research2

The environment and transgenerational effects

The high rate of obesity throughout the modern world has become a public health crisis with an important contribution to morbidity and mortality. Obesity is essential to the development of metabolic syndrome, which includes hypertension, atherosclerosis, and insulin resistance. Data collected prior to 2010 indicate that 69% of adults were overweight and 35% were obese in the US. Although it is clear that changes in diet and activity are important contributors to the obesity epidemic, studies also point to in utero effects that persist in the offspring as significant factors to the increase in body weight. The impact of ancestral environmental effects on the phenotype of descendants in mice and humans has been widely described. For example, the Dutch Hunger Winter Families Study in the Netherlands and the Overkalix cohort in Sweden have reported the effect of diet in the parents on metabolic phenotypes, such as increased cholesterol and triglycerides, in their progeny compared to that of non-exposed siblings. Controlled studies in rats have similarly shown that a high-fat diet in fathers results in impaired insulin metabolism in the female F1 offspring, and that overfeeding of male mice results in a similar alteration of insulin and glucose metabolism in the F1 and F2 generations. Other studies have shown that maternal stress results in depressive-like behaviors in the offspring for two generations, and that paternal traumatic exposure affects the behavior and neuroanatomy of the F1 and F2 offspring. In addition to diet and stress, many environmental compounds, including fungicides, herbicides, pesticides, insecticides, industrial chemicals, detergents, plasticizers, and phytoestrogens, have effects on the development of the fetus when mothers are exposed during a specific window of embryonic development, and some of these effects can be transmitted to the F3 generation. In addition to obesity, these effects include high incidence of breast and testicular cancer, and deficits in social interactions and autism-like symptoms. However, the mechanisms by which these alterations in the epigenome are established and transmitted from parents to subsequent generations in mammals remain largely unknown. Current work in our lab is aimed at understanding the detailed molecular mechanisms by which obesity and behavioral phenotypes induced by exposure to endocrine disruptors are established in the male germline, maintained in the sperm, and passed on to the somatic adult tissues of the F3-F5 generations. Given the importance of epiphenotypes induced by the environment on human health, both for the exposed individuals and their offspring, and the lack of an explanation for how these alterations are set up and maintained, research aimed at dissecting the mechanisms by which the establishment and transmission of epigenetic information through the male germline is of the outmost importance. Understanding the mechanisms underlying these processes is highly significant from the point of view of basic biology and for human health.

The epigenome of mammalian sperm

The lack of a mechanistic understanding of transgenerational inheritance of epiphenotypes through the male germline stems in part from a lack of knowledge of the mammalian sperm epigenome. The sperm nucleus is generally considered a passive vessel that delivers the highly condensed paternal DNA, mostly devoid of histones and complexed with protamines, to the egg during fertilization. DNA methylation has been largely

discounted as a plausible mechanism underlying transgenerational effects because of its erasure and re-establishment during gametogenesis and after fertilization. Approximately 2-10 % of the mouse and human genomes, respectively, remain associated with histones. To explore alternative pathways by which epigenetic information can be altered and transmitted through the paternal germline, we have carried out a comprehensive analysis of the mouse sperm epigenome. The results indicate that sperm promoters are flanked by positioned nucleosomes containing a variety of histone modifications. Furthermore, sperm DNA is bound by CTCF, cohesin, Mediator, FoxA1, estrogen and androgen receptors, and RNA Polymerase II phosphorylated in Ser 5 and Ser2. We also find that the sperm genome is arranged in the 3D nuclear space forming compartmental domains and CTCF loops similar to those found in stem or somatic cells. These findings represent a paradigm shift in sperm biology and open the way for new hypotheses to understand the mechanisms responsible for transgenerational epiphenotypes.

Transmission of epigenetic information from the gametes to the embryo

We have also explored the state of the oocyte epigenome. Oocytes at the prophase GV stage also contain accessible promoters, which remain accessible in the MII metaphase stage. Furthermore, the accessibility state is quantitatively maintained from the gametes to various stages of the preimplantation embryo. It is possible that the apparent maintenance of the transcription complex at these promoters is related to a requirement for their expression in preimplantation embryos. Alternatively, the persistence of promoter accessibility between gametes and the early embryo may be related to the reprograming of DNA methylation of the paternal and maternal genomes after fertilization. In addition to promoters, the oocyte genome, like that of sperm, contains thousands of distal accessible sites that may correspond to regulatory sequences such as enhancers, and their conservation in syntenic regions of the rhesus macaque and human genomes suggests a functional role for these sequences. In agreement with this, analysis of DNA binding motifs at these regions suggest the presence of transcription factors that remain bound to both the sperm and oocyte genomes, including MII oocytes. A subset of these sites persists in the genome of the embryo at different stages up to the time of implantation. Interestingly, the sites that are maintained correspond to those present in both sperm and oocytes. In sperm, where this information is known, sites that persist in the embryo are flanked by nucleosomes containing H3.3, H2A.Z, and high levels of H3K27ac, whereas those that are not maintained lack H3.3 and have lower levels of H2A.Z and H3K27ac. The results suggest a regulated and determined assembly of sperm chromatin with the possible objective of controlling information passed on to the embryo.

The organization of the chromatin fiber in the nuclear space arises as a consequence of interactions between compartmental domains in the same transcriptional state but point-to-point contacts mediated by CTCF and cohesin are also important contributors to the establishment of this organization. Interestingly, most CTCF sites present in sperm are accessible in both GV and MII oocytes, and these sites shared by the gametes persist during early embryogenesis up to the blastocyst stage. In spite of their persistence in MII oocytes, putative CTCF sites present at this stage do not mediate interactions in MII metaphase chromosomes. However, CTCF loops present in the GV oocyte have been re-established in the maternal chromosome of the zygote by the PN5 stage, persist during early embryonic development, and can be observed in adult brain cortex. Interestingly, these interactions are absent from the paternal chromosome in the zygote and 2-cell stage but are established by the 8-cell stage. Similarly, paternal chromosome-specific interactions present in sperm and early embryo are absent in the maternal chromosomes, which become the same as the sperm by the 8-cell stage. The mechanisms by which parent-of-origin specific interactions are established in the sperm or the zygote and converge in the two parental chromosomes by the 8-cell stage are unknown and an important issue for future discovery. In summary, we have shown that sperm and oocytes contain far more information with the potential to encode epigenetic memory than was previously recognized. Specific sites on gamete chromatin are poised with transcription factors despite lack of transcriptional inactivity, and chromatin accessibility at these sites and at distal regulatory elements is maintained in the embryo until at least the ICM stage. Since global remethylation occurs after the ICM stage, persistent sites with bound transcription factors inherited from gametes and retained through early embryogenesis may inhibit remethylation at their binding motifs. These sites may then remain accessible to the transcription machinery later in development as differentiation ensues. These observations open the possibility that transcription factors, whose distribution in the genome may be altered by environmental effects, are the basis for the transmission of epiphenotypes between generations.

Overview of current research interests on 3D organization

We suggest that the environment can alter the interaction of transcription factors with DNA during germline differentiation and that these changes persist in the sperm and can affect transcription in the embryo after fertilization. In addition, transcription factors bound to DNA can guide or protect the erasure or re-establishment of DNA methylation marks during germline differentiation or early embryogenesis. The reciprocal interplay between transcription factor binding and DNA methylation constitutes the basis for the establishment of epigenetic information, and their presence in the genome and mutual reinforcement can explain their persistence between generations. This hypothesis, which has been mostly ignored based on the belief that the sperm genome is covered by protamines with the exception of a few retained nucleosomes, represents a paradigm shift in the field.