Marisa S. Bartolomei - US grants

DESCRIPTION (provided by applicant): A subset of genes in mammals is regulated by genomic imprinting, a process that results in unequal expression of the maternal and paternal alleles of certain genes. As a consequence, deleterious mutations or deletions in the single expressed allele of an imprinted gene will result in the absence of a functional gene product. In humans, disruptions in imprinting and imprinted genes account for the human genetic diseases Beckwith-Wiedemann Syndrome, Prader-Willi Syndrome and Angelman Syndrome, a number of cases of Silver-Russell Syndrome and for cancers such as Wilms'tumor. The objective of this proposal is to investigate the mechanism by which parental identity of imprinted genes is established and maintained. The studies will employ the H19 gene, which is expressed from the maternally-derived allele in mice and humans. The imprinting of H19 and the linked and oppositely imprinted Igf2 gene, is mediated, at least in part, through the 2 kb imprinting control region (ICR) that is located 2 kb upstream from the start of H19 transcription. The ICR, which is also designated the differentially methylated domain (DMD), is hypermethylated on the repressed paternal allele and acts as a methylation-sensitive insulator through CTCF-binding on the maternal allele. This proposal will investigate the mechanism of imprinting at the H19/Igf2 locus through the following experiments: (1) to determine whether ICR/DMD sequence in addition to CTCF binding sites and the proper spacing of the CTCF binding sites are critical for H19/Igf2 imprinting;(2) to determine if CpG mutations outside of the CTCF-binding sites disrupt imprinting;(3) To examine the chromatin structure of the H19 locus in germ cells and embryonic cells and assess the role of these epigenetic modifications in imprinting. In addition to elucidating the mechanism of imprint establishment and maintenance at this locus during development, these experiments will model newly identified mutations in individuals with Beckwith-Wiedemann Syndrome and Silver-Russell Syndrome, providing a better understanding of the etiology of the disease.

DESCRIPTION (provided by applicant): X chromosome inactivation is the chromosome-based silencing mechanism eutherian mammals use to equalize the expression of X-linked genes between males and females early in development. To achieve transcriptional silencing of genes on 1 X chromosome each cell of a female must determine the number of X chromosomes it contains, choose 1 X to inactivate and initiate, propagate and maintain chromosome wide silencing. An elusive component of the X inactivation pathway is the initial choice between the 2 X chromosomes. In the mouse, choice is under the control of the X-controlling element (Xce), a genetically defined locus within the X inactivation center. Xce heterozygotes exhibit skewed, nonrandom inactivation of the X chromosome in somatic tissues. The objective of this proposal is to study X chromosome choice in mice. We previously conducted a phenotype-driven genetic screen involving chemical mutagenesis in the mouse and identified 3 genetically distinct autosomal mutations with dominant effects on X chromosome choice early in embryogenesis. This proposal will investigate the mechanism of X chromosome choice by: (1) Refining the map locations and cloning the genes responsible for the X chromosome choice mutations obtained in the genetic screen; (2) Isolating and characterizing embryonic stem cell lines from each of the mutant strains so that the earliest steps in the X inactivation process can be molecularly and biochemically monitored and manipulated; (3) Fine mapping and further characterizing the Xce. Elucidating the cis-acting sequences and the trans-acting factors involved in X chromosome choice will allow a greater understanding of the complex process of X inactivation. Furthermore, a better understanding of X chromosome choice will improve our knowledge of how differences in allelic ratios can result in significant phenotypic variation, including how unfavorable X chromosome skewing can lead to more severe phenotypes in a variety of X linked diseases.

[unreadable] DESCRIPTION (provided by applicant): Understanding the molecular mechanisms underlying the production of viable eggs and sperm and the initiation of embryonic development is essential for the continued survival of animal species in the 21st century and beyond. Advances in reproductive biology have immediate application, not only for the treatment of human infertility and sub fertility, but also for improvements in developing contraceptive methods, advances in agricultural engineering and novel approaches to wildlife preservation. Moreover, as stem cells, pluripotent cells, gametes and early embryos provide important models for basic research aimed at understanding nuclear programming and cell cycle regulation, as well as transcriptional, translational, and post-translational control mechanisms, these areas have potential significance for a wide variety of biomedical applications. This application requests funding for the 16th Gordon Conference on Mammalian Gametogenesis and Embryogenesis to be held from June 18-23, 2006 at Connecticut College in New London, Connecticut. The objective of the Gordon Research Conference on Mammalian Gametogenesis and Embryogenesis is to provide a forum for the exchange of the latest advances in the field, as well as to promote discussion and debate by providing a relaxed setting that facilitates interaction. A particularly important aspect of the Gordon Research Conference setting is the blending of young and established investigators, as well as national and international scientists to promote exchanges that will generate new ideas and foster new collaborations in the field. The Mammalian Gametogenesis and Embryogenesis Gordon Research Conference has three primary goals: 1) Presentation of cutting-edge and thought-provoking research; 2) Fostering interactions between clinicians and basic scientists; and 3) Fostering career development of junior investigators in this field. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The expression of a subset of genes in mammals is affected by parental origin. These genes, which are designated as imprinted, are expressed from a single parental allele. The existence of imprinted genes is hypothesized to explain why nuclear contributions from both parents are required for normal mammalian development. Furthermore, imprinting plays a role in the transmission of a number of human genetic diseases, including Beckwith-Wiedemann Syndrome, Prader-Willi Syndrome and Angelman Syndrome, in that the sex of the parent that transmits the affected gene(s) determines whether the child will develop the disease. Although greater than 75 imprinted genes have been identified and some of the sequences that mediate their imprinting defined, much less is known about the trans-acting factors that govern imprinted gene expression. These factors are required to establish and maintain imprints, as well as provide long-term stability and memory for the imprints. In this proposal, we will focus on the role of candidate trans-acting factors that likely serve as epigenetic regulators in imprint establishment and maintenance. Specific Aim 1 will examine the general role of the multi-functional protein CTCF, which provides enhancer blocking function and a DNA methylation-free region on the imprinting control region (ICR) of the H19/Igf2 locus. Using CTCF-depleted preimplantation mouse embryos, we will investigate the consequences for imprinting regulation of H19 and other imprinted genes postulated to be regulated by CTCF. Specific Aim 2 will assess the requirement for proteins that bind to methylated CpG dinucleotides, with the hypothesis that these methyl CpG binding proteins are essential to maintaining imprinted gene expression and differential methylation of ICRs of a large number of imprinted genes. Last, Specific Aim 3 will determine whether the EED Polycomb Group protein is required for the establishment of genomic imprinting in the early embryo. Together these experiments will provide a greater understanding of the mechanism of genomic imprinting in early mouse embryos and new insights into the etiology of the loss of imprinting mutations observed in various imprinting syndromes including Beckwith-Wiedemann, Prader-Willi, Angelman and Silver-Russell Syndromes. PUBLIC HEALTH RELEVANCE: The proposed studies will provide new information regarding the mechanism by which genomic imprinting is established and maintained in mouse oocytes and embryos. The results of these studies will also impact our understanding of the loss of imprinting and DNA methylation mutations observed in various human imprinting syndromes including Beckwith-Wiedemann, Prader-Willi, Angelman and Silver-Russell Syndromes. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Project Summary/Abstract: DNA methylation is the most widely studied epigenetic modification to DNA. DNA methylation occurs almost exclusively at the cytosine residue in CpG dinucleotides in many organisms. It is becoming increasingly evident that DNA methylation plays a critical role in development, gene regulation, chromosome stability and the onset and progression of disease. Thus, knowledge of the DNA methylation state of the genome is essential. Although a variety of methods are available to assess DNA methylation status, the study of DNA methylation is compromised by low sensitivity, laborious protocols, excessive time and expensive reagents. While restriction enzyme techniques typically require a large amount of material and do not assay all CpG dinucleotides in the regions of interest, bisulfite mutagenesis/cloning/sequencing technology, which reduces the amount of material and provides a more comprehensive analysis of the regions of interest, is time consuming and low throughput. Pyrosequencing is a fast, simple and quantitative method for analysis of CpG methylation in multiple sites in a single assay. It eliminates the requirement for CpGs in restriction enzyme sites, large amounts of DNA and laborious protocols. It also less prone to bias artifacts generated when traditional bisulfite mutagenesis, cloning and sequencing techniques are used and provides a more quantitative assessment of DNA methylation status. The PyroMark Q96 MD is a highly sensitive and quantitative genetic analysis system based on Pyrosequencing technology. Sample preparation and analysis are very fast and the PyroMark Q96 MD is high throughput: 96 samples can be assayed in less than 1 hour after PCR. Furthermore, this instrument and technology will allow the determination of DNA methylation status in the presence of SNP's, thereby facilitating the assay of allele-specific methylation patterns of imprinted genes as well as the assay of DNA methylation of disease associated genes in combination with polymorphisms. This application describes a wide array of uses for the PyroMark Q96 MD at the University of Pennsylvania and other Philadelphia area institutions. The uses include allele-specific DNA methylation analysis of imprinted gene regulation in animal models, assisted reproduction technology conceptuses and human disease. DNA methylation will also be examined in association with stress and behavior, cancer, environmental exposures, intrauterine growth retardation/low birth, diet-induced obesity, autism and aging. The technology afforded by the PyroMark Q96 MD will allow a more complete, accurate and efficient understanding of the role of DNA methylation changes in development, behavior, environmental exposures and disease.

DESCRIPTION (provided by applicant): In this application, we seek funding for the partial support of the 2011 Gordon Research Conference (GRC) on Epigenetics, one of the most exciting and active areas of biological research today. An explosion of epigenetics research in the past decade reflects the fact that knowledge of complete genome sequences is not sufficient to understand how genomic information is decoded or regulated. Chromosomes are not naked DNA molecules, but DNA-protein complexes known as chromatin. Modification of the chromatin by DNA methylation, post- translational modification of the histones that wrap the DNA, use of alternative histone variants and alterations in histone spacing and chromatin compaction are common aspects of epigenetic regulation throughout eukaryotes. As a result, studies of model organisms have direct relevance to humans and human disease states. Meeting every two years since 1995, the Epigenetics GRC has earned a strong international reputation as the premier meeting in its field, typically drawing more applicants than can be accepted. The conference is known for its cutting edge science and breadth of coverage, providing a forum for the exploration of a wide range of epigenetic phenomena and epigenetic regulatory mechanisms in numerous model organisms, as well as humans. Basic science studies of such intriguing phenomena as X chromosome inactivation, gametic imprinting, position effect variegation, gene dosage control, paramutation, nucleolar dominance, transposon silencing and RNA-mediated DNA methylation will be balanced with medically-relevant research, including the epigenetics of cancer, imprinting disorders, aging, memory and effects of the environment (including diet). The 2011 conference will be co-chaired by mammalian biologist Marisa Bartolomei (University of Pennsylvania), and plant biologist Craig Pikaard (Indiana University), both leaders in the field of epigenetics and long time Epigenetics GRC participants. The 2011 Epigenetics GRC will include a mix of invited established investigators who are leaders in the field, invited young investigators who are doing exciting work and young scientists whose submitted abstracts indicate new breakthroughs at the cutting edge of the field. In this way, the organizers will strive to promote the careers of students, postdoctoral fellows, and new principal investigators, by involving them in both the oral and poster presentations. Special efforts will also be made to support women scientists and include participants who are members of racial/ethnic groups that are underrepresented in science. PUBLIC HEALTH RELEVANCE: Epigenetic regulation is essential for development, controlling such phenomena as inactivation of one X chromosome in somatic cells of female mammals, dictating the whether only maternally or paternally inherited copies of specific genes are expressed in offspring, maintaining transposable elements in a silenced state, or controlling the dosage of repetitive essential genes. Epigenetic misregulation contributes to cancer through silencing of tumor suppressor genes. Disease states including imprinting disorders and Rett syndrome have an epigenetic basis, as do aspects of aging and effects of diet or environmental toxins on gene expression. By advancing the study of epigenetic mechanisms, the Epigenetics Gordon Research Conference will contribute to the understanding and improvement of human health and well-being.

Infertile couples are increasingly turning to Assisted Reproductive Technologies (ART) to treat their infertility. Of growing concern is that ART-conceived children are at increased risk for specific loss-of-imprinting disorders, as well as congenital malformations, intrauterine growth restriction, and preeclampsia. Given the difficulty of conducting studies using human embryos, a mouse model system, which anticipated some risks associated with ART, will be used to assess effects of ART procedures on gene expression, DNA methylation in embryos and extra-embryonic tissues, and physiological and behavioral outcomes in the offspring. Specific Aim 1 will test the hypothesis that (a) expression and epigenetic modification of imprinted genes in embryonic and extra-embryonic tissues are differentially sensitive following ART manipulations, and (b) global patterns in gene expression and promoter DNA methylation are differentially perturbed in extra-embryonic and embryonic tissues. The outcomes of ART procedures will be pursued in Specific Aim 2, which will test the hypothesis that epigenetic changes occurring in response to ART manipulations are linked with adverse outcomes in the offspring as assessed by physiological and behavioral assays. The experimental design will enable us to correlate such changes for a given offspring with molecular changes in that offspring's placenta that are in turn linked to ART manipulations prior to implantation. One-carbon metabolism is central to DNA methylation. Accordingly, Specific Aim 3 will test the hypothesis that folate supplementation of the diet of donor and recipient mothers from which the embryos are derived will alleviate, to some degree, the perturbations in DNA methylation and gene expression observed in Specific Aim 1, as well as restore a more normal physiological profile and behavioral response for those assays conducted in Specific Aim 2. The results will provide a plethora of information regarding molecular mechanisms underlying the linkage between epigenetic changes and gene expression, and whether such changes directly become manifest in offspring. Moreover, our findings may suggest experimental modifications to ART procedures that will minimize the effect of ART manipulations on epigenetic gene regulation.

The Penn Center for the Study of Epigenetics in Reproduction (PennCSER) will elucidate epigenetic mechanisms that govern male and female reproduction, contribute to male infertility and impact development of mouse and human concepti conceived through assisted reproductive technologies (ART). The PennCSER centerpiece is 4 integrated, innovative research projects, spearheaded by experienced leaders in the areas of epigenetics and reproduction. The Center also features an Outreach program that has been in place for more than 5 years; the Penn Academy of Reproductive Sciences uses hands on laboratory experiences and interactive lectures to educate high school students, largely from the Philadelphia area schools, in the reproductive sciences. The clinical project (Project 1, Coutifaris, Sapienza, Mainigi and Senapati) will assess the impact of the periconceptional maternal environment on DNA methylation and gene expression in embryonic (placental vessels and endothelial cells) and extra-embryonic (trophoblasts) tissues in IVF pregnancies following fresh or frozen transfer, pregnancies resulting from unassisted conception and pregnancies following trophectoderm biopsy for pre-implantation genetic testing for aneuploidy (PGT-A). This project will also determine the stability of epigenetic signatures determined at birth into childhood and correlate them with the children?s growth phenotype. Project 2 (Bartolomei, Mainigi, Schultz) will closely parallel Project 1 using a validated mouse model to study the effect of ART laboratory manipulations on epigenetic gene regulation and physiological outcomes in term conceptuses and adults. Project 2 will address the question of whether embryo freezing and transfer to an unstimulated uterus is optimal and evaluate the safety and outcomes associated with trophectoderm biopsy. Project 2 will also test whether decreased expression of Grb10, a growth-regulatory imprinted gene overexpressed in human and mouse ART-derived conceptuses, can rescue ART-associated phenotypes in a mouse model. Project 3 (Berger) will investigate histone modifications during mouse spermatogenesis and determine their conservation in normal human sperm and disruption in abnormal human sperm, as well as in mouse models exhibiting abnormal histone retention. Project 4 (Wang and Masson) will examine the function of TEX15, a protein that is required for meiosis and male fertility, and is a novel epigenetic regulator essential for retrotransposon silencing. Project 4 will also determine whether aberrant retrotransposon activity is associated with male infertility. PennCSER will not only provide training to clinicians, physician scientists, and basic research fellows in the area of epigenetics but also provide PennCSER?s expertise to the NCTRI and associated program members.

a. Objective The objectives of this core are to (a) sustain and enhance the institutional identity of PennCSER; (b) coordinate and integrate all PennCSER components; (c) schedule biweekly meetings of the entire research group and annual meetings of the lAC and EAC; (d) facilitate the Outreach and education activities as needed; (e) organize two annual seminars in collaboration with the CRRWH in which two members from other SCCPIRs are invited to Penn; (f) administer the translational research training; (g) organize travel of PIs to annual steering committee meetings; and (h) prepare all scientific and financial progress reports as requested.

Infertile couples are increasingly turning to Assisted Reproductive Technologies (ART) to treat their infertility. Of growing concern is that ART-conceived children are at increased risk for specific loss-of-imprinting disorders, as well as congenital malformations, intrauterine growth restriction, and preeclampsia. Given the difficulty of conducting studies using human embryos, a mouse model system, which anticipated some risks associated with ART, will be used to assess effects of ART procedures on gene expression, DNA methylation in embryos and extra-embryonic tissues, and physiological and behavioral outcomes in the offspring. Specific Aim 1 will test the hypothesis that (a) expression and epigenetic modification of imprinted genes in embryonic and extra-embryonic tissues are differentially sensitive following ART manipulations, and (b) global patterns in gene expression and promoter DNA methylation are differentially perturbed in extra-embryonic and embryonic tissues. The outcomes of ART procedures will be pursued in Specific Aim 2, which will test the hypothesis that epigenetic changes occurring in response to ART manipulations are linked with adverse outcomes in the offspring as assessed by physiological and behavioral assays. The experimental design will enable us to correlate such changes for a given offspring with molecular changes in that offspring's placenta that are in turn linked to ART manipulations prior to implantation. One-carbon metabolism is central to DNA methylation. Accordingly, Specific Aim 3 will test the hypothesis that folate supplementation of the diet of donor and recipient mothers from which the embryos are derived will alleviate, to some degree, the perturbations in DNA methylation and gene expression observed in Specific Aim 1, as well as restore a more normal physiological profile and behavioral response for those assays conducted in Specific Aim 2. The results will provide a plethora of information regarding molecular mechanisms underlying the linkage between epigenetic changes and gene expression, and whether such changes directly become manifest in offspring. Moreover, our findings may suggest experimental modifications to ART procedures that will minimize the effect of ART manipulations on epigenetic gene regulation.

DESCRIPTION (provided by applicant): Drug addiction is a psychiatric disorder characterized by a transition from recreational to compulsive drug use that continues in spite of severe negative consequences. Despite attempts by individuals to quit, the desire or need to resume drug-taking can last for months or years. The persistence of addiction over time suggests that exposure to drugs results in long-term adaptations in the brain that likely involve alterations in transcription and genetic regulation. In addition to genetic factors, epigenetic mechanisms may also play a role in the maintenance of addictions not only throughout an individual's lifetime, but in his/her descendants. However, the potential impact of drug exposure across generations has not been characterized. To date, no comprehensive study has been undertaken to determine if exposure to drugs of abuse in the parental generation leads to alterations in behavioral or molecular phenotypes in subsequent generations. To address this question, we will examine the phenotypic consequences of two mechanistically different drugs (cocaine and morphine) in three inbred mouse strains (C57BL/6J, DBA/2J, A/J) as well as a multi-generation series of F1 hybrids. In addition to studying behavioral phenotypes across generations, we will characterize RNA expression, DNA methylation, and post-translational histone modifications as both the molecular signature and the mechanistic basis for heritable epigenetic changes. These studies will allow us to determine (1) whether any of 2 mechanistically different drugs of abuse results in transmission of addiction-related phenotypes through multiple generations after treatment, (2) whether these phenotypes are mediated by changes to the epigenome (DNA methylation, post-translational histone modifications, or RNAs), and (3) whether these phenotypes vary according to the sex of exposed parent in an imprinted or allele-specific manner. These experiments will inform future studies aimed at elucidating the mechanisms by which drugs of abuse lead to transgenerational phenotypes.

DESCRIPTION (provided by applicant): There is growing evidence that in utero and early developmental exposures to environmental chemicals may play a role in the development of obesity and related metabolic diseases later in life. Endocrine disrupting compounds are exogenous substances that mimic endogenous hormones in the endocrine pathway. Bisphenol-A (BPA) is a chemical plasticizer and xenoestrogen used in the production of polycarbonate plastics and epoxy resins. Human exposure to BPA is ubiquitous at low levels and occurs primarily through the diet. Studies in our laboratory and other investigators have shown that exposure of rodents to BPA during pregnancy results in increased adiposity and impaired glucose tolerance in adulthood. An important mechanism for in utero and early developmental effects of BPA is thought to be altered epigenetic regulation of gene expression. In support of this hypothesis is our preliminary data in mouse that BPA exposure during pregnancy results in loss of imprinting at two genes critical for development and obesity, Igf2 and Snrpn, in the embryo and placenta. These findings lead us to hypothesize that BPA exposure during development alters the epigenetic profile in key cells including the germline, resulting in altered gene expression, an abnormal phenotype, and transgenerational transmission of the phenotype. Other than the placenta, we cannot obtain target tissues such as islets, liver, or fat from humans during development. Thus an animal model can provide insight into mechanisms that can be later explored in the human. We propose the following specific aims: Specific Aim 1 will assess the epigenome of F1 offspring exposed to BPA in utero as well as the F2-F3 generations. In addition to testing the expression and DNA methylation of imprinted genes, we will assay genome-wide DNA methylation changes and key histone modifications in F1-F3 offspring. The F1-F3 generations will also be metabolically phenotyped and germline of F2 and F3 epigenetically assayed. Specific Aim 2 will ascertain whether BPA exposure of males during puberty and early adulthood alters the germline and leads to transgenerational transmission of an altered epigenotype and abnormal phenotype. In Specific Aim 3 we will determine whether low dose BPA exposure in combination with DEHP, and vinclozolin alters the epigenome in the germline and leads to transgenerational transmission of an abnormal phenotype. Through the comprehensive analyses in the in vivo model, the results of the proposed project will build a solid standard for extrapolation of effects and mode of action to other classes of endocrine disrupting chemicals that are present in food and are identified as potential inducers of obesity and related metabolic diseases later in life. The proof of the principle of prenatal programming by environmental contaminants may change our awareness of critical assessment of the risk of such compounds.

DESCRIPTION (provided by applicant): We propose to continue a flexible interdisciplinary Training Program in Cell and Molecular Biology, currently in its 37th year at the University of Pennsylvania. The training program is a University-wide, interdepartmental and interschool program whose mission is to provide a multifaceted doctoral program to prepare students for careers in cell and molecular biology in academia, industry, or government. The training program's goal is to provide broad-based training in modern methods of cell and molecular biology and in-depth didactic exposure to cell and molecular biology, while at the same time matching the Trainees' specific interests. These goals are achieved through general and specialized courses, literature survey courses, laboratory rotations, a qualifying examination, thesis research with oversight from an advising committee, training in bioethics, and training grant-specific activities. Trainee-specific activities include an annual oral presentation of ongoing thesis research; attending the annual Cell and Molecular Biology Retreat; attending the Annual Trainee Organized Invited Lectures; participating in Alumni Day in which a former Trainee who has completed their Ph.D. and left Penn is invited back to give a seminar and meet with the Trainees; group attendance of Trainees/Trainer at the annual meeting of the American Society for Cell Biology meeting and the Current and Former Trainee Lunch in which 2 former Trainees present a talk on their thesis research. Students completing their second year of graduate studies are appointed for two years and are selected annually by the Executive Committee. Trainers come from the Schools of Medicine, Veterinary Medicine, Arts and Science, and Engineering and Applied Science, and the Wistar Institute and not only have active research programs in cell and molecular biology but also a commitment to graduate education. The training program has formal mechanisms to monitor trainees both during and after their training grant support. In addition, the training program has formal mechanisms to monitor trainers, as well as to resolve any conflicts between trainees and trainers. Last, Trainers participate in a number of efforts to recruit under-represented minorities both locally and nationally. Direct management of the Training Program is done by an Executive Committee that sets and reviews policy and selects trainees. Based on the number of potential trainees, we request support for 14 predoctoral trainees/year for the next 5 years; this number is the same as that currently supported by the training grant.

Abstract Infertile couples are increasingly turning to Assisted Reproductive Technologies (ART) to treat their infertility. Of growing concern is that ART-conceived children are at increased risk for specific loss-of-imprinting disorders, congenital malformations, growth restriction, preeclampsia as well as postnatal cardiac and metabolic disorders. Given the difficulty of conducting studies using human embryos, a mouse model system, which anticipated some risks associated with ART, will be used to assess the effects of ART on placental morphology, imprinted gene regulation, growth, metabolic, cardiac and behavioral phenotypes of the offspring, and gene expression and chromatin structure genome-wide. Specific Aim 1 will assess the phenotypes, including growth, behavior metabolism and cardiovascular function, of ART-offspring in comparison to naturally-conceived controls. We will also interrogate imprinted gene regulation and gene expression, DNA methylation and chromatin structure using gene-specific and high throughput analyses. Moreover, the design of this aim will enable us to isolate, phenotype and match placenta to offspring to determine whether the placental phenotype can accurately predict health of in vitro fertilization (IVF)-derived offspring. Because we have previously reported a low frequency of epigenetic errors in multiple tissues of IVF-conceived offspring, we hypothesize that the germline cells also harbor epigenetic mutations. In Specific Aim 2, we will test this hypothesis by determining whether aberrant phenotypes observed in IVF offspring are transmitted to subsequent generations and, if so, assess the mechanism of this transmission. The result from these experiments will provide a trove of information regarding the linkage between epigenetic changes and health of offspring conceived by ART and whether placental phenotyping can predict offspring health. Our findings may also suggest experimental modifications to ART procedures that can improve offspring outcomes. !

Endocrine disruptors (EDs) are a group of molecules capable of altering normal endocrine function in animals and humans. We and others have demonstrated that in utero exposure to EDs causes obesity and abnormal glucose homeostasis in the offspring. Phthalates are an important group of EDs that are used in a diverse range of industrial applications leading to common exposure risk in humans. One major phthalate is di-(2-ethylhexyl)-phthalate (DEHP), a widely used compound for which dietary exposure (food processing, packaging) likely represents the main source of contamination for the general population. Both human and animal models show a link between phthalate exposure and the development of obesity and the metabolic syndrome. Similar to these studies, our preliminary data show that DEHP exposure through the diet in physiologically relevant doses prior to and during pregnancy and lactation alters DNA methylation in fetal liver, increases body weight in adult offspring in a sex-specific manner. It is becoming increasingly evident that the periconceptional stage also represents a window of susceptibility to environmental exposures on the offspring. While the mechanisms responsible for the transfer of metabolic memory from the oocyte to the offspring remain to be elucidated, 2 plausible possibilities are epigenetic dysregulation and abnormal mitochondria. Further, we and others have shown that epigenetic changes and abnormal mitochondrial function in key organs such as the liver, ß-cell and muscle have been linked to development of obesity and diabetes. Thus, we hypothesize that a preconception exposure to DEHP, similar to a gestational exposure, results in an abnormal metabolic phenotype in the offspring by altering either the epigenetic profile and or mitochondrial function in the oocyte. Thus we propose the following specific aims: Aim 1 will determine whether a preconception exposure to biologically relevant doses of DEHP results in a similar offspring phenotype as a gestational exposure. Aim 2 will determine the molecular and cellular mechanisms by which the two different exposure windows result in a similar or different metabolic phenotype in the offspring. We will profile the transcriptome by RNA-seq and the epigenome by ATACseq and bisulfite-seq in the oocyte and key tissues in the offspring. We will then determine the effects of DEHP on key aspects of mitochondrial function such as respiration, ATP, ROS, and morphology. The proof of the principle of preconception programming by environmental contaminants may change our awareness of critical assessment of the risk of such compounds.

ABSTRACT The overall goal of the Penn Center for the Study of Epigenetics in Reproduction (PennCSER) is to investigate the role of epigenetic gene regulation in reproduction, with a specific emphasis on gametogenesis and early development as they pertain to normal development, implantation and placentation, infertility and Assisted Reproductive Technologies (ART). The Administrative Core will facilitate the successful pursuit of each of the individual projects and enable collaboration among projects. The Core will also organize the solicitation and selection of pilot projects that complement the existing Center program. Finally, the Core will support the Outreach Activities, facilitate training, and assist interactions with the NCTRI network, as well as the internal and external review process.

Abstract Assisted Reproductive Technologies (ART) are invaluable for the increasing number of women who require interventions to treat their infertility. Nevertheless, ART-conceived children are at increased risk for loss-of-imprinting disorders resulting from epigenetic errors, abnormal growth, congenital malformations, and postnatal cardiac and metabolic disorders. Such problems likely arise because ART procedures take place when the mammalian embryo is being epigenetically reprogrammed. Because it is difficult to conduct studies using human embryos, a mouse model system, which anticipated some risks associated with ART, will be used to assess the effect of ART interventions on placental morphology, imprinted gene regulation, growth, metabolic and cardiac phenotypes of the offspring, and epigenetic gene regulation, including DNA methylation and chromatin structure genome-wide. Presently, preliminary evidence suggests that frozen embryos transferred into unstimulated women have less perinatal morbidity than fresh cycles but conflicting data suggest adverse outcomes associated with frozen embryos. Specific Aim 1 will determine the effects of embryo vitrification and maternal hormonal environment on offspring outcome of IVF-generated embryos. Moreover, preimplantation genetic screening (PGS) is being increasingly employed in the absence of research assessing the consequences of blastomere biopsy. Specific Aim 2 will investigate the phenotypes and epigenetic profiles of the placenta and ART offspring derived when PGS is used. Finally, data from our human placental studies demonstrate alterations in DNA methylation in genes critical to early placentation, fetal growth, and adult metabolism. Specific Aim 3 will translate these data to our animal ART model to determine the role of specific genes in adverse outcomes associated with IVF. The role of Grb10 in IVF-associated changes in fetal growth, placentation, and vasculogenesis using a mouse model will be initially assessed. Results of these experiments will provide information regarding the linkage between epigenetic changes and health of offspring conceived by ART. These findings may also suggest experimental modifications to ART procedures that can improve offspring outcomes.

Reproductive health is not only critical for fertility and propagation of the species, but also underlies many key parameters of human health and longevity. As such, our understanding of the genetic and molecular basis of key events in reproduction is critical for our ability to improve reproductive health, fertility, and the physiological well-being of our species. Thus, the primary goal of the current R13 Conference Proposal is to provide a small local two-day annual conference focusing on reproductive biology and infertility. This event has emerged out of similar long-standing symposia held by Cornell University?s Center for Reproductive Genomics (CRG) and by the University of Pennsylvania?s Center for Research in Reproduction and Women?s Health (CRRWH), which make up two of the tripartite group, the third being the Center for Reproductive Genetics and Therapy (CRG&T) at Magee-Womens Research Institute (MWRI) at the University of Pittsburgh. The Cornell and MWRI groups initiated combined symposia in 2018, and over two years, these have been hugely successful. With the inclusion of UPenn, we have renamed this annual two-day symposium Tri-Institutional Symposium on Reproductive Biology & Infertility, or Tri-Repro. The second goal of Tri-Repro is to focus on trainee career development by encouraging participation and leadership from our trainees, and by emphasizing trainee peer-to-peer interactions through the formation of a tri-institutional Trainee Organizing Committee. Trainees will select up to seven invited speakers, including renowned leaders in reproductive science from around the world, but also including new faculty from each of the three institutions. The venue for the symposium will rotate each year, providing a shift in location, but also encouraging changes in emphasis reflecting the varying interests of each Center. Most of the talks, and all of the posters, will feature trainee presenters selected by the committee from submitted abstracts, while a career development lunch forum on day 1 will feature invited speakers who will discuss specific areas of career development for all trainees. The third goal of Tri-Repro is to provide a local environment to promote localized interactions and networking with an aim to forge new collaborations between institutions in the Northeast. This has been the most successful component of our previous symposia: by inviting our neighboring guest institutions to join us at very low cost (the Tri-Repro group provides all registration and food to our guests, leaving them with only modest travel and accommodation costs to cover), we provide a rich environment in which neighboring institutions with interests and expertise in reproductive sciences can come together to discuss collaborative ideas and provide feedback to each other. Such an environment is often more comfortable and intimate than large national conferences, allowing even the most timid of trainees to interact with peers and faculty alike. The fourth goal of Tri-Repro is to champion the inclusion and active participation of women and people from underrepresented groups in our field, a feature that has been a major focus of our previous symposia, and which we will continue to prioritize at future events.