Epigenetic inheritance of DNA methylation changes in fish living in hydrogen sulfide-rich springs.

Environmental factors can promote phenotypic variation through alterations in the epigenome and facilitate adaptation of an organism to the environment. Although hydrogen sulfide is toxic to most organisms, the fish Poecilia mexicana has adapted to survive in environments with high levels that exceed toxicity thresholds by orders of magnitude. Epigenetic changes in response to this environmental stressor were examined by assessing DNA methylation alterations in red blood cells, which are nucleated in fish. Males and females were sampled from sulfidic and nonsulfidic natural environments; individuals were also propagated for two generations in a nonsulfidic laboratory environment. We compared epimutations between the sexes as well as field and laboratory populations. For both the wild-caught (F0) and the laboratory-reared (F2) fish, comparing the sulfidic and nonsulfidic populations revealed evidence for significant differential DNA methylation regions (DMRs). More importantly, there was over 80% overlap in DMRs across generations, suggesting that the DMRs have stable generational inheritance in the absence of the sulfidic environment. This is an example of epigenetic generational stability after the removal of an environmental stressor. The DMR-associated genes were related to sulfur toxicity and metabolic processes. These findings suggest that adaptation of P. mexicana to sulfidic environments in southern Mexico may, in part, be promoted through epigenetic DNA methylation alterations that become stable and are inherited by subsequent generations independent of the environment.

Developmental origins of transgenerational sperm DNA methylation epimutations following ancestral DDT exposure.

Epigenetic alterations in the germline can be triggered by a number of different environmental factors from diet to toxicants. These environmentally induced germline changes can promote the epigenetic transgenerational inheritance of disease and phenotypic variation. In previous studies, the pesticide DDT was shown to promote the transgenerational inheritance of sperm differential DNA methylation regions (DMRs), also called epimutations, which can in part mediate this epigenetic inheritance. In the current study, the developmental origins of the transgenerational DMRs during gametogenesis have been investigated. Male control and DDT lineage F3 generation rats were used to isolate embryonic day 16 (E16) prospermatogonia, postnatal day 10 (P10) spermatogonia, adult pachytene spermatocytes, round spermatids, caput epididymal spermatozoa, and caudal sperm. The DMRs between the control versus DDT lineage samples were determined at each developmental stage. The top 100 statistically significant DMRs at each stage were compared and the developmental origins of the caudal epididymal sperm DMRs were assessed. The chromosomal locations and genomic features of the different stage DMRs were analyzed. Although previous studies have demonstrated alterations in the DMRs of primordial germ cells (PGCs), the majority of the DMRs identified in the caudal sperm originated during the spermatogonia stages in the testis. Interestingly, a cascade of epigenetic alterations initiated in the PGCs is required to alter the epigenetic programming during spermatogenesis to obtain the sperm epigenetics involved in the epigenetic transgenerational inheritance phenomenon.

Epigenetic variation between urban and rural populations of Darwin's finches.

BACKGROUND: The molecular basis of evolutionary change is assumed to be genetic variation. However, growing evidence suggests that epigenetic mechanisms, such as DNA methylation, may also be involved in rapid adaptation to new environments. An important first step in evaluating this hypothesis is to test for the presence of epigenetic variation between natural populations living under different environmental conditions. RESULTS: In the current study we explored variation between populations of Darwin's finches, which comprise one of the best-studied examples of adaptive radiation. We tested for morphological, genetic, and epigenetic differences between adjacent "urban" and "rural" populations of each of two species of ground finches, Geospiza fortis and G. fuliginosa, on Santa Cruz Island in the Galápagos. Using data collected from more than 1000 birds, we found significant morphological differences between populations of G. fortis, but not G. fuliginosa. We did not find large size copy number variation (CNV) genetic differences between populations of either species. However, other genetic variants were not investigated. In contrast, we did find dramatic epigenetic differences between the urban and rural populations of both species, based on DNA methylation analysis. We explored genomic features and gene associations of the differentially DNA methylated regions (DMR), as well as their possible functional significance. CONCLUSIONS: In summary, our study documents local population epigenetic variation within each of two species of Darwin's finches.

SRY induced TCF21 genome-wide targets and cascade of bHLH factors during Sertoli cell differentiation and male sex determination in rats.

Male sex determination is initiated through the testis-determining factor SRY that promotes Sertoli cell differentiation and subsequent gonadal development. The basic helix-loop-helix (bHLH) gene Tcf21 was identified as one of the direct downstream targets of SRY. The current study was designed to identify the downstream targets of TCF21 and the potential cascade of bHLH genes that promote Sertoli cell differentiation. A modified ChIP-Chip comparative hybridization analysis identified 121 direct downstream binding targets for TCF21. The gene networks and cellular pathways potentially regulated by these TCF21 targets were identified. One of the main bHLH targets for TCF21 was the bHLH gene scleraxis (Scx). An embryonic ovarian gonadal cell culture was used to examine the functional role of Sry, Tcf21, and Scx to promote an in vitro sex reversal and induction of Sertoli cell differentiation. SRY and TCF21 were found to induce the initial stages of Sertoli cell differentiation, whereas SCX was found to induce the later stages of Sertoli cell differentiation associated with pubertal development using transferrin gene expression as a marker. Therefore, a cascade of SRY followed by TCF21 followed by SCX appears to promote, in part, Sertoli cell fate determination and subsequent differentiation. The current observations help elucidate the initial molecular events involved in the induction of Sertoli cell differentiation and testis development.

SRY directly regulates the neurotrophin 3 promoter during male sex determination and testis development in rats.

Neurotrophin 3 (Ntf3) is expressed in Sertoli cells and acts as a chemo-attractant for cell migration from the mesonephros into the developing testis, a process critical to the early morphological events of testis cord formation. The male sex-determining gene Sry initiates the process of testicular development. Sox9 is a key regulator of male sex determination and is directly regulated by SRY. Information on other downstream target genes of SRY is limited. The current study demonstrates an interaction of SRY with the Ntf3 promoter both in vitro and in vivo. The Ntf3 promoter in both rat and mouse contains at least one putative SRY binding site in the -0.6 kb promoter region. In a luciferase reporter assay system, both SRY and SOX9 stimulated the Ntf3 promoter in vitro through an interaction with this SRY-binding motif. In an immunoprecipitation-based pull-down assay, recombinant SRY protein bound the Ntf3 promoter fragment containing an intact SRY binding site, whereas the same protein did not interact with the fragment containing a mutated SRY motif. Specific antibodies against SRY were used in a chromatin immunoprecipitation (ChIP) assay of embryonic testis and were found to precipitate the Ntf3 promoter region. The SRY ChIP assay confirmed the direct interaction between SRY and the Ntf3 promoter in vivo during male sex determination. Observations suggest that SRY physically interacts with the Ntf3 promoter during male sex determination to coordinate cell migration in the testis to form testis cords.

Genome-Wide Mapping of DNA Methylation 5mC by Methylated DNA Immunoprecipitation (MeDIP)-Sequencing.

Methylated DNA immunoprecipitation is a large scale purification technique. It enables the isolation of methylated DNA fragments for subsequent locus-specific or genome-wide analysis. Here we describe an immunoprecipitation protocol using a monoclonal mouse anti 5-methyl-cytidine antibody followed by next-generation sequencing (MeDIP-Seq).

Differential DNA methylation in somatic and sperm cells of hatchery vs wild (natural-origin) steelhead trout populations.

Environmental factors such as nutrition, stress, and toxicants can influence epigenetic programming and phenotypes of a wide variety of species from plants to humans. The current study was designed to investigate the impacts of hatchery spawning and rearing on steelhead trout (Oncorhynchus mykiss) vs the wild fish on a molecular level. Additionally, epigenetic differences between feeding practices that allow slow growth (2 years) and fast growth (1 year) hatchery trout were investigated. The sperm and red blood cells (RBC) from adult male slow growth/maturation hatchery steelhead, fast growth/maturation hatchery steelhead, and wild (natural-origin) steelhead were collected for DNA preparation to investigate potential alterations in differential DNA methylation regions (DMRs) and genetic mutations, involving copy number variations (CNVs). The sperm and RBC DNA both had a large number of DMRs when comparing the hatchery vs wild steelhead trout populations. The DMRs were cell type specific with negligible overlap. Slow growth/maturation compared to fast growth/maturation steelhead also had a larger number of DMRs in the RBC samples. A number of the DMRs had associated genes that were correlated to various biological processes and pathologies. Observations demonstrate a major epigenetic programming difference between the hatchery and wild natural-origin fish populations, but negligible genetic differences. Therefore, hatchery conditions and growth/maturation rate can alter the epigenetic developmental programming of the steelhead trout. Interestingly, epigenetic alterations in the sperm allow for potential epigenetic transgenerational inheritance of phenotypic variation to future generations. The impacts of hatchery exposures are not only important to consider on the fish exposed, but also on future generations and evolutionary trajectory of fish in the river populations.

Epigenome association study for DNA methylation biomarkers in buccal and monocyte cells for female rheumatoid arthritis.

Genetics (i.e., mutations) has been assumed to be the major factor in rheumatoid arthritis (RA) etiology, but accounts for a minority of the variance in disease risk for RA. In contrast to genetics, the environment can have dramatic impacts on epigenetics that associate with disease etiology. The current study used buccal cells and purified blood monocytes from two different clinical cohorts involving Caucasian or African American female populations with or without arthritis. The differential DNA methylation regions (DMRs) between the control and RA populations were identified with an epigenome-wide association study. The DMRs (i.e., epimutations) identified in the buccal cells and monocytes were found to be distinct. The DMR associated genes were identified and many have previously been shown to be associated with arthritis. Observations demonstrate DNA methylation epimutation RA biomarkers are cell type specific and similar findings were observed with the two racial background populations. Rheumatoid arthritis susceptibility epigenetic diagnosis appears feasible and may improve the clinical management of RA and allowpreventative medicine considerations.

Sperm DNA methylation epimutation biomarker for paternal offspring autism susceptibility.

BACKGROUND: Autism spectrum disorder (ASD) has increased over tenfold over the past several decades and appears predominantly associated with paternal transmission. Although genetics is anticipated to be a component of ASD etiology, environmental epigenetics is now also thought to be an important factor. Epigenetic alterations, such as DNA methylation, have been correlated with ASD. The current study was designed to identify a DNA methylation signature in sperm as a potential biomarker to identify paternal offspring autism susceptibility. METHODS AND RESULTS: Sperm samples were obtained from fathers that have children with or without autism, and the sperm then assessed for alterations in DNA methylation. A genome-wide analysis (> 90%) for differential DNA methylation regions (DMRs) was used to identify DMRs in the sperm of fathers (n = 13) with autistic children in comparison with those (n = 13) without ASD children. The 805 DMR genomic features such as chromosomal location, CpG density and length of the DMRs were characterized. Genes associated with the DMRs were identified and found to be linked to previously known ASD genes, as well as other neurobiology-related genes. The potential sperm DMR biomarkers/diagnostic was validated with blinded test sets (n = 8-10) of individuals with an approximately 90% accuracy. CONCLUSIONS: Observations demonstrate a highly significant set of 805 DMRs in sperm that can potentially act as a biomarker for paternal offspring autism susceptibility. Ancestral or early-life paternal exposures that alter germline epigenetics are anticipated to be a molecular component of ASD etiology.

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.

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.

Sperm DNA Methylation Epimutation Biomarkers for Male Infertility and FSH Therapeutic Responsiveness.

Male factor infertility is increasing and recognized as playing a key role in reproductive health and disease. The current primary diagnostic approach is to assess sperm quality associated with reduced sperm number and motility, which has been historically of limited success in separating fertile from infertile males. The current study was designed to develop a molecular analysis to identify male idiopathic infertility using genome wide alterations in sperm DNA methylation. A signature of differential DNA methylation regions (DMRs) was identified to be associated with male idiopathic infertility patients. A promising therapeutic treatment of male infertility is the use of follicle stimulating hormone (FSH) analogs which improved sperm numbers and motility in a sub-population of infertility patients. The current study also identified genome-wide DMRs that were associated with the patients that were responsive to FSH therapy versus those that were non-responsive. This novel use of epigenetic biomarkers to identify responsive versus non-responsive patient populations is anticipated to dramatically improve clinical trials and facilitate therapeutic treatment of male infertility patients. The use of epigenetic biomarkers for disease and therapeutic responsiveness is anticipated to be applicable for other medical conditions.

Epigenetic transgenerational inheritance of testis pathology and Sertoli cell epimutations: generational origins of male infertility.

Male reproductive health has been in decline for decades with dropping sperm counts and increasing infertility, which has created a significant societal and economic burden. Between the 1970s and now, a general decline of over 50% in sperm concentration has been observed in the population. Environmental toxicant-induced epigenetic transgenerational inheritance has been shown to affect testis pathology and sperm count. Sertoli cells have an essential role in spermatogenesis by providing physical and nutritional support for developing germ cells. The current study was designed to further investigate the transgenerational epigenetic changes in the rat Sertoli cell epigenome and transcriptome that are associated with the onset of testis disease. Gestating female F0 generation rats were transiently exposed during the period of fetal gonadal sex determination to the environmental toxicants, such as dichlorodiphenyltrichloroethane (DDT) or vinclozolin. The F1 generation offspring were bred (i.e. intercross within the lineage) to produce the F2 generation grand-offspring that were then bred to produce the transgenerational F3 generation (i.e. great-grand-offspring) with no sibling or cousin breeding used. The focus of the current study was to investigate the transgenerational testis disease etiology, so F3 generation rats were utilized. The DNA and RNA were obtained from purified Sertoli cells isolated from postnatal 20-day-old male testis of F3 generation rats. Transgenerational alterations in DNA methylation, noncoding RNA, and gene expression were observed in the Sertoli cells from vinclozolin and DDT lineages when compared to the control (vehicle exposed) lineage. Genes associated with abnormal Sertoli cell function and testis pathology were identified, and the transgenerational impacts of vinclozolin and DDT were determined. Alterations in critical gene pathways, such as the pyruvate metabolism pathway, were identified. Observations suggest that ancestral exposures to environmental toxicants promote the epigenetic transgenerational inheritance of Sertoli cell epigenetic and transcriptome alterations that associate with testis abnormalities. These epigenetic alterations appear to be critical factors in the developmental and generational origins of testis pathologies and male infertility.

Transgenerational sperm DNA methylation epimutation developmental origins following ancestral vinclozolin exposure.

A number of environmental factors from nutrition to toxicants have been shown to promote the epigenetic transgenerational inheritance of disease and phenotypic variation. This requires alterations in the germline (sperm or egg) epigenome. Previously, the agricultural fungicide vinclozolin was found to promote the transgenerational inheritance of sperm differential DNA methylation regions (DMRs) termed epimutations that help mediate this epigenetic inheritance. The current study was designed to investigate the developmental origins of the transgenerational DMRs during gametogenesis. Male control and vinclozolin lineage F3 generation rats were used as a source of embryonic day 13 (E13) primordial germ cells, embryonic day 16 (E16) prospermatogonia, postnatal day 10 (P10) spermatogonia, adult pachytene spermatocytes, round spermatids, caput epididymal spermatozoa, and caudal sperm. The DMRs between the control versus vinclozolin lineage samples were determined for each developmental stage. The top 100 statistically significant DMRs for each stage were compared. The developmental origins of the caudal epididymal sperm DMRs were assessed. The chromosomal locations and genomic features of the different stage DMRs were investigated. In addition, the DMR associated genes were identified. Previous studies have demonstrated alterations in the DMRs of primordial germ cells (PGCs). Interestingly, the majority of the DMRs identified in the current study for the caudal sperm originated during the spermatogenic process in the testis. A cascade of epigenetic alterations initiated in the PGCs appears to be required to alter the epigenetic programming during spermatogenesis to modify the sperm epigenome involved in the transgenerational epigenetic inheritance phenomenon.

Environmental Toxicant Induced Epigenetic Transgenerational Inheritance of Prostate Pathology and Stromal-Epithelial Cell Epigenome and Transcriptome Alterations: Ancestral Origins of Prostate Disease.

Prostate diseases include prostate cancer, which is the second most common male neoplasia, and benign prostatic hyperplasia (BPH), which affects approximately 50% of men. The incidence of prostate disease is increasing, and some of this increase may be attributable to ancestral exposure to environmental toxicants and epigenetic transgenerational inheritance mechanisms. The goal of the current study was to determine the effects that exposure of gestating female rats to vinclozolin has on the epigenetic transgenerational inheritance of prostate disease, and to characterize by what molecular epigenetic mechanisms this has occurred. Gestating female rats (F0 generation) were exposed to vinclozolin during E8-E14 of gestation. F1 generation offspring were bred to produce the F2 generation, which were bred to produce the transgenerational F3 generation. The transgenerational F3 generation vinclozolin lineage males at 12 months of age had an increased incidence of prostate histopathology and abnormalities compared to the control lineage. Ventral prostate epithelial and stromal cells were isolated from F3 generation 20-day old rats, prior to the onset of pathology, and used to obtain DNA and RNA for analysis. Results indicate that there were transgenerational changes in gene expression, noncoding RNA expression, and DNA methylation in both cell types. Our results suggest that ancestral exposure to vinclozolin at a critical period of gestation induces the epigenetic transgenerational inheritance of prostate stromal and epithelial cell changes in both the epigenome and transcriptome that ultimately lead to prostate disease susceptibility and may serve as a source of the increased incidence of prostate pathology observed in recent years.

Sperm epimutation biomarkers of obesity and pathologies following DDT induced epigenetic transgenerational inheritance of disease.

Dichlorodiphenyltrichloroethane (DDT) has previously been shown to promote the epigenetic transgenerational inheritance of adult onset disease in rats. The current study investigated the potential that sperm epimutation biomarkers can be used to identify ancestral induced transgenerational obesity and associated pathologies. Gestating F0 generational female rats were transiently exposed to DDT during fetal gonadal sex determination, and the incidence of adult-onset pathologies was assessed in the subsequent F1, F2, and F3 generations. In addition, sperm differential DNA methylation regions (DMRs) that were associated with specific pathologies in the transgenerational F3 generation males were investigated. There was an increase of testis disease and early-onset puberty in the F2 generation DDT lineage males. The F3 generation males and females had significant increases in the incidence of obesity and multiple disease. The F3 generation DDT males also had significant increases in testis disease, prostate disease, and late onset puberty. The F3 generation DDT females had increases in ovarian and kidney disease. Epigenetic alterations of the germline are required for the transgenerational inheritance of pathology. Therefore, the F3 generation sperm was collected to examine DMRs for the ancestrally exposed DDT male population. Unique sets of DMRs were associated with late onset puberty, prostate disease, kidney disease, testis disease, obesity, and multiple disease pathologies. Gene associations with the DMR were also identified. The epigenetic DMR signatures identified for these pathologies provide potential biomarkers for transgenerationally inherited disease susceptibility.

Assessment of Glyphosate Induced Epigenetic Transgenerational Inheritance of Pathologies and Sperm Epimutations: Generational Toxicology.

Ancestral environmental exposures to a variety of factors and toxicants have been shown to promote the epigenetic transgenerational inheritance of adult onset disease. One of the most widely used agricultural pesticides worldwide is the herbicide glyphosate (N-(phosphonomethyl)glycine), commonly known as Roundup. There are an increasing number of conflicting reports regarding the direct exposure toxicity (risk) of glyphosate, but no rigorous investigations on the generational actions. The current study using a transient exposure of gestating F0 generation female rats found negligible impacts of glyphosate on the directly exposed F0 generation, or F1 generation offspring pathology. In contrast, dramatic increases in pathologies in the F2 generation grand-offspring, and F3 transgenerational great-grand-offspring were observed. The transgenerational pathologies observed include prostate disease, obesity, kidney disease, ovarian disease, and parturition (birth) abnormalities. Epigenetic analysis of the F1, F2 and F3 generation sperm identified differential DNA methylation regions (DMRs). A number of DMR associated genes were identified and previously shown to be involved in pathologies. Therefore, we propose glyphosate can induce the transgenerational inheritance of disease and germline (e.g. sperm) epimutations. Observations suggest the generational toxicology of glyphosate needs to be considered in the disease etiology of future generations.

Regulation of granulosa and theca cell transcriptomes during ovarian antral follicle development.

Coordinated interactions between ovarian granulosa and theca cells are required for female endocrine function and fertility. To elucidate these interactions the regulation of the granulosa and theca cell transcriptomes during bovine antral follicle development were investigated. Granulosa cells and theca cells were isolated from small (<5 mm), medium (5-10 mm), and large (>10 mm) antral bovine follicles. A microarray analysis of 24,000 bovine genes revealed that granulosa cells and theca cells each had gene sets specific to small, medium and large follicle cells. Transcripts regulated (i.e., minimally changed 1.5-fold) during antral follicle development for the granulosa cells involved 446 genes and for theca cells 248 genes. Only 28 regulated genes were common to both granulosa and theca cells. Regulated genes were functionally categorized with a focus on growth factors and cytokines expressed and regulated by the two cell types. Candidate regulatory growth factor proteins mediating both paracrine and autocrine cell-cell interactions include macrophage inflammatory protein (MIP1 beta), teratocarcinoma-derived growth factor 1 (TDGF1), stromal derived growth factor 1 (SDF1; i.e., CXCL12), growth differentiation factor 8 (GDF8), glia maturation factor gamma (GMFG), osteopontin (SPP1), angiopoietin 4 (ANGPT4), and chemokine ligands (CCL 2, 3, 5, and 8). The current study examined granulosa cell and theca cell regulated genes associated with bovine antral follicle development and identified candidate growth factors potentially involved in the regulation of cell-cell interactions required for ovarian function.

Environmentally induced epigenetic transgenerational inheritance of disease.

Ancestral environmental exposures such as toxicants, abnormal nutrition or stress can promote the epigenetic transgenerational inheritance of disease and phenotypic variation. These environmental factors induce the epigenetic reprogramming of the germline (sperm and egg). The germline epimutations can in turn increase disease susceptibility of subsequent generations of the exposed ancestors. A variety of environmental factors, species and exposure specificity of this induced epigenetic transgenerational inheritance of disease is discussed with a consideration of generational toxicology. The molecular mechanisms and processes involved in the ability of these inherited epimutations to increase disease susceptibility are discussed. In addition to altered disease susceptibility, the potential impact of the epigenetic inheritance on phenotypic variation and evolution is considered. Observations suggest environmentally induced epigenetic transgenerational inheritance of disease is a critical aspect of disease etiology, toxicology and evolution that needs to be considered.

Epigenetic Transgenerational Inheritance of Altered Sperm Histone Retention Sites.

A variety of environmental toxicants and factors have been shown to induce the epigenetic transgenerational inheritance of disease and phenotypic variation. Epigenetic alterations in the germline (sperm or egg) are required to transmit transgenerational phenotypes. The current study was designed to investigate the potential role of histones in sperm to help mediate the epigenetic transgenerational inheritance. The agricultural fungicide vinclozolin and the pesticide DDT (dichlorodiphenyltrichloroethane) were independently used to promote the epigenetic transgenerational inheritance of disease. Purified cauda epididymal sperm were collected from the transgenerational F3 generation control and exposure lineage male rats for histone analysis. A reproducible core of histone H3 retention sites was observed using an H3 chromatin immunoprecipitation (ChIP-Seq) analysis in control lineage sperm. Interestingly, the same core group of H3 retention sites plus additional differential histone retention sites (DHRs) were observed in the F3 generation exposure lineage sperm. Although new histone H3 retention sites were observed, negligible change in histone modification (methylation of H3K27me3) was observed between the control and exposure lineages. Observations demonstrate that in addition to alterations in sperm DNA methylation and ncRNA previously identified, the induction of differential histone retention sites (DHRs) also appear to be involved in environmentally induced epigenetic transgenerational inheritance.