@article{jima_skaar_planchart_motsinger-reif_cevik_park_cowley_wright_house_liu_et al._2022, title={Genomic map of candidate human imprint control regions: the imprintome}, volume={6}, ISSN={["1559-2308"]}, url={https://doi.org/10.1080/15592294.2022.2091815}, DOI={10.1080/15592294.2022.2091815}, abstractNote={Imprinted genes – critical for growth, metabolism, and neuronal function – are expressed from one parental allele. Parent-of-origin-dependent CpG methylation regulates this expression at imprint control regions (ICRs). Since ICRs are established before tissue specification, these methylation marks are similar across cell types. Thus, they are attractive for investigating the developmental origins of adult diseases using accessible tissues, but remain unknown. We determined genome-wide candidate ICRs in humans by performing whole-genome bisulphite sequencing (WGBS) of DNA derived from the three germ layers and from gametes. We identified 1,488 hemi-methylated candidate ICRs, including 19 of 25 previously characterized ICRs (https://humanicr.org/). Gamete methylation approached 0% or 100% in 332 ICRs (178 paternally and 154 maternally methylated), supporting parent-of-origin-specific methylation, and 65% were in well-described CTCF-binding or DNaseI hypersensitive regions. This draft of the human imprintome will allow for the systematic determination of the role of early-acquired imprinting dysregulation in the pathogenesis of human diseases and developmental and behavioural disorders.}, journal={EPIGENETICS}, author={Jima, Dereje D. and Skaar, David A. and Planchart, Antonio and Motsinger-Reif, Alison and Cevik, Sebnem E. and Park, Sarah S. and Cowley, Michael and Wright, Fred and House, John and Liu, Andy and et al.}, year={2022}, month={Jun} }
 @article{jirtle_2022, title={The science of hope: an interview with Randy Jirtle}, ISSN={["1750-192X"]}, DOI={10.2217/epi-2022-0048}, abstractNote={In this interview, Professor Randy L Jirtle speaks with Storm Johnson, Commissioning Editor for Epigenomics, on his work on genomic imprinting, environmental epigenomics and the fetal origins of disease susceptibility. Professor Randy Jirtle joined the Duke University Department of Radiology in 1977 and headed the Epigenetics and Imprinting Laboratory until 2012. He is now Professor of Epigenetics in the Department of Biological Sciences at North Carolina State University, Raleigh, NC, USA. Jirtle's research interests are in epigenetics, genomic imprinting and the fetal origins of disease susceptibility. He is known for his groundbreaking studies linking environmental exposures early in life to the development of adult diseases through changes in the epigenome and for determining the evolutionary origin of genomic imprinting in mammals. He has published over 200 peer-reviewed articles as well as the books Liver Regeneration andCarcinogenesis: Molecular and Cellular Mechanisms, Environmental Epigenomics in Health and Disease: Epigenetics and Disease Origins and Environmental Epigenomics in Health andDisease: Epigenetics and Complex Diseases. He was honored in 2006 with the Distinguished Achievement Award from the College of Engineering at the University of Wisconsin-Madison. In 2007, he was a featured scientist on the NOVA television program on epigenetics titled 'Ghost in Your Genes' and was nominated for Time Magazine's 'Person of the Year'. He was the inaugural recipient of the Epigenetic Medicine Award in 2008 and received the STARS Lecture Award in Nutrition and Cancer from the National Cancer Institute in 2009. Jirtle was presented the Linus Pauling Award from the Institute of Functional Medicine in 2014. In 2017, ShortCutsTV produced the English documentary 'Are You What Your Mother Ate? The Agouti Mouse Study' based on his pioneering epigenetic research. He received the 2018 Northern Communities Health Foundation Visiting Professorship Award at the University of Adelaide, Australia. The Personalized Lifestyle Medicine Institute presented Jirtle with the Research and Innovation Leadership Award in 2019. Dr Jirtle was also given the Alexander Hollaender Award in 2019 at the 50th annual meeting of the Environmental Mutagenesis and Genomics Society.}, journal={EPIGENOMICS}, author={Jirtle, Randy L.}, year={2022}, month={Mar} }
 @article{skaar_dietze_alva-ornelas_ann_schones_hyslop_sistrunk_zalles_ambrose_kennedy_et al._2021, title={Epigenetic Dysregulation of KCNK9 Imprinting and Triple-Negative Breast Cancer}, volume={13}, ISSN={["2072-6694"]}, DOI={10.3390/cancers13236031}, abstractNote={Genomic imprinting is an inherited form of parent-of-origin specific epigenetic gene regulation that is dysregulated by poor prenatal nutrition and environmental toxins. KCNK9 encodes for TASK3, a pH-regulated potassium channel membrane protein that is overexpressed in 40% of breast cancer. However, KCNK9 gene amplification accounts for increased expression in <10% of these breast cancers. Here, we showed that KCNK9 is imprinted in breast tissue and identified a differentially methylated region (DMR) controlling its imprint status. Hypomethylation at the DMR, coupled with biallelic expression of KCNK9, occurred in 63% of triple-negative breast cancers (TNBC). The association between hypomethylation and TNBC status was highly significant in African-Americans (p = 0.006), but not in Caucasians (p = 0.70). KCNK9 hypomethylation was also found in non-cancerous tissue from 77% of women at high-risk of developing breast cancer. Functional studies demonstrated that the KCNK9 gene product, TASK3, regulates mitochondrial membrane potential and apoptosis-sensitivity. In TNBC cells and non-cancerous mammary epithelial cells from high-risk women, hypomethylation of the KCNK9 DMR predicts for increased TASK3 expression and mitochondrial membrane potential (p < 0.001). This is the first identification of the KCNK9 DMR in mammary epithelial cells and demonstration that its hypomethylation in breast cancer is associated with increases in both mitochondrial membrane potential and apoptosis resistance. The high frequency of hypomethylation of the KCNK9 DMR in TNBC and non-cancerous breast tissue from high-risk women provides evidence that hypomethylation of the KNCK9 DMR/TASK3 overexpression may serve as a marker of risk and a target for prevention of TNBC, particularly in African American women.}, number={23}, journal={CANCERS}, author={Skaar, David A. and Dietze, Eric C. and Alva-Ornelas, Jackelyn A. and Ann, David and Schones, Dustin E. and Hyslop, Terry and Sistrunk, Christopher and Zalles, Carola and Ambrose, Adrian and Kennedy, Kendall and et al.}, year={2021}, month={Dec} }
 @article{jirtle_2021, title={Memorial Tribute to Kelly H. Clifton IN MEMORY}, volume={195}, ISSN={["1938-5404"]}, DOI={10.1667/RADE-21-000KHC}, number={2}, journal={RADIATION RESEARCH}, author={Jirtle, Randy L.}, year={2021}, month={Feb}, pages={218–219} }
 @article{gomih_smith_north_hudgens_brewster_huang_skaar_valea_bentley_vidal_et al._2018, title={DNA methylation of imprinted gene control regions in the regression of low-grade cervical lesions}, volume={143}, ISSN={["1097-0215"]}, DOI={10.1002/ijc.31350}, abstractNote={The role of host epigenetic mechanisms in the natural history of low-grade cervical intraepithelial neoplasia (CIN1) is not well characterized. We explored differential methylation of imprinted gene regulatory regions as predictors of the risk of CIN1 regression. A total of 164 patients with CIN1 were recruited from 10 Duke University clinics for the CIN Cohort Study. Participants had colposcopies at enrollment and up to five follow-up visits over 3 years. DNA was extracted from exfoliated cervical cells for methylation quantitation at CpG (cytosine-phosphate-guanine) sites and human papillomavirus (HPV) genotyping. Hazard ratios (HR) and 95% confidence intervals (CI) were estimated using Cox regression to quantify the effect of methylation on CIN1 regression over two consecutive visits, compared to non-regression (persistent CIN1; progression to CIN2+; or CIN1 regression at a single time-point), adjusting for age, race, high-risk HPV (hrHPV), parity, oral contraceptive and smoking status. Median participant age was 26.6 years (range: 21.0-64.4 years), 39% were African-American, and 11% were current smokers. Most participants were hrHPV-positive at enrollment (80.5%). Over one-third of cases regressed (n = 53, 35.1%). Median time-to-regression was 12.6 months (range: 4.5-24.0 months). Probability of CIN1 regression was negatively correlated with methylation at IGF2AS CpG 5 (HR = 0.41; 95% CI = 0.23-0.77) and PEG10 DMR (HR = 0.80; 95% CI = 0.65-0.98). Altered methylation of imprinted IGF2AS and PEG10 DMRs may play a role in the natural history of CIN1. If confirmed in larger studies, further research on imprinted gene DMR methylation is warranted to determine its efficacy as a biomarker for cervical cancer screening.}, number={3}, journal={INTERNATIONAL JOURNAL OF CANCER}, author={Gomih, Ayodele and Smith, Jennifer S. and North, Kari E. and Hudgens, Michael G. and Brewster, Wendy R. and Huang, Zhiqing and Skaar, David and Valea, Fidel and Bentley, Rex C. and Vidal, Adriana C. and et al.}, year={2018}, month={Aug}, pages={552–560} }
 @misc{leak_calabrese_kozumbo_gidday_johnson_mitchell_ozaki_wetzker_bast_belz_et al._2018, title={Enhancing and Extending Biological Performance and Resilience}, volume={16}, ISSN={["1559-3258"]}, DOI={10.1177/1559325818784501}, abstractNote={Human performance, endurance, and resilience have biological limits that are genetically and epigenetically predetermined but perhaps not yet optimized. There are few systematic, rigorous studies on how to raise these limits and reach the true maxima. Achieving this goal might accelerate translation of the theoretical concepts of conditioning, hormesis, and stress adaptation into technological advancements. In 2017, an Air Force-sponsored conference was held at the University of Massachusetts for discipline experts to display data showing that the amplitude and duration of biological performance might be magnified and to discuss whether there might be harmful consequences of exceeding typical maxima. The charge of the workshop was “to examine and discuss and, if possible, recommend approaches to control and exploit endogenous defense mechanisms to enhance the structure and function of biological tissues.” The goal of this white paper is to fulfill and extend this workshop charge. First, a few of the established methods to exploit endogenous defense mechanisms are described, based on workshop presentations. Next, the white paper accomplishes the following goals to provide: (1) synthesis and critical analysis of concepts across some of the published work on endogenous defenses, (2) generation of new ideas on augmenting biological performance and resilience, and (3) specific recommendations for researchers to not only examine a wider range of stimulus doses but to also systematically modify the temporal dimension in stimulus inputs (timing, number, frequency, and duration of exposures) and in measurement outputs (interval until assay end point, and lifespan). Thus, a path forward is proposed for researchers hoping to optimize protocols that support human health and longevity, whether in civilians, soldiers, athletes, or the elderly patients. The long-term goal of these specific recommendations is to accelerate the discovery of practical methods to conquer what were once considered intractable constraints on performance maxima.}, number={3}, journal={DOSE-RESPONSE}, author={Leak, Rehana K. and Calabrese, Edward J. and Kozumbo, Walter J. and Gidday, Jeffrey M. and Johnson, Thomas E. and Mitchell, James R. and Ozaki, C. Keith and Wetzker, Reinhard and Bast, Aalt and Belz, Regina G. and et al.}, year={2018}, month={Aug} }
 @article{jirtle_2018, title={Epigenetic Responses to Low Dose Ionizing Radiation}, volume={124}, ISSN={["1873-4596"]}, DOI={10.1016/j.freeradbiomed.2018.05.014}, abstractNote={Two epigenetically regulated subsets of genes that potentially link environmental exposures early in development to adult diseases are imprinted genes and those with metastable epialleles. Genes with metastable epialleles have highly variable expressions because of stochastic allelic modifications in the epigenome. Genomic imprinting is an unusual epigenetic form of gene regulation that results in monoallelic expression in a parent-of-origin dependent manner. The viable yellow agouti (Avy) mouse harbors a metastable Agouti gene because of an upstream insertion of a transposable element. We previously used this animal model to demonstrate that nutritional and chemical toxicant exposures during early development induce persistent epigenetic changes at the Avy locus that result in alterations in coat color and adult disease susceptibility. We also showed that low doses of ionizing radiation (}, journal={FREE RADICAL BIOLOGY AND MEDICINE}, author={Jirtle, Randy L.}, year={2018}, month={Aug}, pages={559–559} }
 @article{golden_yu_meilleur_blakeley_duff_karton_vrielink_2017, title={An Extended n-h bond, driven by a conserved second-order interaction, orients the flavin n5 orbital in cholesterol oxidase}, volume={7}, journal={Scientific Reports}, author={Golden, E. and Yu, L. J. and Meilleur, F. and Blakeley, M. P. and Duff, A. P. and Karton, A. and Vrielink, A.}, year={2017} }
 @misc{vaiserman_koliada_jirtle_2017, title={Non-genomic transmission of longevity between generations: potential mechanisms and evidence across species}, volume={10}, ISSN={["1756-8935"]}, DOI={10.1186/s13072-017-0145-1}, abstractNote={Accumulating animal and human data indicate that environmental exposures experienced during sensitive developmental periods may strongly influence risk of adult disease. Moreover, the effects triggered by developmental environmental cues can be transgenerationally transmitted, potentially affecting offspring health outcomes. Increasing evidence suggests a central role of epigenetic mechanisms (heritable alterations in gene expression occurring without changes in underlying DNA sequence) in mediating these effects. This review summarizes the findings from animal models, including worms, insects, and rodents, and also from human studies, indicating that lifespan and longevity-associated characteristics can be transmitted across generations via non-genetic factors.}, journal={EPIGENETICS & CHROMATIN}, author={Vaiserman, Alexander M. and Koliada, Alexander K. and Jirtle, Randy L.}, year={2017}, month={Jul} }
 @article{skaar_jirtle_hoyo_2016, title={Environmentally Induced Alterations in the Epigenome Affecting Obesity and Cancer in Minority Populations}, ISBN={["978-3-319-41608-3"]}, DOI={10.1007/978-3-319-41610-6_5}, abstractNote={The obesity epidemic of the last 30–40 years is may be linked to increased environmental chemical exposures with endocrine disrupting potential. The increases in obesity prevalence and severity coincide with increases in several adenocarcinomas at a time when cancer incidence has been generally declining, with disproportionate effects in different ethnic groups. Despite demonstrated associations between such exposures with obesity, and obesity with these cancers, an association between exposure to these environmental chemicals and adenocarcinomas has been difficult to demonstrate in part due to limits in exposure assessment. Exposure to these compounds elicits stable epigenetic responses; thus, if these alterations to the epigenome can be fully characterized, they can be exploited to improve exposure ascertainment. We summarize in this chapter evidence for the influence of environmental exposures on obesity and how epigenetic alterations may contribute to cancers that disproportionately affect minority populations exhibit disparities in incidence and mortality.}, journal={EPIGENETICS, ENERGY BALANCE, AND CANCER}, author={Skaar, David A. and Jirtle, Randy L. and Hoyo, Cathrine}, year={2016}, pages={109–146} }
 @article{li_xie_murphy_skaar_nye_vidal_cecil_dietrich_puga_jirtle_et al._2016, title={Lead Exposure during Early Human Development and DNA Methylation of Imprinted Gene Regulatory Elements in Adulthood}, volume={124}, ISSN={["1552-9924"]}, DOI={10.1289/ehp.1408577}, abstractNote={Lead exposure during early development causes neurodevelopmental disorders by unknown mechanisms. Epidemiologic studies have focused recently on determining associations between lead exposure and global DNA methylation; however, such approaches preclude the identification of loci that may alter human disease risk.The objective of this study was to determine whether maternal, postnatal, and early childhood lead exposure can alter the differentially methylated regions (DMRs) that control the monoallelic expression of imprinted genes involved in metabolism, growth, and development.Questionnaire data and serial blood lead levels were obtained from 105 participants (64 females, 41 males) of the Cincinnati Lead Study from birth to 78 months. When participants were adults, we used Sequenom EpiTYPER assays to test peripheral blood DNA to quantify CpG methylation in peripheral blood leukocytes at DMRs of 22 human imprinted genes. Statistical analyses were conducted using linear regression.Mean blood lead concentration from birth to 78 months was associated with a significant decrease in PEG3 DMR methylation (β = -0.0014; 95% CI: -0.0023, -0.0005, p = 0.002), stronger in males (β = -0.0024; 95% CI: -0.0038, -0.0009, p = 0.003) than in females (β = -0.0009; 95% CI: -0.0020, 0.0003, p = 0.1). Elevated mean childhood blood lead concentration was also associated with a significant decrease in IGF2/H19 (β = -0.0013; 95% CI: -0.0023, -0.0003, p = 0.01) DMR methylation, but primarily in females, (β = -0.0017; 95% CI: -0.0029, -0.0006, p = 0.005) rather than in males, (β = -0.0004; 95% CI: -0.0023, 0.0015, p = 0.7). Elevated blood lead concentration during the neonatal period was associated with higher PLAGL1/HYMAI DMR methylation regardless of sex (β = 0.0075; 95% CI: 0.0018, 0.0132, p = 0.01). The magnitude of associations between cumulative lead exposure and CpG methylation remained unaltered from 30 to 78 months.Our findings provide evidence that early childhood lead exposure results in sex-dependent and gene-specific DNA methylation differences in the DMRs of PEG3, IGF2/H19, and PLAGL1/HYMAI in adulthood.Li Y, Xie C, Murphy SK, Skaar D, Nye M, Vidal AC, Cecil KM, Dietrich KN, Puga A, Jirtle RL, Hoyo C. 2016. Lead exposure during early human development and DNA methylation of imprinted gene regulatory elements in adulthood. Environ Health Perspect 124:666-673; http://dx.doi.org/10.1289/ehp.1408577.}, number={5}, journal={ENVIRONMENTAL HEALTH PERSPECTIVES}, author={Li, Yue and Xie, Changchun and Murphy, Susan K. and Skaar, David and Nye, Monica and Vidal, Adriana C. and Cecil, Kim M. and Dietrich, Kim N. and Puga, Alvaro and Jirtle, Randy L. and et al.}, year={2016}, month={May}, pages={666–673} }
 @article{king_darrah_money_meentemeyer_maguire_nye_michener_murtha_jirtle_murphy_et al._2015, title={Geographic clustering of elevated blood heavy metal levels in pregnant women}, volume={15}, ISSN={["1471-2458"]}, DOI={10.1186/s12889-015-2379-9}, abstractNote={Cadmium (Cd), lead (Pb), mercury (Hg), and arsenic (As) exposure is ubiquitous and has been associated with higher risk of growth restriction and cardiometabolic and neurodevelopmental disorders. However, cost-efficient strategies to identify at-risk populations and potential sources of exposure to inform mitigation efforts are limited. The objective of this study was to describe the spatial distribution and identify factors associated with Cd, Pb, Hg, and As concentrations in peripheral blood of pregnant women.Heavy metals were measured in whole peripheral blood of 310 pregnant women obtained at gestational age ~12 weeks. Prenatal residential addresses were geocoded and geospatial analysis (Getis-Ord Gi* statistics) was used to determine if elevated blood concentrations were geographically clustered. Logistic regression models were used to identify factors associated with elevated blood metal levels and cluster membership.Geospatial clusters for Cd and Pb were identified with high confidence (p-value for Gi* statistic <0.01). The Cd and Pb clusters comprised 10.5 and 9.2 % of Durham County residents, respectively. Medians and interquartile ranges of blood concentrations (μg/dL) for all participants were Cd 0.02 (0.01-0.04), Hg 0.03 (0.01-0.07), Pb 0.34 (0.16-0.83), and As 0.04 (0.04-0.05). In the Cd cluster, medians and interquartile ranges of blood concentrations (μg/dL) were Cd 0.06 (0.02-0.16), Hg 0.02 (0.00-0.05), Pb 0.54 (0.23-1.23), and As 0.05 (0.04-0.05). In the Pb cluster, medians and interquartile ranges of blood concentrations (μg/dL) were Cd 0.03 (0.02-0.15), Hg 0.01 (0.01-0.05), Pb 0.39 (0.24-0.74), and As 0.04 (0.04-0.05). Co-exposure with Pb and Cd was also clustered, the p-values for the Gi* statistic for Pb and Cd was <0.01. Cluster membership was associated with lower education levels and higher pre-pregnancy BMI.Our data support that elevated blood concentrations of Cd and Pb are spatially clustered in this urban environment compared to the surrounding areas. Spatial analysis of metals concentrations in peripheral blood or urine obtained routinely during prenatal care can be useful in surveillance of heavy metal exposure.}, number={1}, journal={BMC PUBLIC HEALTH}, publisher={Springer Science and Business Media LLC}, author={King, Katherine E. and Darrah, Thomas H. and Money, Eric and Meentemeyer, Ross and Maguire, Rachel L. and Nye, Monica D. and Michener, Lloyd and Murtha, Amy P. and Jirtle, Randy and Murphy, Susan K. and et al.}, year={2015}, month={Oct} }
 @article{jirtle_2014, title={The Agouti mouse: A biosensor for environmental epigenomics studies investigating the developmental origins of health and disease}, volume={6}, DOI={10.2217/epi.14.58}, abstractNote={EpigenomicsVol. 6, No. 5 EditorialFree AccessThe Agouti mouse: a biosensor for environmental epigenomics studies investigating the developmental origins of health and diseaseRandy L JirtleRandy L JirtleE-mail Address: jirtle@geneimprint.comDepartment of Sport & Exercise Sciences, University of Bedfordshire, Bedford, UKDepartment of Oncology; University of Wisconsin-Madison; Madison, WI, USADepartment of Biological Sciences; NC State University; Raleigh, NC, USASearch for more papers by this authorPublished Online:28 Nov 2014https://doi.org/10.2217/epi.14.58AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail Keywords: Avy mouseDNA methylationdietenvironmental epigenomicsepigeneticsparityFigure 1. Avy mouse coat color classification.Mouse coat color is grouped into one of five categories based on the proportion of brown-to-yellow fur: yellow (<5% brown), slightly mottled (between 5 and 50% brown), mottled (50% brown), heavily mottled (between 50 and 95% brown) and pseudoagouti (>95% brown). The isogeneic Avy mice shown are of the same age and sex.Reproduced with permission from [8].Figure 2. Avy mouse as a biosensor for environmentally induced alterations in the epigenome.Maternal exposure to methyl donors [7], genistein [8], ethanol [9], low-dose ionizing radiation [12], bisphenol A (BPA) [10] and in vitro culturing [11] cause coat-color changes in Avy offspring by hypermethylating or hypomethylating an intracisternal A particle inserted into the Agouti locus (Reproduced with permission from [1]). Boxes: coat color.The field of epigenetics continues to grow exponentially, doubling approximately every 2 to 3 years [1]. Concomitant with this growth is an increased interest in using the agouti viable yellow (Avy) isogenic mouse for environmental epigenomic studies investigating the developmental origins of health and disease. The Agouti gene in this mouse model is metastable because of a retroviral intracisternal A particle (IAP) insertion upstream of the Agouti transcription start site [1,2]. Metastable genes are highly variable in their expression. This results from stochastic allelic changes in the epigenome rather than mutations in the genome.The degree of IAP methylation at this alternative promoter in the proximal end of the IAP varies dramatically among individual animals, causing a wide distribution in coat color, ranging from brown (i.e., methylated IAP) to yellow (i.e., unmethylated IAP); mottled Avy mice are epigenetically mosaic (Figure 1) [3,4]. Furthermore, mice with any yellow fur become obese because of ectopic production of the agouti protein. They are also more prone to developing diabetes and cancer than the brown pseudo agouti mice that are of normal weight [1,5,6].This mouse model has been successfully used as a biosensor for detecting the effect of maternal diet [7,8], chemicals [9–11] and physical agents [12] on the epigenome (Figure 2). Because of its exquisite sensitivity to environmental exposures, careful attention must be given to experimental design for its optimum use. Environmental conditions as subtle as diet [7,8,10,12] and parity [13] can change the epigenome, thereby altering the epigenetic effects of test compounds. Thus, to appropriately interpret the results of environmental epigenomics studies utilizing the Avy mouse, and to maximize its sensitivity, careful attention must be given to the experimental conditions employed.Avy mouse modelThe Avy mutation initially arose spontaneously in C3H/HeJ mice in 1962 [14]. Animals carrying this mutation were backcrossed with C57BL/6J mice, followed by more than 220 generations of sibling mating. This has resulted in generation of Avy mice with a genetically invariant background that is 93% C57BL/6J [15].Coat color classificationCoat color assessment should always be done by the same individual, and performed when the offspring are weaned at 22 days of age. A five coat color classification system is required to accurately assess the effect of environmental factors on the epigenome (Figure 1). Collapsing the five coat color classes into three, as some investigators have done [13], fails to make biological sense since the lean pseudoagouti mice are inappropriately grouped with the obese heavily mottled animals (Figure 1). It also reduces the sensitivity of the assay since the coat color shifts, in response to environmental exposures, are seen most significantly in the tails of the coat color distribution (i.e., the pure yellow and pure pseudoagouti mice). Thus, contracting the coat color classification system to three groups markedly reduces the ability to assess effects of the environment on coat color distribution.Epigenetic measurementsDNA methylation [7,8,10,12] and/or histone modifications [16] in the promoter region of the IAP upstream of the transcription start site for the Agouti gene should always be assessed concomitantly with coat color classification. For example, the measurement of DNA methylation in Avy/a mice quantifies the percentage of cells methylated, and provides an independent and unbiased corroboration of observed coat color shifts [7,8]. Furthermore, unlike coat color data, which are limited to categorical statistical analysis, calculating percent methylation allows for more sophisticated statistical tests to be performed due to the continuous percent methylation variable [7,8,10,12].Animal breedingMale Avy/a mice of varying coat colors always need to be mated with female virgin a/a mice 8 to 10 weeks of age. The Avy allele is passed through the paternal lineage since the epigenotype is effectively reset with paternal, but not with maternal transmission of the Avy locus [17].The female a/a mice should be bred only once! There are significant parity effects on the epigenome in a/a mice, and multiple pregnancies increase the incidence of brown offspring [13]. Importantly, this parity effect would be expected to reduce the ability to detect the hypomethylating effect of test compounds, as seen with bisphenol A (BPA) [13]. There are even parity or birth order effects in people with autism [18,19] and schizophrenia [20,21] that could also result from epigenetic dysregulation of genes involved in their etiology.For optimal statistical analyses, 10 to 15 pregnant dams are required for each experimental dose. This results in 40 to 60 Avy/a offspring per group since half of the mice have an a/a genotype. To utilize all the mice produced following perinatal exposures, the a/a offspring can be used for genome-wide methylation studies [22] or followed for life course health effects [23,24].Matings also need to be performed within a short time span to minimize potential seasonal effects on the epigenome, and changes in the diets that could occur due to batch effects and/or food aging. If this is not possible, appropriate controls must be performed throughout the complete time span for the experimental studies. Quality control measures such as ensuring that caging, bedding and water are free of contamination from the test compounds are also imperative.Animal exposureThis in vivo epigenetic mouse bioassay is sensitive to many environmental factors, including diet composition [7,8]. For example, we demonstrated that the phytoestrogenic compound, genistein, found in soya products results in a significant increase in DNA methylation at the Avy locus, with a concomitant increase in the frequency of brown offspring [7,8]. Thus, to reduce the exposure to genistein, a/a females serving as controls receive a modified AIN-93G diet (i.e., diet 95092 with 7% corn oil substituted for 7% soybean oil, Harlan Teklad, WI, USA) rather than standard mouse chow. In contrast, dams exposed to chemical compounds receive a modified AIN-93G diet supplemented with the test compound. All diets are provided to the a/a females 2 weeks before mating with Avy/a males, and throughout pregnancy and lactation. If a single dose is to be given to the a/a females, as in our low-dose radiation study [12], the optimal time for exposure is at the blastocyst stage of development (i.e., 4.5 days after fertilization) because the repertoire of epigenetic marks in the embryonic stem cells are being reset at this time during gestation.Tissue collectionWhen the mice are weaned at 22 days after birth, all offspring should be weighed, digitally photographed and rated for coat color phenotype prior to sacrifice. Tissue from the three germ layers (e.g., brain, liver and kidney) and tail tissue are collected, flash frozen in liquid nitrogen and stored at -80ºC until DNA methylation and histone modifications are assessed.ConclusionIt is important to be meticulous when using the Avy mouse to investigate the effects of the maternal environment on the epigenome in the offspring, otherwise sensitivity and interpretability will be lost. If the following suggestions are adhered to, this epigenetic biosensor will be useful in determining the effects of maternal diet [7,8], chemicals [9–11] and physical agents [12] on the epigenome of embryonic stem cells in vivo: • Use a five coat color classification system;• Determine DNA methylation and/or histone modifications at the Avy locus;• Use only virgin a/a female mice to avoid parity effects on the epigenome;• Introduce the Avy allele through the male mouse to eliminate epigenetic transgenerational inheritance at this locus;• Use 10–15 dams (i.e., 40–60 Avy offspring) per experimental dose;• Use a single cohort of animals for controls and exposure groups, or match controls and exposure groups across all cohorts to avoid seasonal effects;• Use the modified AIN-93G diet to eliminate the epigenetic effect of genistein;• Ensure caging, bedding and water are free of contamination from the experimental agent or compounds known to alter the epigenome (e.g., BPA).Financial & competing interests disclosureThe author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties.No writing assistance was utilized in the production of this manuscript.References1 Jirtle RL. Epigenetics: how genes and environment interact. In: Environmental Epigenomics in Health and Disease: Epigenetics and Disease Origins. Jirtle RL, Tyson FL (Eds.), Springer-Verlag, Heidelberg, Germany, 3–30 (2013).Crossref, Google Scholar2 Duhl DM, Vrieling H, Miller KA et al. Neomorphic agouti mutations in obese yellow mice. Nat. Genet. 8(1), 59–65 (1994).Crossref, Medline, CAS, Google Scholar3 Miltenberger RJ, Mynatt RL, Wilkinson JE et al. The role of the agouti gene in the yellow obese syndrome. J. Nutr. 127(9), 1902S–1907S (1997).Crossref, Medline, CAS, Google Scholar4 Morgan HD, Sutherland HGE, Martin DIK et al. Epigenetic inheritance at the agouti locus in the mouse. Nat. 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This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties.No writing assistance was utilized in the production of this manuscript.PDF download}, number={5}, journal={Epigenomics}, author={Jirtle, R. L.}, year={2014}, pages={447–450} }