Abstract
The main objective of this conference was to provide solid evidence that environmental exposures during early development can affect faithful reproduction of individual parental epigenomes without changing DNA sequence in the offspring. No doubt, this important goal has been successfully achieved owing to the high quality of presented epidemiological and experimental studies and engaging discussions of many yet to be published results. Compelling data suggested a strong causal link between prenatal vulnerability of future parental epigenomes to damaging environmental factors aggravated by abnormal socio-cultural conditions (including, for instance, malnutrition and chronic stress) and the alarming risk of developing heritable complex medical conditions later in life, such as asthma, autism, cancer, cardiovascular disease, diabetes, obesity, schizophrenia and a whole range of rare neuromuscular pathologies. It was concluded that modern epigenetic research promises to markedly improve our ability to diagnose, prevent and treat these and other pathological conditions of humans. However, the complex heritability pattern of ‘epigenetic syndromes‘ also introduces unique legal and ethical issues that were discussed at the end of this outstanding meeting.
The symposium was organized by Randy Jirtle (Duke University, USA), Moshe Szyf (McGill University, Canada) and Frederick Tyson (NIEHS/NIH, USA), who gathered an outstanding group of scientists with a wide range of interests spanning from investigating the basic principles of genome and epigenome organization and function, to clinicians studying the effects of environmental pollutants on the genome and epigenome and their link to heritable syndromes and predisposition to complex diseases. A total of 53 renowned speakers from Europe, Asia, Australia and North America presented their research. More than 300 delegates attended this event. The symposium was opened with a rather unconventional Keynote Address entitled: ‘Being Fit in Four Dimensions‘, provided by Eva Jablonka from Tel-Aviv University, Israel. Using a rather unorthodox approach, she first combined a historical essay with philosophical reasoning to prove the inevitability of epigenetics emerging as a ‘new era in genetics‘ of different origin. She cited the growing evidence for suggesting that environmental exposures, especially during early mammalian development, can have a persistent or ‘transgenerational‘ effect on the epigenome, thereby capable of not only affecting organisms that have been exposed to the environmental challenges but also nonexposed progeny of future generations. She argued that owing to the complexity of defining primary targets, generating effects from environmental stimuli multiplied by the complexity of such resulting diseases as autism and other neurologic syndromes that affect not only cognition but emotional and conscious control mechanisms of the human brain, as well as considering the lack of simpler animal (mouse) models, there is an urgent necessity to develop new research programs whereby scientists of many dissimilar disciplines focused on human aspects of molecular, developmental and evolutionary biology, as well as ecologists, sociologists, psychologists and several other specialties, could directly interact with each other in a joint effort to achieve the next level in understanding of sophisticated concepts of this emerging field, and be prepared to share this with the rest of the molecular and medical research communities.
Fetal origins of adult disease susceptibility
This first session was devoted to the link between fetal exposures and adult disease susceptibility. Dr Ezra Susser (Columbia University, NY, USA) presented a largely epidemiological analysis of two historically well-documented famine events, the Dutch hunger winter and the Chinese famine, which provided him with a rare opportunity to prove that higher incidences of schizophrenia occur in progeny exposed to hunger in utero. He also presented unpublished data obtained in collaboration with a team of scientists from Johns Hopkins University (MA, USA) that revealed a stunning correlation between DNA methylation inside the CTCF site 3 of the H19 imprinting control region (ICR) and cerebellum weights.
Next, more evidence for a significant influence of paternal inheritance on several epidemiological cohorts was presented by Prof Marcus Pembrey (University College London, UK) in a talk about ‘Human Transgenerational Responses‘ that was focused on: revealing a clear association of either longevity or diabetic death with the quality of paternal ancestors‘ food supply during their mid-childhood spent in the Överkalix hunger cohorts in Sweden; a similar study of an Avon Longitudinal Study of Parents and Children (ALSPAC) cohort of fathers who began smoking before the age of 11 years which manifested a strong correlation with a much greater body mass index in their grandsons at age 9, but not in their granddaughters; and, finally, a third study of a Taiwan cohort representing paternal betel nut chewing which correlated well with an early onset of metabolic syndrome in the nonchewing progeny. The speaker acknowledged that for all three categories, no molecular mechanisms of transgenerational paternal inheritance are known with any certainty yet. Nevertheless, he suggested considering a possibility that “the nonrecombining region of the Y chromosome (and possibly the X) could preferentially transmit environmentally induced states to the next generation(s)”. Dr Gudrun E Moore (Institute of Child Health, UK) gave another talk, titled ‘Epigenetic Deregulation of Imprinting Influences Birth Weight and Disease Susceptibility in Humans‘, where she presented a careful study of several imprinted genes including maternally expressed PHLDA2. She found that expression of imprinted genes in a child, placenta and in both parents shows a good correlation with bodyweight and head circumference in a child. As a result, she found 15p copy number variants affecting imprinted gene expression and she also discussed the developmental and long-term disease consequences of deregulation of imprinted genes studied.
Next, Dr Claudine Junien (Institut National de la Recherche Agronomique [INRA], France) spoke on ‘Nutritional Programming: Sexual Dimorphism Epigenetics‘. Analyzing as to how gene expression and epigenetic changes in the placenta of day E15.5 could depend on the sex of the embryo and on a high-fat diet, she found that not only male and female placenta showed significant differences in gene expression and in their epigenomes, but that they also use remarkably different pathways to cope with high-fat diets.
Epigenetic mechanisms of gene regulation
To begin this session, Dr Ian Wood (Leeds University, UK) described the crosstalk of histone modifications in switching from active to repressive states, and emphasized the role of the REST protein (which is known to be an important modulator of vascular smooth muscle cell proliferation) in the recruitment of molecular complexes with histone-modifying enzymes to explain this process. Next, an interesting talk about ‘Sequencing of Epigenomes of Plants and People‘ was given by Dr Joseph R Ecker (Salk Institute, CA, USA), who has elegantly demonstrated the association of Arabidopsis thaliana epigenomes with their wide range of phenotypes by sequencing a wide range of variants of Arabidopsis thaliana methylomes and transcriptomes. He also analyzed the methylome of human embryonic stem (ES) and induced pluripotent stem (iPS) cells and found 25% of non-CpG methylation (mainly CpApG), as well as partially methylated domains that correlated with repressive histone modifications. In addition, Dr Ecker discussed a number of intriguing pathways linking RNA-directed DNA methylation with the interplay of genetic, epigenetic and transcriptional variations of phenotype. Dr V Lobanenkov (NIAID/NIH, USA) outlined over 25 years of work on the identification and cloning of CTCF and BORIS genes, and described how and why finding seven CpGs inside of the first name-giving CCCTC-binding site allowed CTCF to be linked to epigenetic regulation through CpG islands. Then, a wide range of functional implications was mentioned for numerous specific CTCF-target sites mapped and characterized since 1986 for distinct structure–functional properties. Many more recent CTCF ChIP-Seq and 3C/5C genome-wide studies have quantitatively expanded most of the CTCF characteristic features described before, but also placed CTCF, acting as a ‘master weaver of the genome‘, in charge of nearly every known chromatin function. It appears that CTCF/BORIS-binding sites are universally engaged in epigenetic maintenance of global 3D organization of the whole chromatin by coordinating ‘in time and in space‘ its structural compartmentalization with all levels of transcriptional regulation, both through mitosis in somatic cells and through meiosis in germ cells. Replacement of CTCF occupancy on DNA by its cancer/testis-specific paralog BORIS in vivo appears to be a novel common mechanism of acquired loss of function by many cancer-related CTCF sites as exemplified by the hTERT site, and by additional properties of BORIS splice isoforms. Many CTCF/BORIS sites hit by deletions, epimutations or by SNPs are found in association with multiple epigenetic pathologies besides cancer. For example, a mysterious C(-43)G SNP mapped in the XIST promoter from families with a skewed X-inactivation syndrome was shown to affect CTCF binding. In conclusion, he suggested that one of the major normal functions of CTCF/BORIS sites in germ cells might involve surveillance and protection of individual maternal and paternal epigenomes against detrimental gain of epimutations through aberrant cytosine remethylation cycles, as well as from aberrant microdeletions and repeat expansions that could occur upon recombination during spermatogenesis before fertilization.
In vitro fertilization, cloning & stem cells
In this session, Dr James A Thompson (University of Wisconsin, WI, USA) compared human ES cells with human iPS cells in his talk about ‘The Epigenetic State and Genetic Stability of Human iPS Cells‘. His main conclusion was that the cell types are very similar but have subtle differences detected using genome-wide genetic and epigenetic sequencing. While the significance of the differences found provides an invaluable resource for numerous future studies, the entire approach needs to be re-analyzed separately for clinical applications. In continuation of this topic, Dr Lorraine Young (Nottingham University, UK) described an automated system developed in her laboratory for the culture and differentiation of human ES cells, allowing the screening of multiple drugs and of other treatments based on this automated process. Dr Lousie Laurent (University of California, CA, USA) gave an overview of potential uses of ES and iPS cells in drug toxicity tests. She investigated the epigenetic instability of iPS cells and found that iPS cells tend to lose X-inactivation with passaging and relaxation of imprinting. The six-letter genetic code – that is, the 5-hydroxymethylcytidine (5hmC) – was introduced by Dr Wolf Reik (Babraham Institute, UK) in his talk about ‘Mechanisms and Functions of Epigenetic Reprogramming in Mammalian Development‘. He convincingly demonstrated an association of active demethylation with the paternal genome in the fertilized zygotes, while the maternal genome stained exclusively with anti-mC antibodies. Dr Reik has also shown that presence of the 5-hmC is associated with active chromatin, and that conversion of the 5-mC into the 5-hmC in growing ES cells is required to maintain chromatin in an ‘open state‘. Upon induced ES cell differentiation, the enzyme Tet-1 is reduced, to keep stable DNA methylation patterns in resulting differentiated somatic cells. The main part of this session was closed by the talk delivered by Dr Toshi Shioda (Massachussets General Hospital, MA, USA) who reported his outstanding results on the importance of methylation status of Glt2/Meg3 intragenic differentially methylated region in murine iPS cells for in vitro derivation of germline cells. While some of the major data that he presented have been either already published or submitted, this talk caused a lot of questions and was one of the most memorable events by the end of first day of this symposium.
Postnatal epigenetic programing in the brain
This session began with the talk on ‘Early Life Environment and the Epigenome‘ given by Dr Moshe Szyf from the McGill University, Canada. He reported his studies on humans and nonhuman primates showing the association between early-life adversity and DNA methylation in cortical neurons and T-lymphocytes. This association is organized structurally and functionally, suggesting highly regulated epigenetic responses. The next talk was given by Dr EB Keverne (Cambridge University, UK) and was about ‘Epigenetics, Brain Evolution and Behavior‘. He outlined the process of coadaptation between epigenome in the brain and the epigenome in the placenta during development. Using a mouse knockout of the Peg-3 imprinted gene, he was able to show the disturbance of this coadaptation in both placenta and developing hypothalamus. Moreover, this coadaptation positively shapes the next generation‘s mothering capabilities. Dr David Sweatt from the University of Alabama (AL, USA) presented ‘Epigenetic Mechanisms of Memory Formation‘. Using contextual fear conditioning in rats, he was able to visualize histone acetylation undergoing alterations in the hippocampus in concert with changing DNA methylation of specific promoters in the cortex, serving presumably as an associative signal for long-term memory. He concluded that long-term memory can indeed be strongly influenced by both HDAC and DNMT inhibitors, which opens novel avenues for translational studies in the immediate future.
Epigenetics & complex diseases
This session was started by Dr Maria Berdasco (Bellvitge Biomedical Research Institute [IDIBELL], Spain) by presenting a talk on ‘Human Cancer Epigenetics‘ detailing the high-throughput CpG methylation status analysis as well as genome-wide histone modifications and miRNA expression profiling in multiple human cancers. She demonstrated frequent transition from active histone modifications to a passive one in the promoters of tumor suppressor genes, and also found that multiple miRNAs have been affected especially in metastatic disease accompanied with a gain of novel frameshift mutations in HDAC-2 and aberrant methylation of histone methyltransferase gene NSDI in gliomas and neuroblastomas. She rightly emphasized that knowledge of genome-wide cancer epigenomes will help to develop new therapies for various cancers. Dr F Perera (Columbia University, NY, USA) presented a talk on ‘Environmental Exposure, Epigenetics and Human Health‘, where she reported the clinical findings of a positive correlation of exposure to bisphenol A BPA and polyhydroxyalkanoate (in mothers and children) with asthma and neurological disorders. Dr Dana Dolinoy (University of Michigan, MI, USA) gave a short talk about her studies of the response to BPA in both animal models and humans. She came to the conclusion that epigenetic alterations induced by BPA varies among species and across dose, and claimed that it should be considered in the disease risk assessment. Dr Cheryl L Walker (MD Anderson Cancer Center, TX, USA) in her talk about ‘Fetal and Early Postnatal Environmental Exposures‘ discussed developmental reprogramming of susceptibility to adult disease. Using a rat model of ovarian tumor where the mutant tumor suppressor gene TSC2 results in up to 65% penetrance of tumorigenesis in vivo, she showed that by exposure to exogenous estrogens for 3–5 days during embryo development cancer penetrance is enhanced by up to 100%. Therefore, she suggested that hormone-dependent epigenetic mechanisms are responsible for this conversion.
Epigenetic transgenerational inheritance
This important session was opened by the talk given by Dr Emma Whitelaw (Queensland Institute of Medical Research, Australia) about ‘Transgenerational Epigenetic Inheritance‘. She described mouse models for research of transgenerational epigenetic inheritance that include the widely published Agouti mice, as well as a newly developed model for Fetal Alcohol Spectrum Disorder, and her approach to screen for the ‘epigenetic modifier‘ genes. She showed how careful one should be in interpreting results as progeny of severe phenotypes frequently carry an extra chromosome – that is, by having a defect of clear genetic origin. In the conclusion of her talk, Dr Whitelaw kindly offered to provide every laboratory willing to continue studies with her published elsewhere ‘Momme‘ phenotypes, each a strain of her mice carrying heterozygous mutations in three ‘epigenetic modifier‘ genes, including the loss-of-function mutations in Snf 2h, Dnmt 3L and Dnmt-1 genes – a rare offer that received a loud appreciation from the attending audience. Next, Dr Michael K Skinner (Washington State University, WA, USA) gave a talk on ‘Epigenetic Transgenerational Inheritance following Exposure to Endocrine Disruptors‘, where he described experiments based on treating experimental rodents first with vinclozoline acting as antiandrogenic agent, and then with other compounds such as pesticides and herbicides given during the period of embryonal sex-determination. Treatment during this period of ontogenesis led to transgenerational (observed throughout F1–F4 generations) spermatogenic defects due to permanent DNA methylation and transcriptome alterations. Using comparative MeDIP-ChIP analysis for profiling aberrant DNA methylation in sperm DNA from F1 and F3 generations, Dr Skinner identified preferential regions hit by epimutations induced in pregnancy upon vinclozoline exposure, and mapped a common AT-rich motif that may be responsible for transgenerational paternal transmission. Thus, he concluded that environmental factors can reprogram the germ line, inducing transgenerational inheritance and this should be considered in both disease predisposition and evolutionary biology. Finally, Dr Douglas Ruden (Wayne State University, MI, USA) discussed transgenerational effects in insects including Drosophila and the honey bee as well as other species such as snails and mammals. Using the next-generation sequencing technologies to analyze both DNA methylation and chromatin modifications, he tried to find the correlation between epigenome and phenotype. One of his important conclusions was that 5-hydroymethyl-C is an important part of the process of 5-methyl-C at every site.
The session of the last day focused on the role of epigenetic mechanisms in the development of neurological disorders and disease susceptibility. Analysis of epigenetic twin differences and similarities is a powerful tool for examining complex non-Mendelian diseases such as autism, schizophrenia, bipolar disorder and asthma. Dr Art Petronis (University of Toronto, Canada) discussed the epigenetic differences in monozygotic and dizygotic twins in neurologic disorders which cannot be accounted for by SNPs or by other DNA sequence differences only, and are therefore more likely to manifest some transgenerationally inherited epigenetic marks. Dr Janine M LaSalle (University of California, CA, USA), emphasized the role of MECP2, as a key epigenetic regulator of gene expression in brain development and its involvement in a spectrum of neurodevelopmental disorders, including autism. Using a Mecp2-truncated mouse model, Dr LaSalle demonstrated impaired learning, changes in fertility and XCI patterns in mice perinatally exposed to organic pollutants PCB 95 and PBDE 47. Histone methylation in memory was investigated by Dr Farah Lubin (University of Alabama, AL, USA). In response to contextual fear conditioning, trimethylation of H3K4 in the hippocampus temporarily increases together with the promoter methylation of the Zif 268 gene, essential for hippocampus-dependent memory formation. An unusually entertaining talk given by Dr David Haig (Harvard University, MA, USA) summarized historical, medical and psychological factors correlating to the rapid decrease in the age of menarche onset. Genomic imprinting in early puberty onset was examined as a genetic battle between maternally and paternally expressed genes. The discussion about the balance of maternal and paternal allele expression was continued in the fascinating presentation of Dr Christopher Badcock (University of London, UK) who reviewed imprinting-related disorders in correlation with sex-battle genes. He suggested the bias toward expression of paternally expressed genes may lead to autism, while the bias toward expression of maternal genes may lead to psychotic and mental disorders. Dr David A Skaar (Duke University, NC, USA), introduced by Dr Randy L Jirtle, closed the session with his work on the function of Igf2r and identification of a SNP in exon 34 leading to alternative splicing and subsequent truncation of downstream exons. It was long suspected that the function of Igf2r contributes to cognitive function and memory. Water-maze tests performed on mice bearing the SNP suggest that Igf2r indeed plays a role in cognitive brain functioning.
Short talks and poster sessions contributed significant new data to the field. Overall, the meeting showed that responses to environmental exposures reveal a strong connection to acquisition and heritability of epigenetic traits, potentially having a significant role in complex disease susceptibility. Understanding the underlying mechanisms of the epigenetic changes can be crucial in developing future therapies of complex diseases.
Financial & competing interests disclosure
The authors have 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 prduction of this manuscript.