Epigenetic Inheritance, Trauma and the Holocaust

 

By Alison Bernstein, PhD

Since my research interests focus on environmental impacts on health and how epigenetic processes mediate those effects, my mother sent me this article, “Study of Holocaust survivors finds trauma passed on to children’s genes“, from The Guardian. This article reports the recent paper, “Holocaust exposure induced intergenerational effects on FKBP5 methylation“, in Biological Psychiatry. I get overly excited by teachable moments so I decided to take the opportunity to teach some more epigenetics (see my pages on Facebook or Google+ for my Intro to Epigenetics series).

Epigenetics literally means “over the genome”. It encompasses all meiotically and mitotically heritable changes in gene expression that are not coded in the DNA sequence itself. If we break that down, there are some key points to note:

  • “Not coded in the DNA”: There is no change in the DNA sequence. Thus, for these to be heritable, there must be mechanisms of inheritance besides DNA replication.
  • “Changes in gene expression”: The underlying assumption of all epigenetic studies should be that these changes alter gene expression (or change how inducible or repressible gene expression is, but that’s harder to measure).
  • “Meiotically and mitotically heritable”: This means heritable through cell division, but not necessarily heritable from parent to offspring.

Epigenetics generally refers to 4 mechanisms: DNA methylation (and other modifications to cytosine), histone modifications, non-coding RNAs, and long-range chromatin interactions (3D structure of chromosomes). In this paper, the authors focused on DNA methylation and identified changes in DNA methylation that occur in people who were in a Nazi concentration camp, witnessed or experienced torture, or hid from the Nazis during World War II. Similar changes were seen in their children. This transmission of a trait from parents to children is called intergenerational inheritance.

The effects of severe stress and other exposures has been shown to be inherited intergenerationally, multigenerationally (to grandchildren) and sometimes even transgenerationally (to great-grandchildren), both in animals and in people. The Dutch famine of 1944 and the polybrominated biphenyl exposure in Michigan in 1978 have provided evidence that exposures that occur prior to conception and in utero can have lasting effects on subsequent generations. However, it is difficult to separate out the different mechanisms that contribute to the inheritance of traits to subsequent generations. Thus, it is an important research question to ask how the effects of trauma, stress and other exposures are passed from generation to generation. This is the question the scientists wanted to address in this paper: is there an epigenetic component to the intergenerational inheritance of the effects of trauma?

Epigenetic Inheritance

This paper provides direct evidence in humans that the epigenetic effects of pre-conception stress can be seen in both parents and offspring. The authors looked at one specific gene only – FKBP5 – because it is known to be involved in the response to high glucocorticoid levels (a biological signal for stress) and is a possible novel target for antidepressant medication. They looked for changes in DNA methylation in glucocorticoid response elements within this gene. Response elements are sequences of DNA that bind to specific transcription factors and regulate transcription of genes. In this case, glucocorticoid response elements are bound by glucocorticoid hormones and their receptors to regulate expression of the gene containing the response element. They found changes in DNA methylation in these specific elements of the specific FKBP5 gene in Jewish Holocaust survivors and their children, but not in other Jewish people of similar age. This observed change in DNA methylation of the FKBP5 gene was in the opposite direction in parents and offspring, yet we do not yet have an explanation as to why this change would be different in parents and offspring. Thus, it is actually impossible to say from the results of this paper if these epigenetic changes are due to direct effects of stress and high glucocorticoid levels (or other shared environmental factors) or to inheritance of epigenetic marks.

Let’s say a woman or girl lived through the Holocaust. She and her eggs were exposed to high glucocorticoid levels, and other effects, due to stress. If a woman was pregnant during this time, she, her eggs and her in utero daughters’ eggs were exposed. So that’s 2, and possibly, 3 generations directly exposed to the stress. Until you get to the 4th generation, there is still a possibility of direct exposure. It might be epigenetic, but it is also possible that it’s still a result of direct exposure. Changes must be observed in the generation the great-grandchildren to definitively say that they are epigenetically inherited and not a result of direct exposure. In general, the great-grandchildren are the first generation that was definitely not directly exposed to the stressor. However, in this case, they looked at preconception stress, so looking at the 3rd generation (grandchildren) would be sufficient to differentiate between epigenetic inheritance and direct exposure.

This paper only looks at parents and their children. So the eggs that produced ALL those children were directly exposed (since females are born with all their eggs) to the trauma. It’s possible that high glucocorticoid levels directly affect the methylation of FKBP5 in the eggs as well in cells of the parent. The discussion of the paper itself goes into this, but the article overlooked this point and it’s a really important point to understand if you are interested in epigenetic inheritance.

From the discussion section of the paper:

“The main finding in this study is that Holocaust survivors and their offspring have methylation changes on the same site in a functional intronic region of the FKBP5 gene, a GR binding sequence in intron 7, but in the opposite direction. To our knowledge, these results provide the first demonstration of transmission of preconception stress effects resulting in epigenetic changes in both exposed parents and their offspring in adult humans. Bin 3/site 6 methylation was not associated with the FKBP5 risk-allele, and could not be attributed to the offspring’s own trauma exposure, their own psychopathology, or other examined characteristics that might independently affect methylation of this gene. Yet, it could be attributed to Holocaust exposure in the F0.

It is not possible to infer mechanisms of transmission from these data. It was not possible to disentangle the influence of parental gender, including in utero effects, since both Holocaust parents were survivors. Epigenetic effects in maternal or paternal gametes are a potential explanation for epigenetic effects in offspring, but blood samples will not permit ascertainment of gamete dependent transmission. What can be detected in blood samples is parental and offspring experience-dependent epigenetic modifications. Future prospective, longitudinal studies of high risk trauma survivors prior to conception, during pregnancy and postpartum may uncover sources of epigenetic influences.”

The paper reports evidence that the epigenetic effects of stress and trauma can be seen in both parents and offspring. However, there are a lot of variables that may cause similar epigenetic changes in parents and offspring. Further studies are needed to really know what the mechanism of these shared epigenetic marks are, before we can confidently assert that the epigenetic changes observed in parents and offspring are due to epigenetic inheritance. As with all good science, this paper answers a question while, at the same time, raising additional questions for future research.

This article was originally published on The Sound of Science blog in August 2015.

Leaving Your Mark on the World

By Danielle Gerhard

 

The idea that transgenerational inheritance of salient life experiences exists has only recently entered the world of experimental research. French scientist Jean-Baptiste Lamarck proposed the idea that acquired traits throughout an organism’s life could be passed along to offspring. This theory of inheritance was originally disregarded in favor of Mendelian genetics, or the inheritance of phenotypic traits isn’t a blending of the traits but instead a specific combination of alleles to form a unique gene encoding the phenotypic trait. However, inheritance is much more complicated than either theory allows for. While Lamarckian inheritance has largely been negated by modern genetics, recent findings in the field of genetics have caused some to revisit l’influence des circonstances, or, the influence of circumstances.

 

Over the past decade, efforts have shifted towards understanding the mechanisms underlying the non-Mendelian inheritance of experience-dependent information. While still conserving most of the rules of Mendelian inheritance, new discoveries like epigenetics and prions challenge the central dogma of molecular biology. Epigenetics is the study of heritable changes in gene activity as a result of environmental factors. These changes do not affect DNA sequences directly but instead impact processes that regulate gene activity such as DNA methylation and histone acetylation.

 

Epigenetics has transformed how psychologists approach understanding the development of psychological disorders. The first study to report epigenetic effects on behavior came from the lab of Michael Meany and Moshe Szyf at McGill University in the early 2000s. In a 2004 Nature Neuroscience paper they report differential DNA methylation in pups raised by high licking and grooming mothers compared to pups raised by low licking and grooming mother. Following these initial findings, neuroscientists have begun using epigenetic techniques to better understand how parental life experiences, such as stress and depression, can shape the epigenome of their offspring.

 

Recent research coming out from the lab of Tracy Bale of the University of Pennsylvania has investigated the heritability of behavioral phenotypes. A 2013 Journal of Neuroscience paper found that stressed males went on to produce offspring with blunted hypothalamic pituitary (HPA) axis responsivity. In simpler terms, when the offspring were presented with a brief, stressful event they had a reduction in the production of the stress hormone corticosterone (cortisol in humans), symptomatic of a predisposition to psychopathology. In contrast, an adaptive response to acute stressors is a transient increase in corticosterone that signals a negative feedback loops to subsequently silence the stress response.

 

The other key finding from this prior study is the identification of nine small non-coding RNA sperm microRNAs (miRs) increased in stressed sires. These findings begin to delve into how paternal experience can influence germ cell transmission but does not explain how selective increases in these sperm miRs might effect oocyte development in order to cause the observed phenotypic and hormonal deficits seen in adult offspring.

 

A recent study from the lab published in PNAS builds off of these initial findings to further investigate the mechanisms underlying transgenerational effects of paternal stress. Using the previously identified nine sperm miRs, the researchers performed a multi-miR injection into single-cell mouse zygotes that were introduced into healthy surrogate females. To confirm that all nine of the sperm miRs were required to recapitulate the stress phenotype, another set of single-cell mouse zygotes were microinjected with a single sperm miR. Furthermore, a final set of zygotes received none of the sperm miRs. Following a normal rearing schedule, the adult offspring were briefly exposed to an acute stressor and blood was collected to analyze changes in stress hormones. As hypothesized, male and female adult offspring from the multi-miR group had a blunted stress response relative to both controls.

 

To further investigate potential effects on neural development, the researchers dissected out the paraventricular nucleus (PVN) of the hypothalamus, a region of the brain that has been previously identified by the group to be involved in regulation of the stress response. Using RNA sequencing and gene set enrichment analysis (GSEA) techniques they found a decrease in genes involved in collagen formation and extracellular matrix organization which the authors go on to hypothesize could be modifying cerebral circulation and blood brain barrier integrity.

 

The final experiment in the study examined the postfertilization effects of multi-miR injected zygotes. Specifically, the investigators were interested in the direct, combined effect of the nine identified sperm miRs on stored maternal mRNA. Using a similar design as the initial experiment, the zygote mRNA was collected and amplified 24 hours after miR injection in order to examine differential gene expression. The researchers found that microinjection of the nine sperm miRs reduced stored maternal mRNA of candidate genes.

 

This study is significant as it has never been shown that paternally derived miRs play a regulatory role in zygote miR degradation. In simpler terms, these findings contradict the conventional belief that zygote development is solely maternally driven. Paternal models of transgenerational inheritance of salient life experiences are useful as they avoid confounding maternal influences in development. Studies investigating the effects of paternal drug use, malnutrition, and psychopathology are ongoing.

 

Not only do early life experiences influence the epigenome passed down to offspring but recent work out of the University of Copenhagen suggests that our diet may also have long-lasting, transgenerational effects. A study that will be published in Cell Metabolism next year examined the effects of obesity on the epigenome. They report differential small non-coding RNA expression and DNA methylation of genes involved in central nervous system development in the spermatozoa of obese men compared to lean controls. Before you start feeling guilty about the 15 jelly donuts you ate this morning, there is hope that epigenetics can also work in our favor. The authors present data on obese men who have undergone bariatric surgery-induced weight loss and they show a remodeling of DNA methylation in spermatozoa.

 

Although still a nascent field, epigenetics has promise for better understanding intergenerational transmission of risk to developing a psychopathology or disease. The ultimate goal of treatment is to identify patterns of epigenetic alternations across susceptible or diagnosed individuals and develop agents that aim to modify epigenetic processes responsible for regulating genes of interest. I would argue that it will one day be necessary for epigenetics and pharmacogenetics, another burgeoning field, to come into cahoots with one another to not only identify the epigenetic markers of a disease but to identify the markers on an person by person basis. However, because the fields of epigenetics and pharmacogenetics are still in the early stages, the tools and techniques currently available limit them. As a result, researchers are able to extract correlations in many of their studies but unable to determine potential causality. Therefore, longitudinal, transgenerational studies like those from the labs of Tracy Bale and others are necessary to provide insight into the lability of our epigenome in response to lifelong experiences.

The Epigenetics of Metabolic Reprogramming

 

By John McLaughlin

One of the greatest health issues facing the US is adult and childhood obesity, which exerts a huge human and economic cost on the healthcare system; therefore, its underlying causes are of enormous interest. While environmental factors such as diet and exercise are obviously major contributors, what roles do genetic variation or epigenetic effects play in predisposing individuals to obesity or affecting metabolism in general? Addressing these questions in simple, genetically tractable model systems is a time-tested method for pursuing the answers.

 

In recent studies on gene expression, especially related to disease, there has been growing interest in the role that epigenetic regulation plays in development and metabolism. On the web page of its “Roadmap Epigenomics Project,” the NIH defines “epigenetics” as referring to “…both heritable changes in gene activity and expression (in the progeny of cells or of individuals) and also stable, long-term alterations in the transcriptional potential of a cell that are not necessarily heritable.” Although the precise working definition of “epigenetic” is still contested, its usage by biologists generally includes phenomena such as DNA methylation and chemical modification of histones, which can cause changes in the transcriptional capacity of chromatin without altering DNA sequence.

 

In a recent Cell article, Öst and colleagues describe a phenomenon in Drosophila melanogaster which they term “intergenerational metabolic reprogramming” (IGMR). The study is intriguing because it adds to a growing body of work demonstrating intergenerational metabolic effects in a variety of organisms, including rats, flies and worms. An intergenerational effect, as described in this work, results when an environmental stimulus is applied to an organism and elicits a defined response in its offspring, which were not exposed to the original stimulus. Specifically, this study examined intergenerational effects on metabolism that are transmitted paternally; therefore, the mediators of the effect are presumably in the sperm cells at the time of egg fertilization.

 

Their IGMR experimental paradigm is straightforward: male flies are fed diets of varying sugar levels, mated to normally fed females, and their offspring characterized with regards to different metabolic features. The experiments were controlled to ensure that rearing conditions, such as diet and fly density, were identical among all groups of offspring. Interestingly, the progeny of both high-sugar and low-sugar fed fathers exhibited a similar phenotype: increased triglycerides, lipid droplet size, and food intake compared to offspring whose fathers had moderate sugar diets. In addition, this IGMR response was highly specific in causing metabolic phenotypes, as no general developmental effects were observed such as altered wing size, offspring number, or timing of adult eclosure. In other words, the male fly’s diet had a specific and significant impact on its offsprings’ metabolism.

 

An obvious question followed these results: what were the changes in gene regulation mediating this phenotypic response? The IGMR effect correlated with increased gene expression from X chromosome heterochromatin, suggesting an epigenetic process such as chromatin remodeling was at work. This idea was reinforced by an observed decrease in H3K9me3 staining, a histone mark typical of heterochromatin, in the fat bodies of IGMR flies. Although the authors didn’t identify the precise molecular mechanisms of paternal-diet-induced epigenetic reprogramming, they did several analyses comparing gene expression levels of flies fathered by control and IGMR males. RNA sequencing and computational analysis showed that several hundred genes, whose upregulation was correlated with the paternal high-sugar IGMR phenotype, are involved in known metabolic pathways in flies and other organisms.

 

So, what purpose might this type of intergenerational regulation serve? One possibility is that it increases the adaptiveness of offspring to local environments. Parents can “signal” to their offspring, through epigenetic mechanisms, the nutritional state of their surroundings, and thus the next generation of flies will be more metabolically primed to deal with the new environment. Of course, this one study should not be extrapolated to draw unwarranted conclusions about metabolic reprogramming in humans or other animals; much more work remains to be done on the subject. However, it is still exciting to ponder the myriad processes, both known and unknown, that work in the complexities of development. As our understanding of all the facets of gene regulation continues to progress, there will most likely be more amazing surprises to come.

 

 

 

Family Fear?

 

 

All You Need To Know About Epigenetic Inheritance of Fear Conditioning

 

Celine Cammarata

A new paper has exploded into the neuroscience world this week with the claim that conditioned fear of an odor can be passed down from father to son – remember Lamarck’s idea of how giraffe’s pass on their elongated necks?  The research has investigators’ knickers in a twist – some are excited, some are skeptical, and many are both.  So, what’s the scoop on this paper?

 

The Research

Emory investigators Brian Dias and Kerry Ressler showed intriguing evidence that fear memories can be inherited.  Sexually naive adult male mice were conditioned, using shock paired with odor exposure, to be fearful toward one of two scents, or, as a control, were left in their home cages.  Subsequently, these males were bred to naive females, and the lo and behold the male offspring showed increased sensitivity specifically to the odor that their fathers had been conditioned with, but not others.  The same held true for the conditioned males’ grandchildren, pups that were raised by other parents, and pups born from IVF, strengthening the argument that genetics were underlying this inherited fear.  The offspring of odor conditioned males also showed enlargements of the glomerulus corresponding to the odor sensory neurons that detect the conditioned scent and increased numbers of these neurons.  In the conditioned males’ sperm, the gene for the odor receptor of the conditioned scent showed reduced methylation of some regions, suggesting that this hypomethylation might underlie the increased receptor expression in offspring

 

The Context

This isn’t the first suggestion of heritable epigenetics.  A Science paper earlier this year revealed how methlyation, specifically, is “erased” in precursor cells that will become gametes, but also demonstrated that some epigenetic changes are able to escape erasure. Nor is this the first indication that parental experiences can shape offspring.  Researchers at the Mount Sinai School of Medicine showed that [mouse] fathers who experience social defeat leading to learned helplessness can pass on depression to their children, and a 2012 paper from U Penn and Mass General argued for heritable resistance to cocaine based.

 

The Buzz

Of course, the paper leaves some crucial questions open, most notably: how do the conditioned male’s sperm cells know what’s happening in the olfactory bulb?  The authors make some guesses, but largely leave this blank.  The investigators also never directly correlate the enlarge glomerulus in offspring to their enhanced sensitivity to their fathers’ conditioned odor.  And while the work is exciting, not everyone is ready to jump on board.  Some are dubious that the methylation seen in sperm would have the kind of effect shown in offspring, or reject the notion of such a high level of malleability in the genome; others point out that it’s difficult to determine whether it’s truly a fearful memory that’s been inherited, or just an increased sensitivity.  Nonetheless, nearly everyone has something to say on the topic – so take your newfound knowledge and get ready to join the conversation!