association study (EWAS) to identify potential sperm DNA methylation epimutation biomarkers for specific transgenerational disease. METHODS: Outbred Sprague Dawley gestating female rats (F0) were transiently exposed during fetal gonadal sex determination to the pesticide combination including Permethrin and DEET. The F3 generation great-grand offspring within the pesticide lineage were aged to 1 year. The transgenerational adult male rat sperm were collected from individuals with single and multiple diseases and compared to non-diseased animals to identify differential DNA methylation regions (DMRs) as biomarkers for specific transgenerational disease. RESULTS: The exposure of gestating female rats to a permethrin and DEET pesticide combination promoted transgenerational testis disease, prostate disease, kidney disease, and the presence of multiple disease in the subsequent F3 generation great-grand offspring. The disease DMRs were found to be disease specific with negligible overlap between different diseases. The genomic features of CpG density, DMR length, and chromosomal locations of the disease specific DMRs were investigated. Interestingly, the majority of the disease specific sperm DMR associated genes have been previously found to be linked to relevant disease specific genes. CONCLUSIONS: Observations demonstrate the EWAS approach identified disease specific biomarkers that can be potentially used to assess transgenerational disease susceptibility and facilitate the clinical management of environmentally induced pathology.

Epigenome-wide association study for transgenerational disease sperm epimutation biomarkers following ancestral exposure to jet fuel hydrocarbons.

Jet fuel hydrocarbons is the generic name for aviation fuels used in gas-turbine engine powered aircraft. The Deepwater Horizon oil rig explosion created the largest environmental disaster in U.S. history, and the second largest oil spill in human history with over 800 million liters of hydrocarbons released into the Gulf of Mexico over a period of 3 months. Due to the widespread use of jet fuel hydrocarbons, this compound mixture has been recognized as the single largest chemical exposure for military personnel. Previous animal studies have demonstrated the ability of jet fuel (JP-8) exposure to promote the epigenetic transgenerational inheritance of disease susceptibility in subsequent generations. The diseases observed include late puberty, kidney, obesity and multiple disease pathologies. The current study is distinct and was designed to identify potential sperm DNA methylation biomarkers for specific transgenerational diseases. Observations show disease specific differential DNA methylation regions (DMRs) called epimutations in the transgenerational F3 generation great-grand-offspring male rats ancestrally exposed to jet fuel. The potential epigenetic DMR biomarkers were identified for late puberty, kidney, obesity, and multiple diseases, and found to be predominantly disease specific. These disease specific DMRs have associated genes that were previously shown to be linked with each of these specific diseases. Therefore, the germline (i.e. sperm) has environmentally induced ancestrally derived epimutations that have the potential to transgenerationally transmit disease susceptibilities to subsequent generations. Epigenetic biomarkers for specific diseases could be developed as medical diagnostics to facilitate clinical management of disease, and allow preventative medicine therapeutics.

Between-Generation Phenotypic and Epigenetic Stability in a Clonal Snail.

Epigenetic variation might play an important role in generating adaptive phenotypes by underpinning within-generation developmental plasticity, persistent parental effects of the environment (e.g., transgenerational plasticity), or heritable epigenetically based polymorphism. These adaptive mechanisms should be most critical in organisms where genetic sources of variation are limited. Using a clonally reproducing freshwater snail (Potamopyrgus antipodarum), we examined the stability of an adaptive phenotype (shell shape) and of DNA methylation between generations. First, we raised three generations of snails adapted to river currents in the lab without current. We showed that habitat-specific adaptive shell shape was relatively stable across three generations but shifted slightly over generations two and three toward a no-current lake phenotype. We also showed that DNA methylation specific to high-current environments was stable across one generation. This study provides the first evidence of stability of DNA methylation patterns across one generation in an asexual animal. Together, our observations are consistent with the hypothesis that adaptive shell shape variation is at least in part determined by transgenerational plasticity, and that DNA methylation provides a potential mechanism for stability of shell shape across one generation.

Epigenome-wide association study (EWAS) for potential transgenerational disease epigenetic biomarkers in sperm following ancestral exposure to the pesticide methoxychlor.

Environmental exposures such as chemical toxicants can alter gene expression and disease susceptibility through epigenetic processes. Epigenetic changes can be passed to future generations through germ cells through epigenetic transgenerational inheritance of increased disease susceptibility. The current study used an epigenome-wide association study (EWAS) to investigate whether specific transgenerational epigenetic signatures of differential DNA methylation regions (DMRs) exist that are associated with particular disease states in the F3 generation great-grand offspring of F0 generation rats exposed during gestation to the agricultural pesticide methoxychlor. The transgenerational epigenetic profiles of sperm from F3 generation methoxychlor lineage rats that have only one disease state were compared to those that have no disease. Observations identify disease specific patterns of DMRs for these transgenerational rats that can potentially serve as epigenetic biomarkers for prostate disease, kidney disease, obesity, and the presence of multiple diseases. The chromosomal locations, genomic features, and gene associations of the DMRs are characterized. Disease specific DMR sets contained DMR-associated genes that have previously been shown to be associated with that specific disease. Future epigenetic biomarkers could potentially be developed and validated for humans as a disease susceptibility diagnostic tool to facilitate preventative medicine and management of disease.

Regional epigenetic variation in asexual snail populations among urban and rural lakes.

Epigenetic variation has the potential to influence environmentally dependent development and contribute to phenotypic responses to local environments. Environmental epigenetic studies of sexual organisms confirm the capacity to respond through epigenetic variation. An epigenetic response could be even more important in a population when genetic variation is lacking. A previous study of an asexual snail, Potamopyrgus antipodarum, demonstrated that different populations derived from a single clonal lineage differed in both shell phenotype and methylation signature when comparing lake versus river populations. Here, we examine methylation variation among lakes that differ in environmental disturbance and pollution histories. Snails were collected from a more pristine rural Lake 1 (Lake Lytle), and two urban lakes, Lake 2 (Capitol Lake) and Lake 3 (Lake Washington) on the Northwest Pacific coast. DNA methylation was assessed for each sample population using methylated DNA immunoprecipitation, MeDIP, followed by next-generation sequencing. The differential DNA methylation regions (DMRs) identified among the different lake comparisons suggested a higher number of DMRs and variation between rural Lake 1 and one urban Lake 2, and between the two urban Lakes 2 and 3, but limited variation between the rural Lake 1 and urban Lake 3. DMR genomic characteristics and gene associations were investigated. The presence of site-specific differences between each of these lake populations suggest an epigenetic response to varied environmental factors. The results do not support an effect of geographic distance in these populations. The role of dispersal distance among lakes, population history, environmental pollution and stably inherited methylation versus environmentally triggered methylation in producing the observed epigenetic variation are discussed. Observations support the proposal that epigenetic alterations may associate with phenotypic variation and environmental factors and history of the different lakes.

Epigenetics and adaptive phenotypic variation between habitats in an asexual snail.

In neo-Darwinian theory, adaptation results from a response to selection on relatively slowly accumulating genetic variation. However, more rapid adaptive responses are possible if selectable or plastic phenotypic variation is produced by epigenetic differences in gene expression. This rapid path to adaptation may prove particularly important when genetic variation is lacking, such as in small, bottlenecked, or asexual populations. To examine the potential for an epigenetic contribution to adaptive variation, we examined morphological divergence and epigenetic variation in genetically impoverished asexual populations of a freshwater snail, Potamopyrgus antipodarum, from distinct habitats (two lakes versus two rivers). These populations exhibit habitat specific differences in shell shape, and these differences are consistent with adaptation to water current speed. Between these same habitats, we also found significant genome wide DNA methylation differences. The differences between habitats were an order of magnitude greater than the differences between replicate sites of the same habitat. These observations suggest one possible mechanism for the expression of adaptive shell shape differences between habitats involves environmentally induced epigenetic differences. This provides a potential explanation for the capacity of this asexual snail to spread by adaptive evolution or plasticity to different environments.