Multigenerational temperature effects on paternal epigenetic inheritance in European sea bass

Andrea Cupp made a serendipitous discovery when she was a postdoctoral fellow at Washington State University: While investigating how chemicals affect sex determination in embryonic animals, she bred the offspring of pregnant rats that had been dosed with an insecticide called methoxyclor. When the males from that litter grew into adults, they had decreased sperm counts and higher rates of infertility. Cupp had seen these same abnormalities in the animals’ fathers, which had been exposed to methoxyclor in the womb. But this latest generation hadn’t been exposed that way, which suggested that methoxyclor’s toxic effects had carried over generations. “At first I couldn’t believe it,” says Cupp’s advisor, Michael Skinner, a biochemist and Washington State professor. “But then we repeated the breeding experiments and found that the results held up.” Skinner and Cupp, who is now a professor at the University of Nebraska–Lincoln, published their findings in 2005.1 Since that paper—which showed that reproductive effects not just from methoxyclor but also from the fungicide vinclozolin persisted for at least four generations—the number of published articles reporting similar transgenerational findings has increased steadily. “In the last year and half there’s been an explosion in studies showing transgenerational effects from exposure to a wide array of environmental stressors,” says Lisa Chadwick, a program administrator at the National Institute of Environmental Health Sciences (NIEHS). “This is a field that’s really starting to take off.” According to Chadwick, the new findings compel a reevaluation of how scientists perceive environmental health threats. “We have to think more long-term about the effects of chemicals that we’re exposed to every day,” she says. “This new research suggests they could have consequences not just for our own health and for that of our children, but also for the health of generations to come.” Figure 1 Glossary The NIEHS recently issued requests for applications totaling $3 million for research on transgenerational effects in mammals.2 Chadwick says funded studies will address two fundamental data needs, one pertaining to potential transgenerational mechanisms and another to the number of chemicals thought to exert these effects. These studies will extend to what’s known as the F3 generation—the great-grandchildren of the originally exposed animal. That’s because chemicals given to pregnant females (the F0 generation) interact not only with the fetal offspring (the F1 generation) but also the germ cells developing within those offspring, which mature into the sperm and eggs that give rise to the F2 generation. Thus, the F3 animals are the first generation to be totally unexposed to the original agent. Effects that extend to the F2 generation are known as “multigenerational,” whereas those that extend to the F3 generation are known as “transgenerational.”3 Transgenerational effects have now been reported for chemicals including permethrin, DEET, bisphenol A, certain phthalates, dioxin, jet fuel mixtures, nicotine, and tributyltin, among others. Most of these findings come from rodent studies.4,5,6,7 But preliminary evidence that chemical effects can carry over generations in humans is also emerging, although no F3 data have been published yet. Given the challenges of tracking effects over multiple human lifespans, the evidence is more difficult to interpret, particularly with respect to potential mechanisms, says Tessa Roseboom, a professor of early development and health at the Academic Medical Center in Amsterdam, the Netherlands. Still, some reports have linked nutritional deficiencies from famine and exposure to diethylstilbestrol (DES)—a nonsteroidal estrogen used to protect against miscarriage from the 1940s to the 1970s—to effects that persist among the grandchildren of exposed women.8,9,10,11,12,13

Environmental compounds are known to promote epigenetic transgenerational inheritance of adult onset disease in subsequent generations (F1–F3) following ancestral exposure during fetal gonadal sex determination. The current study was designed to determine if a mixture of plastic derived endocrine disruptor compounds bisphenol-A (BPA), bis(2-ethylhexyl)phthalate (DEHP) and dibutyl phthalate (DBP) at two different doses promoted epigenetic transgenerational inheritance of adult onset disease and associated DNA methylation epimutations in sperm. Gestating F0 generation females were exposed to either the “plastics” or “lower dose plastics” mixture during embryonic days 8 to 14 of gonadal sex determination and the incidence of adult onset disease was evaluated in F1 and F3 generation rats. There were significant increases in the incidence of total disease/abnormalities in F1 and F3 generation male and female animals from plastics lineages. Pubertal abnormalities, testis disease, obesity, and ovarian disease (primary ovarian insufficiency and polycystic ovaries) were increased in the F3 generation animals. Kidney and prostate disease were only observed in the direct fetally exposed F1 generation plastic lineage animals. Analysis of the plastics lineage F3 generation sperm epigenome previously identified 197 differential DNA methylation regions (DMR) in gene promoters, termed epimutations. A number of these transgenerational DMR form a unique direct connection gene network and have previously been shown to correlate with the pathologies identified. Observations demonstrate that a mixture of plastic derived compounds, BPA and phthalates, can promote epigenetic transgenerational inheritance of adult onset disease. The sperm DMR provide potential epigenetic biomarkers for transgenerational disease and/or ancestral environmental exposures.

Environmental compounds including fungicides, plastics, pesticides, dioxin and hydrocarbons can promote the epigenetic transgenerational inheritance of adult-onset disease in future generation progeny following ancestral exposure during the critical period of fetal gonadal sex determination. This study examined the actions of the pesticide methoxychlor to promote the epigenetic transgenerational inheritance of adult-onset disease and associated differential DNA methylation regions (i.e. epimutations) in sperm. Gestating F0 generation female rats were transiently exposed to methoxychlor during fetal gonadal development (gestation days 8 to 14) and then adult-onset disease was evaluated in adult F1 and F3 (great-grand offspring) generation progeny for control (vehicle exposed) and methoxychlor lineage offspring. There were increases in the incidence of kidney disease, ovary disease, and obesity in the methoxychlor lineage animals. In females and males the incidence of disease increased in both the F1 and the F3 generations and the incidence of multiple disease increased in the F3 generation. There was increased disease incidence in F4 generation reverse outcross (female) offspring indicating disease transmission was primarily transmitted through the female germline. Analysis of the F3 generation sperm epigenome of the methoxychlor lineage males identified differentially DNA methylated regions (DMR) termed epimutations in a genome-wide gene promoters analysis. These epimutations were found to be methoxychlor exposure specific in comparison with other exposure specific sperm epimutation signatures. Observations indicate that the pesticide methoxychlor has the potential to promote the epigenetic transgenerational inheritance of disease and the sperm epimutations appear to provide exposure specific epigenetic biomarkers for transgenerational disease and ancestral environmental exposures.

Introduction Haitians diagnosed with breast cancer in Haiti experience a significantly worse outcome than immigrants in Miami that appears to be related to 1) more advanced stage, 2) younger age, 3) more ER-negative tumors, and 4) lack of timely effective treatments including restricted access to trastuzumab and radiation. The objective of the study was to evaluate the epigenetic differences by DNA methylation between women of African descent who have breast cancer and are native to Haiti, the USA or who have emigrated from Haiti to the USA. Methods The study was IRB approved and conducted between 2017-2019 at University of Miami (n=50), Ministry of Health Haiti and Innovating Health International (n=35) and Moffit Cancer Center (n=14). DNA were extracted from saliva (DNAGenotek PrepIT-LP), FFPE breast tumor and normal adjacent breast epithelia (QIAmp DNA FFEP kit) when available. Reduced representation bisulfite sequencing was performed by BGI Inc (Cambridge MA) with EZ DNA metylation gold kit (Zymo) and followed by directional BS-sequence library preparation on HISeq x Ten, PE150. Data was processed in methylKit package and analyzed in a two-step filtering process to 1) include 200bp tiled regions to maximize CpG coverage, > 10 read coverage, <400 read coverage, >3 shared CpGs per group or all samples if the number of samples was less than 3 in the group, q <0.05 and 2) study DMRs only in ER+ breast tumors. Results The mean age of Haitian natives (H) (49.4 years), Haitian Immigrants (HI) (51.9 years) and African American (AA) (48.6 years). The H were in the US for a range of 5-47 years. 64.7% of H, 63% of HI and 42.9% of AA were diagnosed at stages III/IV. Differentially methylated regions (DMRs) were assessed between groups for germline, tumor and normal breast epithelial. There were significantly more DMRs in germline DNA than tumor for each comparison group. The DMRs identified in germline DNA; AA vs HI=54,150 DMRs, HI vs H=45,031 and H vs AA=40,461 DMRs. In the tumors, there were more DMRs between HI and AA (556 DMRs) than H vs AA (33 DMRs) and H vs AA (3 DMRs). Pathway analysis of DMRs identified in germline DNA amongst the three groups revealed common tumor molecular mechanisms of cancer, AMPK and CREB signaling. Whereas Wnt3a, EZH2, PI3K and molecules involved in DNA damage and cell cycle were distinct effectors in tumors of each group. Conclusion This work provides data for exploring the biological basis of breast cancer subtypes within the US Black sub-populations stratified by time and nativity. Haitian immigrants and US born Black women with breast cancer have more epigenetic differences and represent unique pathways which drive tumor biology. Citation Format: Vincent DeGennaro, Kaliyah Brown, Danielle A Cerbon, Daniel Karl, Sofia Palacio, Jennifer Garcia, Camille Ragin, Tuya Pal, Susan Vadaparampil, Carmen Gomez, Judith Hurley, Sophia HL George. Short-term effects of immigration on the development of breast cancer in women of African descent [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B086.

Integrin-β7 is part of an extensive family of glycoproteins and is overexpressed in the MF subgroups in Multiple Myeloma (MM), contributing to drug resistance and poor survival. ITGB7 promotes adhesion and proliferation of myeloma cells in bone-marrow stroma by supplementing growth factors like VEGF. Here, we investigate the possible epigenetic mechanism of ITGB7 overexpression in the MF subgroup, comprising of t(14;16) and t(14;20) myeloma. Newly diagnosed MM (NDMM) patient bone marrow aspirates underwent CD138 cell selection to enrich tumor cells to >98%. Samples consisted of those with a t(14;16) (n=17), t(14;20) (n=7), t(4;14) (n=9), t(11;14) (n=10) and hyperdiploidy (n=19, separated into D1 (n=12) or D2 (n=7 subgroups). NDMM patient samples were compared to plasma cells isolated from age-matched healthy donors (n=4), to determine differential changes in epitranscripts. We performed unbiased genome-wide reduced representation bisulfite sequencing (RRBS) to identify differentially methylated regions (DMRs) in CpG islands, which were validated by Infinium MethylationEPIC arrays (Illumina) in conjunction with gene expression array data (U133 Plus 2.0, Affymetrix) to determine the epitranscriptomic profile in all samples. RRBS was performed using 75 bp reads to a minimum of 20 M reads per sample. Data from MM cells at the Blueprint consortium were also used for annotation of epigenetic marks. Using the RRBS data we identified 26 hypomethylated, overexpressed genes in the MF group samples, which were not present in the other subgroups. In comparison, only 1 gene, FUT7, was hypermethylated and down-regulated in the MF cluster. Most interestingly, we identified ITGB7 amongst the hypomethylated, overexpressed genes. With an in-depth analysis we identified 4 significant (p<0.05) DMRs across intragenic regions of ITGB7, within a 2.5 kb region. Mean methylation across the DMRs reduced from 58% to 22% (p<0.01) in t(14;16) and 39% (p=0.02) in t(14;20) with concomitant overexpression of ITGB7 in the t(14;16), 19.5-fold increase, and the t(14;20), 23.4-fold increase, subgroups. These 4 DMRs belong to CpG islands, annotated to be the part of possible intragenic enhancer site with promoter like activity, and enriched for open chromatin structures. The DMRs also align with the hotspot of H3K4me1 marks and a putative binding site of activating transcription factors such as AP1/2 or Sp1. This suggests that the identified DMRs could be within the enhancer that regulates ITGB7 overexpression through DNA-hypomethylation. ITGB7 is a known oncogenic factor in high-risk MM, contributing to cell adhesion, migration and homing. Here we show using combined DNA methylation and expression data that ITGB7 is regulated through hypomethylation of the an enhancer region in the MF subgroup in MM. Citation Format: Samrat Roy Choudhury, Cody Ashby, Ruslana Tytarenko, Yan Wang, Purvi H. Patel, Aneta Mikulasova, Michael Bauer, Shayu Deshpande, Faith E. Davies, Gareth J. Morgan, Brian A. Walker. Intragenic DNA-hypomethylation promotes overexpression of ITGB7 in MF subgroup of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5324.

BackgroundSex in fish is plastic and in several species can be influenced by environmental factors. In sensitive species, elevated temperatures have a masculinizing effect. Previous studies on the effects of temperature on gene expression have been restricted to a few cognate genes, mostly related to testis or ovarian development, and analyzed in gonads once they had completed the process of sex differentiation. However, studies on the effect of temperature at the whole gonadal transcriptomic level are scarce in fish and, in addition, temperature effects at the time of sex differentiation at the transcriptomic level are also unknown. Here, we used the European sea bass, a gonochoristic teleost with a polygenic sex determination system influenced by temperature, and exposed larvae to elevated temperature during the period of early gonad formation. Transcriptomic analysis of the gonads was carried out about three months after the end of temperature exposure, shortly after the beginning of the process of sex differentiation.ResultsElevated temperature doubled the number of males with respect to untreated controls. Transcriptomic analysis of early differentiating female gonads showed how heat caused: 1) an up-regulation of genes related to cholesterol transport (star), the stress response (nr3c1) and testis differentiation (amh, dmrt, etc.), 2) a decrease in the expression of genes related to ovarian differentiation such as cyp19a1a, and 3) an increase in the expression of several genes related to epigenetic regulatory mechanisms (hdac11, dicer1, ehmt2, jarid2a, pcgf2, suz12, mettl22).ConclusionsTaken together, the results of this study contribute to the understanding of how the early environment sets permanent changes that result in long-lasting consequences, in this case in the sexual phenotype. Results also show the usefulness of comparing the effects of heat on the behavior of cognate genes related to sex differentiation as well as that of genes involved in establishing and maintaining cell identity through epigenetic mechanisms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1862-0) contains supplementary material, which is available to authorized users.

The ENCODE project has funded the generation of a diverse collection of methylation profiles using reduced representation bisulfite sequencing (RRBS) technology, enabling the analysis of epigenetic variation on a genomic scale at single-site resolution. A standard application of RRBS experiments is in the location of differentially methylated regions (DMRs) between two sets of samples. Despite numerous publications reporting DMRs identified from RRBS datasets, there have been no formal analyses of the effects of experimental and biological factors on the performance of existing or newly developed analytical methods. These factors include variable read coverage, differing group sample sizes across genomic regions, uneven spacing between CpG dinucleotide sites, and correlation in methylation levels among sites in close proximity. To better understand the interplay among technical and biological variables in the analysis of RRBS methylation profiles, we have developed an algorithm for the generation of experimentally realistic RRBS datasets. Applying insights derived from our simulation studies, we present a novel procedure that can identify DMRs spanning as few as three CpG sites with both high sensitivity and specificity. Using RRBS data from muscle vs. non-muscle cell cultures as an example, we demonstrate that our method reveals many more DMRs that are likely to be of biological significance than previous methods.

ABSTRACTA 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.

Ancestral TCDD exposure promotes epigenetic transgenerational inheritance of imprinted gene Igf2: Methylation status and DNMTs

Epigenomic Signatures in Myelodysplastic Syndrome Patients As Predictors of Donor Compatibility and Transplant Outcome

Developmental origins of transgenerational sperm histone retention following ancestral exposures

Epigenetic modifications, including DNA methylation, regulate gene expression and contribute to the differentiation of cells. Tissues are characterized by methylation patterns that reflect their specific functions and origin. Currently, there is limited knowledge of the epigenetic patterns of the heart. To address this gap, we generated methylation profiles for all anatomical regions of the human heart. We hypothesize that unique differentially methylated regions (DMRs) of DNA exist for each cardiac region and that these patterns are disrupted in heart failure. Using non-diseased cardiac tissue and reduced representation bisulfite sequencing on a Illumina platform, we generated genome-wide methylomes for the human right atrium (n=4), left atrium (n=4), right ventricle (n=4), left ventricle (n=4), aorta (n=3), pulmonary artery (n=2), mitral valve (n=3), tricuspid valve (n=3), aortic valve (n=3) and pulmonary valve (n=3). DMRs, defined as regions with significantly different mean methylation differences, were identified using Metilene. For each tissue we identified between 10-20 million reads covering 8-10 million CpG methylation sites and 4-228 tissue-specific DMRs. There were relatively few (4) different DMRs between the left and right ventricles but 228 unique DMRs were found in the vessels (aorta and pulmonary artery) compared to the ventricles including regions upstream of BMP3 and FOXC1 , genes implicated in cardiogenesis. We then applied this approach and normal data to the analysis of disease. We generated additional left ventricular methylomes from adult (n=3) and pediatric (n=3) patients with heart failure. We identified 19 unique DMRs in the adult group and 107 in the pediatric group. Genes associated with pediatric HF DMRs included ROCK1 and FBLN2 that have been previously implicated in contraction and remodelling. We have created an atlas of the human heart based upon differences in DNA methylation between the anatomical regions of the human heart. This data will help increase our understanding of cardiac development, identify new disease biomarkers and have application to a wide range of cardiac diseases

Ancestral environmental exposures to a variety of environmental toxicants and other factors have been shown to promote the epigenetic transgenerational inheritance of adult onset disease. The current study examined the potential transgenerational actions of the herbicide atrazine. Atrazine is one of the most commonly used herbicides in the agricultural industry, in particular with corn and soy crops. Outbred gestating female rats were transiently exposed to a vehicle control or atrazine. The F1 generation offspring were bred to generate the F2 generation and then the F2 generation bred to generate the F3 generation. The F1, F2 and F3 generation control and atrazine lineage rats were aged and various pathologies investigated. The male sperm were collected to investigate DNA methylation differences between the control and atrazine lineage sperm. The F1 generation offspring (directly exposed as a fetus) did not develop disease, but weighed less compared to controls. The F2 generation (grand-offspring) was found to have increased frequency of testis disease and mammary tumors in males and females, early onset puberty in males, and decreased body weight in females compared to controls. The transgenerational F3 generation rats were found to have increased frequency of testis disease, early onset puberty in females, behavioral alterations (motor hyperactivity) and a lean phenotype in males and females. The frequency of multiple diseases was significantly higher in the transgenerational F3 generation atrazine lineage males and females. The transgenerational transmission of disease requires germline (egg or sperm) epigenetic alterations. The sperm differential DNA methylation regions (DMRs), termed epimutations, induced by atrazine were identified in the F1, F2 and F3 generations. Gene associations with the DMRs were identified. For the transgenerational F3 generation sperm, unique sets of DMRs (epimutations) were found to be associated with the lean phenotype or testis disease. These DMRs provide potential biomarkers for transgenerational disease. The etiology of disease appears to be in part due to environmentally induced epigenetic transgenerational inheritance, and epigenetic biomarkers may facilitate the diagnosis of the ancestral exposure and disease susceptibility. Observations indicate that although atrazine does not promote disease in the directly exposed F1 generation, it does have the capacity to promote the epigenetic transgenerational inheritance of disease.

Transgenerational epigenetic inheritance and immunity in chickens that vary in Marek's disease resistance

Understanding the consequences of thermal and chemical variations in aquatic habitats is of importance in a scenario of global change. In ecology, the sex ratio is a major population demographic parameter. So far, research that measured environmental perturbations on fish sex ratios has usually involved a few model species with a strong genetic basis of sex determination, and focused on the study of juvenile or adult gonads. However, the underlying mechanisms at the time of gender commitment are poorly understood. In an effort to elucidate the mechanisms driving sex differentiation, here we used the European sea bass, a fish species where genetics and environment (temperature) contribute equally to sex determination. Here, we analyzed the transcriptome of developing gonads experiencing either testis or ovarian differentiation as a result of thermal and/or exogenous estrogen influences. These external insults elicited different responses. Thus, while elevated temperature masculinized genetic females, estrogen exposure was able to override thermal effects and resulted in an all-female population. A total of 383 genes were differentially expressed, with an overall downregulation in the expression of genes involved in both in testicular and ovarian differentiation when fish were exposed to Estradiol-17s through a shutdown of the first steps of steroidogenesis. However, once the female phenotype was imposed, gonads could continue their normal development, even taking into account that some of the resulting females were fish that otherwise would have developed as males. The data on the underlying mechanisms operating at the molecular level presented here contribute to a better understanding of the sex ratio response of fish species subjected to a combination of two of the most common environmental perturbations and can have implications in future conservational policies.