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Archive for the ‘Epigenetics’ Category

Your Inner Fish by Neil Shubin – 2008

You might all be wondering why I’ve added this book to my collection of Informative Books regarding the synthetic chemical revolution. This is in many ways where epigenetics began. This book documents the very beginning of our understanding of epigenetics. This is where geneticists began learning about gene expression and the triggers that silence them or make them active. It is also where they first discovered the method by which birth defects occur. This is a groundbreaking book with information not yet in our textbooks. The discovery of the Sonic hedgehog gene led to the understanding of how birth defects occur in limb development. You will understand why this book is so critically important to understanding the potential impacts of the synthetic revolution in evolutionary terms regarding humans. Humans share many genes with other species. Our development is extraordinarily similar. You will understand the implications of this field of study after reading the following excerpt.

HANDY GENES

We begin with an apparent puzzle. Our body is made up of hundreds of different kinds of cells. This cellular diversity gives our tissues and organs their distinct shapes and functions. The cells that make our bones, nerves, guts, and so on look and behave entirely differently. Despite these differences, there is a deep similarity among every cell inside our bodies: all of them contain exactly the same DNA. If DNA contains the information to build our bodies, tissues, and organs, how is it that cells as different as those found in muscle, nerve, and bone contain the same DNA?

The answer lies in understanding what pieces of DNA (the genes) are actually turned on in every cell. A skin cell is different from a neuron because different genes are active in each cell. When a gene is turned on, it makes a protein that can affect what the cell looks like and how it behaves. Therefore, to understand what makes a cell in the eye different from a cell in the bones of the hand, we need to know about the genetic switches that control the activity of genes in each cell and tissue.

Here’s an important fact: these genetic switches help to assemble us. At conception, we start as a single cell that contains all the DNA needed to build our body. The plan for that entire body unfolds via instructions contained in this single microscopic cell. To go from this generalized egg cell to a complete human, with trillions of specialized cells organized in just the right way, whole batteries of genes need to be turned on and off at just the right stages of development. Like a concerto composed of individual notes played by many instruments, our bodies are a composition of individual genes turning on and off inside each cell during our development…

When we compare the ensemble of genes active in the development of a fish fin to those active in the development of a human hand, we can catalogue the genetic differences between fins and limbs. This kind of comparison gives us some likely culprits–the genetic switches that may have changed during the origin of limbs. We can then study what these genes are doing in the embryo and how they might have changed. We can even do experiments in which we manipulate the genes to see how bodies actually change in response to different conditions or stimuli…..We will begin by looking at the structure of our limbs, and zoom all the way down to the tissues, cells, and genes that make it.

MAKING HANDS

Our limbs exist in three dimensions: they have a top and a bottom, a pinky side and a thumb side, a base and a tip. The bones at the tips, in our fingers, are different from the bones at our shoulder. Likewise, our hands are different from one side to the other. Our pinkies are shaped differently from one side to the other. The Holy Grail of our developmental research is to understand what genes differentiate the various bones of our limb, and what controls development in these three dimensions. What DNA actually makes a pinky different from a thumb? What makes our fingers distinct from our arm bones? If we understand the genes that control such patterns, we will be privy to the recipe that builds us.

All the genetic switches that make our fingers, arm bones, and toes do their thing during the third to eighth week after conception. Limbs begin their development as tiny buds that extend from our embryonic bodies. The buds grow over two weeks, until the tip forms a paddle. Inside this paddle are millions of cells which will ultimately give rise to the skeleton, nerves, and muscles that we’ll have for the rest of our lives.

To study how this pattern emerges, we need to look at embryos and sometimes interfere with their development to assess what happens when things go wrong. Moreover, we need to look at mutants and at their internal structures and genes, often by making whole mutant populations through careful breeding. Obviously, we cannot study humans in these ways. The challenge for the pioneers in this field was to find the animals that could be useful windows into our own development. The first experimental embryologists interested in limbs in the 1930s and 1940s faced several problems. They needed an organism in which the limbs were accessible for observation and experiment. The embryo had to be relatively large, so that they could perform surgical procedures on it. Importantly, the embryo had to grow in a protected place, in a container that sheltered it from jostling and other environmental disturbances. Also, and critically, the embryos had to be abundant and available year-round. the obvious solution to this scientific need is at your local grocery store: chicken eggs.

In the 1950s and 1960s a number of biologists, including Edgar Zwilling and John Saunders, did extraordinary creative experiments on chicken eggs to understand how the pattern of the skeleton forms. This was an era of slice and dice. Embryos were cut up and various tissues moved about to see what effect this had on development. The approach involved very careful microsurgery, manipulating patches of tissue no more than a millimeter think. In that way, by moving tissues about in the developing limb, Saunders and Zwilling uncovered some of the key mechanisms that build limbs as different as bird wings, whale flippers, and human hands.

They discovered that two little patches of tissue essentially control the development of the pattern of bones inside limbs. A strip of tissue at the extreme end of the limb bud is essential for all limb development. Remove it, and development stops. Remove it early, and we are left with only an upper arm, or a piece of an arm. Remove it slightly later, and we end up with an upper arm and a forearm. Remove it even later, and the arm is almost complete, except that the digits are short and deformed.

Another experiment, initially done by Mary Gasseling in John Saunder’s laboratory, led to a powerful new line of research. Take a little patch of tissue from what will become the pinky side of a limb bud, early in development, and transplant it on the opposite side, just under where the first finger will form. Let the chick develop and form a wing. The result surprised nearly everybody. The wing developed normally except that it also had a full duplicate set of digits. Even more remarkable was the pattern of the digits: the new fingers were mirror images of the normal set. Obviously, something inside that patch of tissue, some molecule or gene, was able to direct the development of the pattern of the fingers. This result spawned a blizzard of new experiments, and we learned that this effect can be mimicked by a variety of other means. For example, take a chicken embryo and dab a little vitamin A (retinoic acid) on its limb bud, or simply inject vitamin A into the egg. and let the embryo develop. If you supply the vitamin A at the right concentration and at the right stage, you’ll get the same mirror-image duplication that Gasseling, Saunders, and Zwilling got from the grafting experiments. This patch of tissue was named the zone of polarizing activity (ZPA). Essentially, the ZPA is a patch of tissue that causes the pinky side to be different from the thumb side. Obviously chicks do not have a pinky and a thumb. The terminology we use is to the number of digits, with our pinky corresponding to digit five of other animals and our thumb corresponding to digit one.

The ZPA drew interest because it appeared, in some way, to control the formation of fingers and toes. But how? Some people believed that the cells in the ZPA made a molecule that then spread across the limb to instruct cells to make different fingers. The key proposal was that it was the concentration of this named molecule that was the important factor. In areas close to the ZPA, where there is a high concentration of this molecule, cells would respond by making a pinky. In the opposite side of the developing hand, farther from the ZPA so that the molecule was more diffused, the cells would respond by making a thumb. Cells in the middle would each respond according to the concentration of this molecule to make the second, third, and fourth fingers.

This concentration-dependent idea could be tested. In 1979, Denis Summerbell placed an extremely small piece of foil between the ZPA patch and the rest of the limb. The idea was to use this barrier to prevent any kind of molecule from diffusing from the ZPA to the other side. Summerbell studied what happened to the cells on each side of the barrier. Cells on the opposite side often did not form digits; if they did, the digits were badly malformed. The conclusion was obvious. Something was emanating from the ZPA that controlled how the digits formed and what they looked like. To identify that something, researchers needed to look at DNA.

THE DNA RECIPE

That project was left to a new generation of scientists. Not until the 1990s, when new molecular techniques became available, was the genetic control for the ZPA’s operation unraveled.

A major breakthrough happened in 1993, when Cliff Tabin’s laboratory at Harvard started hunting for the genes that control the ZPA. Their prey was the molecular mechanisms that gave the ZPA its ability to make our pinky different from our thumb. By this time his group started to work in the early 1990s, a number of experiments like the ones I’ve described had led us to believe that some sort of molecule caused the whole thing. This was a grand theory, but nobody knew what this molecule was. People would propose one molecule after another, only to find that none was up to the job. Finally, the Tabin lab came up with a novel notion, and one very relevant to the theme of this book. Look to flies for the answer.

Genetic experiments in the 1980s had revealed the wonderful pattern of gene activity that sculpts the body of a fly from a single-celled egg. The body of a fruit fly is organized from front to back, with the head at the front and the wings at the back. Whole batteries of genes are turned on and off during fly development, and this pattern of gene activity serves to demarcate the different regions of the fly.

Tabin didn’t know at the time, but two other laboratories–those of Andy MacMahon and Phil Ingham–had already come up with the same general idea independently. What emerged was a remarkably successful collaboration among three different lab groups. One of the fly genes caught the attention of Tabin, McMahon, and Ingham. They noted that this gene made one end of a body segment look different from the other. Fly geneticists named it hedgehog. Doesn’t the function of hedgehog in the fly body–to make one region different from another–sound like what ZPA does in making the pinky different from the thumb? That parallel was not lost on the three labs. So off they went, looking for a hedgehog gene in creatures like chickens, mice, and fish.

Because the lab groups knew the structure of the fly’s hedgehog gene, that had a search image to help them single out the gene in chickens. Each gene sequence; using a number of molecular tools, the researchers could scan a chicken’s DNA for the hedgehog sequence. After a lot of trial and error, they found a chicken hedgehog gene.

Just as paleontologists get to name a new species, geneticists get to name new genes. The fly geneticists who discovered hedgehog had named it that because the flies with a mutation in the gene had bristles that reminded them of a little hedgehog. Tabin, McMahon, and Ingham named the chicken version of the gene Sonic hedgehog, after the Sega Genesis video game.

Now came the fun question: What does Sonic hedgehog actually do in the limb? The Tabin group attached a dye to a molecule that would stick to the gene, enabling them to visualize where the gene is active in the limb. To their great surprise, they found that only cells in a tiny patch of the limb had gene activity: the ZPA.

So the next steps were obvious. The patterns of activity in the Sonic hedgehog gene could mimic those of the ZPA tissue itself. Recall when you treat the limb with retinoic acid, a form of vitamin A, you get a ZPA active on the opposite side. Guess what happens when you treat a limb with retinoic acid, then map where Sonic hedgehog is active? Sonic hedgehog is active on both sides–pinky and thumb–just as the ZPA does when it is treated with retinoic acid.

Knowing the structure of the chicken Sonic hedgehog gave other researchers the tools to look for it in everything else that has fingers, from frogs to humans. Every limbed animal has the Sonic hedgehog gene. And in every single animal we have studied, Sonic hedgehog is active in ZPA tissue. If Sonic hedgehog hadn’t turned on properly during the eight week of your own development, then you either would have extra fingers or your pinky and thumb would look alike. Occasionally, when things go wrong with Sonic hedgehog, the hand ends up looking like a broad paddle with as many as twelve fingers that all look alike.

We now know that Sonic hedgehog is one of dozens of genes that act to sculpt our limbs from shoulder to fingertip by turning on and off at the right time. Remarkably, work in chickens, frogs, and mice was telling us the same thing. The DNA recipe to build upper arms, forearms, wrists, and digits is virtually identical in every creature that has limbs.

How far back can we trace Sonic hedgehog and other bits of DNA that build limbs? Is this stuff active in building the skeleton of fish fins? Or are hands genetically completely different from fish fins? We saw an inner fish in the anatomy of our arms and hands. What about the DNA that builds it? ….

The title of the book indicates the answers to those questions

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Ancestral ghosts in your genome | Michael Skinner | TEDxRainier


Epigenetic Transgenerational Actions of Endocrine Disruptors by Dr. Michael K. Skinner & Matthew D. Anway

Epigenetic Transgenerational Actions of Endocrine Disruptors

Matthew D. Anway and Michael K. Skinner

Center for Reproductive Biology, School of Molecular Biosciences, Washington State University, Pullman, Washington 99164

Address all correspondence and requests for reprints to: Michael K. Skinner, Center for Reproductive Biology, School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4231. E-mail: Skinner@mail.wsu.edu.

Abstract

Endocrine disruptors have recently been shown to promote an epigenetic transgenerational phenotype involving a number of disease states (e.g. male infertility). The anti-androgenic fungicide vinclozolin was found to act transiently at the time of embryonic sex determination to promote in the F1 generation a spermatogenic cell defect and subfertility in the male. When the animals were allowed to age up to 1 yr, a number of other disease states developed. This phenotype was transferred through the male germ line to all subsequent generations analyzed (F1–F4). The ability of an environmental factor (i.e. endocrine disruptor) to promote an epigenetic transgenerational phenotype impacts the potential hazards of environmental toxins, mechanisms of disease etiology, and evolutionary biology. The biological importance of the epigenetic actions of environmental agents is reviewed in the context of the primordial germ cell and development of epigenetic transgenerational phenotypes.

GENOMIC DNA CONTAINS the core of genetic information of the cell. There is a distinct pattern of gene expression throughout mammalian development that is heritable from parents to offspring. Epigenetics is defined as the molecular phenomena that regulate gene expression without alterations to the DNA sequence (1). The most studied epigenetic modification is DNA methylation of CpG nucleotides that are essential for mammalian development (2, 3, 4, 5). DNA methylation of CpG sites is used by mammals to regulate transcription of genes, alter chromosomal positioning, influence X-chromosome inactivation, control imprinted genes, and repress parasitic DNAs (1, 5, 6, 7, 8, 9). Alterations in the DNA methylation state can lead to multiple disease states including cancers (10, 11), Rett syndrome, and Prader-Willi/Angelman syndrome (11, 12, 13), male infertility (14), autism (12), and Angelman and Beckwith-Wiedemann syndromes (13). Both chemical and environmental toxins have been shown to alter DNA methylation patterns resulting in epigenetic phenotypes (14, 15).

DNA methylation patterns are established at two times during development: the lineage-specific pattern during gastrulation and the germ-line-specific pattern in the gonad after sex determination (16). The lineage-specific pattern establishes the DNA methylation for somatic cell development after fertilization. This epigenetic reprogramming is based on the genetic material transferred from the egg and sperm. Alterations in the lineage-specific epigenetic reprogramming results in developmental defects or embryonic lethality (13, 16). The germ-line DNA methylation pattern is established during gonadal development and is sex specific (16, 17, 18). Epigenetic reprogramming of the germ line is critical for imprinting (19, 20, 21, 22). Unlike the lineage-specific reprogramming, alterations in the germ-line epigenetic reprogramming can alter the heritable epigenetic information, resulting in a transgenerational phenotype (15) (Fig. 1). The embryonic period is the most sensitive for chemical and environmental effects on the epigenetics of the male germ line (15, 21, 22).

Recent investigations of the DNA methylation state of the primordial germ cells have indicated that as primordial germ cells migrate down the genital ridge, a demethylation (i.e. erasure of methylation) starts, and upon colonization in the early gonad, a complete demethylation is achieved (21, 22, 23). This has been primarily observed through the analysis of specific imprinted genes (24). During the period of sex determination in the gonad, the germ cells undergo a remethylation involving a sex-specific determination of the germ cells (Fig. 2). Although the demethylation may not require the gonad somatic cells (21), the remethylation of the germ line appears to be dependent on association with the somatic cells in the gonads (22, 23). Because of this unique property of the germ cells to undergo a demethylation and remethylation during the period of sex determination in the developing gonad, the ability of an environmental agent such as an endocrine disruptor to influence through an epigenetic process the germ line is postulated. This epigenetic effect on the germ line could reprogram the germ cell through an event such as altered DNA imprinting (25, 26). This epigenetic effect could cause a transgenerational effect on subsequent generations through the germ line. Because the remethylation of the germ line appears dependent upon the gonadal somatic cells, an alteration in somatic cell function by an agent such as an endocrine disruptor could indirectly influence the germ cell remethylation (Fig. 2). Epigenetic alterations that lead to transgenerational transmission of specific genetic traits or molecular events (e.g. imprinting) have recently been identified (6, 7, 27). These observations have led to the conclusion that a reprogramming through altered epigenetics of the male germ line is possible (15). The impact this has on human health and evolutionary biology is significant (6, 27).

Transgenerational Phenomena and Environmental Factors
Environmental effects of irradiation, chemical treatments (e.g. chemotherapy), and environmental toxins such as endocrine disruptors have been observed over the past decade. The majority of observations are simply the effects of the agent on the gestating mother (F0) and subsequent actions on the offspring associated with the F1 generation (28, 29, 30). Examples of environmental factors during embryonic development that influence the F1 generation include the effects of heavy metals causing cancer (31), abnormal nutrition that causes diabetic and uterine defects (32, 33, 34), chemical exposure (i.e. ethosuximide and benzpyrene) causing brain and endocrine defects (35, 36), and endocrine disruptors such as diethylstilbestrol (37, 38), phthalates, and dithiothreitol causing reproductive tract and endocrine defects (39, 40, 41). Environmental factors have effects on the F1 generation of a number of species including insects (42, 43, 44), fish (45, 46), birds (47), and other species (48). Therefore, exposure to a number of environmental factors in utero can cause abnormal phenotypes in the F1 generation in a number of different species. Because the F1 generation is exposed to the environmental factor, the F1 effect is not a transgenerational phenotype.

Transgenerational effects of environmental factors require effects minimally on the F3 generation (15, 49) (Fig. 1). This is because the F3 generation is the first generation not directly exposed to the environmental factor. The ability of an external agent to induce a transgenerational phenotype requires a genetic (i.e. DNA sequence) or an epigenetic (i.e. DNA methylation) phenomenon mediated through the germ line (50, 51, 52, 53). Transgenerational inheritance of an epigenetic state has been shown to occur using several mouse genetic lines and markers (6, 27, 54) and more recently with the use of monozygotic twins with epigenetic differences (55). Irradiation exposure was one of the first transgenerational phenomena observed to be transmitted through the germ line to multiple generations, often associated with mutagenesis and tumor formation (50, 51, 52, 53). The chemotherapeutic treatment of cancers has been shown to cause F1 generation effects (31, 35, 46), but the transmission to multiple generations has not been thoroughly investigated. Environmental factors do appear to promote a transgenerational susceptibility to cancer (56, 57). Gestating nutritional deficiency effects on the F1 generation have been observed (34), and recently these nutritional effects on a diabetic condition and growth defects have been shown to be transgenerational to the F2 generation (58, 59, 60). Several environmental chemical exposures have also been shown to transgenerationally affect the F2 generation including benzpyrene (36, 61), orthoaminoasotoluol (62), and dioxin (63). Environmental toxins such as endocrine disruptors have also been shown to influence the F1 generation after parental exposure (39, 46, 64, 65, 66, 67), but few have demonstrated transgenerational effects on multiple generations (15). Some evidence that diethylstilbestrol has effects in the F2 generation have been reported (68).

Endocrine Disruptors and Reproductive Toxicology

Many reports have suggested that environmental endocrine disruptors, which act to mimic estrogens or act as antiestrogens or antiandrogens, are detrimental to reproduction and may promote abnormalities such as a decrease in sperm count, an increase in testicular cancer (69, 70), and an increase in abnormalities in sex determination for many species (71). Examples of environmental endocrine disruptors that have been targeted for adverse effects on reproductive systems in humans and other animals are pesticides [e.g. dichlorodiphenyltrichloroethane (DDT) and methoxychlor] (72), fungicides (e.g. vinclozolin) (15, 73), insecticides (e.g. trichlorfon) (74), herbicides (e.g. atrazine) (75), plastics (e.g. phthalates) (76), and a range of xenoestrogens (77). Most of these chemicals are ubiquitous in the environment, resulting in daily exposure for humans and other animals. Many of these compounds and endocrine disruptors can be metabolized into both estrogenic and antiandrogenic activities (78). Recently, methoxychlor and vinclozolin have been used (66, 67) as model endocrine disruptors (72) that have estrogenic, antiestrogenic, and antiandrogenic metabolites (78).

Many environmental endocrine disruptors are weakly estrogenic and elicit their actions through the estrogen receptors. The two mammalian receptors for estrogen (ER- and ER-ß) are widely distributed throughout the reproductive tract (79, 80). ER-ß is present in higher concentrations within the fetal testis and ovary, whereas ER- is present mainly within the uterus (81, 82). During fetal testis development, ER-ß is expressed in Sertoli and myoid cells after seminiferous cord formation (83). In rats, ER-ß has also been localized to prespermatogonia, which may explain the proliferative actions of estrogen on early postnatal gonocyte cultures (84). The importance of ER- was delineated when knockout mice (85) and human males (86) lacking expression of this gene were found to be sterile. Fetal development of the testis in these experiments was not altered; however, fetal testis morphology in a double knockout remains to be examined (87). Neonatal exposure to estrogen alters the ER- and ER-ß expression during postnatal testis and hypothalamic/pituitary development (88, 89). Interestingly, neonatal exposure to the estrogenic compound diethylstilbestrol promotes abnormal testis and male reproductive tract development (90) and leads to changes in gene expression (91). Therefore, actions of estrogenic endocrine disruptors on estrogen receptors may impair normal fetal gonadal development and lead to infertility. Although the estrogen receptors are thought to have a role in testis development (92, 93, 94), the specific functions remain to be elucidated. Treatment of males with estrogens during early fetal life may alter responsiveness to androgens by changing androgen receptor (AR) expression patterns (95, 96) and/or Leydig cell function (91).

Antiandrogenic endocrine disruptors can also influence fetal gonad development. AR expression is very similar to ER-ß expression in the developing testis (82, 97). AR is detected in Sertoli, myoid, and prespermatogonial cells just after cord formation (98) and in interstitial cells late in fetal development. It is proposed that AR is present in cells that migrate from the mesonephros and enables cord formation to occur (98). Therefore, inappropriate expression or actions of AR through treatment by endocrine disruptors may affect the process of morphological sex differentiation (i.e. cord formation). Antiandrogens such as flutamide (99) or cyproterone acetate (100) administered to pregnant rats at different ages of gestation impair fertility in the male offspring. Both flutamide and cyproterone acetate block the ability of androgens and epidermal growth factor to stabilize the Wolffian duct (101). Therefore, perturbation of AR may also cause inappropriate expression and action of growth factors in the testis. A commonly used antiandrogenic endocrine disruptor is vinclozolin, which is used as a fungicide in the wine industry (102, 103). Vinclozolin has been shown to act as an environmental antiandrogen and influence gonad development and fertility (15, 67).

Epigenetic Transgenerational Actions of Endocrine Disruptors

A recent observation demonstrated that the exposure of a pregnant rat transiently to endocrine disruptors caused a spermatogenic cell defect and subfertility in the F1 generation and all subsequent generations examined (F1–F4) (15) (Fig. 1). The endocrine disruptors used were the antiandrogenic fungicide vinclozolin used in the fruit (e.g. wine) industry (73) and the pesticide methoxychlor used to replace dichlorodiphenyltrichloroethane (DDT) (78). The critical exposure period was at the time of sex determination, and the transgenerational phenotype was transmitted through the male germ line (15) (Fig. 1). The phenotype of increased spermatogenic cell apoptosis and decreased sperm numbers and sperm motility was observed in greater than 90% of all males of all the generations examined. When the animals were allowed to age up to 1 yr, additional diseases developed including cancer, prostate disease, kidney disease, and immune cell defects (Anway, M. D., and M. K. Skinner, submitted for publication). A high frequency of transmission was observed in all generations examined for all the disease states.

The frequency of the transgenerational phenotype was such that a DNA sequence mutational event could not be involved. The random nature of a DNA sequence mutation has a phenotype typically less than 1%, and this often declines in subsequent generations (50, 104). An epigenetic mechanism is involved because of the frequency of the phenotype. To support these conclusions, two genes were identified in the sperm that had altered methylation patterns associated with the transgenerational phenotype discussed (15). Therefore, the endocrine disruptors appear to induce an epigenetic transgenerational disease condition for four generations through the male germ line (15) (Fig. 1). The epigenetics appears to involve altered DNA methylation. Although most genes get reset in early embryonic development, a subset of genes called imprinted genes maintains their DNA methylation pattern that appears to be permanently programmed. In contrast to all somatic cells, the primordial germ cells undergo a demethylation during migration and early colonization of the embryonic gonad, followed by a remethylation starting at the time of sex determination in a sex-specific manner (23, 24, 105). The exposure of the pregnant mother at the time of sex determination appears to have altered the remethylation of the germ line and permanently reprogrammed the imprinted pattern of DNA methylation (15). This provides a unique epigenetic mechanism to promote a transgenerational phenotype induced by an environmental factor.

Summary

The observations that an environmental toxin (e.g. endocrine disruptor) can have an epigenetic effect on the germ line and cause a transgenerational effect on male reproduction significantly impacts our understanding of the potential hazards of these compounds to human health as well as all other mammalian species (15). These studies establish a novel mechanism of action not previously appreciated on how environmental toxins may act on a gestating mother to influence her grandchildren and subsequent generations. Elucidation of this phenomenon will allow us to better understand the true hazards of environmental toxins, identify the specific causal agents, and develop appropriate preventative and therapeutic approaches. Independent of the specific compound or agent of interest, the establishment of this potential mechanism of action is critical to our insight into the effects of environmental factors that influence embryonic development and adult reproduction.

The level of endocrine disruptors used in the recent studies (15, 66, 67, 106) (Anway, M. D., and M. K. Skinner, submitted for publication) is higher than anticipated in the environment, such that conclusions regarding the toxicology of these endocrine disruptors are not possible. However, the important factor is the identification of this novel phenomenon, that an environmental factor can promote an epigenetic transgenerational phenotype (15). Because of this observation, the potential hazards of environmental factors need to be carefully evaluated. If the exposure of your grandmother at midgestation to environmental toxins can cause a disease state in you with no exposure, and you will pass it on to your grandchildren, the potential hazards of environmental toxins need to be rigorously assessed. Transgenerational studies need to be performed in evaluating the toxicology of environmental compounds.

The epigenetic transgenerational phenotype also provides critical insights into disease etiology. Because a number of common disease states are induced (Anway, M. D., and M. K. Skinner, submitted for publication), an epigenetic component of disease now needs to be seriously considered. The fetal basis of adult-onset disease could be a result of epigenetic factors (107, 108). In the event a major epigenetic component exists, the epigenetic background of an individual may be a significant factor in susceptibility to disease development. Therefore, identification of the genes involved with altered methylation may provide essential new diagnostics to assess onset of disease. These epigenetic factors may influence the outcomes of current medical therapies such as assisted reproductive procedures (109, 110). Further analysis of the epigenetic transgenerational phenotypes and identification of specific epigenetic changes will allow new therapeutic targets and therapies to be developed to potentially prevent the onset of disease. This is a new paradigm in disease etiology that needs to be considered.

In a broader biological perspective, the ability of an environmental factor to cause a permanent genetic trait in all subsequent progeny of an effected individual can significantly impact our understanding of evolutionary biology. Currently, a DNA sequence mutation event that allows an adaptation and natural selection is considered the driving factor in evolutionary biology. However, the frequency of specific evolutionary events (110, 111) and regional influences on evolution suggest an additional epigenetic mechanism should be considered (112, 113, 114, 115). Although a DNA sequence mutational event will be important for evolutionary biology, an epigenetic component influenced by an environmental factor needs to be considered as an alternate factor that will help explain some aspects of evolutionary biology. Epigenetics is the next layer of complexity beyond the DNA sequence.

Acknowledgments

We acknowledge the assistance of Ms. Jill Griffin in the preparation of this manuscript.

Footnotes

This research was supported in part by grants from the U.S. Environmental Protection Agency, EPA STAR program, and from the National Institute of Environmental Health Sciences (to M.K.S.).

None of the authors have any conflicts of interest regarding the current publication.

First Published Online May 11, 2006

Abbreviation: AR, Androgen receptor.

Received August 19, 2005.

Accepted for publication October 14, 2005.

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Epigenetic Transgenerational Actions of Endocrine Disruptors by Dr. Michael K. Skinner & Matthew D. Anway

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Dr. Michael Skinner is a leading Principal Investigator in the field of epigenetics research. His discoveries will alter the way we view the origin and nature of disease.

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PBS Nova – Ghost in Your Genes

This is a profound & enlightening program covering the breaking field of epigenetics. It’s important to note that Dow Chemical helped fund this program but the only way you would know this is through watching it because it is mentioned at the beginning and the ending of the program. I got a DVD copy from an educator through the Howard Hughes Medical Institute. The public cannot access copies through HHMI though. This is one of the only Nova episodes that is not available to watch online. Here is why I’m guessing that Dow Chemical has not released this program for sale. The following is taken from the transcripts and has serious implications.

But in Washington state, Michael Skinner seems to have found compelling additional evidence by triggering a similar effect with commonly used pesticides. Skinner wanted to see how these chemicals would affect pregnant rats and their offspring.

MICHAEL SKINNER: And so I treated the animals, the pregnant mother, with these compounds, and then we started seeing, between six months to a year, a whole host of other diseases that we didn’t expect. And this ranged between tumors, such as breast and skin tumors, prostate disease, kidney disease and immune dysfunction.

NARRATOR: He checked that there were no genetic mutations and then proceeded to breed the rats.

MICHAEL SKINNER: The next step was for us to go to the next generation. And the same disease state occurs. So after we did several repeats, and got the third generation showing it and then a fourth generation, we sat back and realized that the phenomenon was real. We started seeing these major diseases occur in approximately 85 percent of all the animals of every single generation.

NARRATOR: His discoveries were a revelation.

MICHAEL SKINNER: We knew that if an individual was exposed to an environmental toxin that they can get a disease state, potentially. The new phenomenon is that environmental toxin no longer affects just the individual exposed but two or three generations down the line.

I knew that epigenetics existed. I knew that it was a controlling factor for DNA activity—whether genes are silenced or not—but to say that epigenetics would have a major role in disease development, so…I had no concept for that. The fact that this could have such a huge impact and could explain a whole host of things we couldn’t explain before took a while to actually sink in.

NARRATOR: Further work has revealed changed epigenetic marks in 25 segments of the affected rat’s DNA. The implications, if they apply to humans, are sobering.

The program description version of this extraordinary discovery is significantly downplayed and the name of the toxin is completely omitted from the program. It was vinclozolin (One of the most common fungicides utilized in agriculture).


Program Description

“In Washington State, a researcher finds that a toxin given to rats still affects their offspring four generations later, without producing any changes in their genes.”


Ghost in Your Genes – “Some NOVA programs are not for sale because the rights are not available.” I’m thinking that Dow Chemical owns the rights of this program. You will not find the specifics of Dr. Skinner’s research regarding pesticides and their impact on epigenetics and germ lines in the program description. (I personally don’t think that’s an accident.)

It is unbalanced in it’s emphasis on “lifestyle” choices but does cover Dr. Michael Skinner’s research on pesticide exposure and impacts on epigenetics and germ lines (transgenerational impacts). This episode also explores the research being completed on identical twins. This research is critical to understanding genetics versus environmental influence on genetic expression. This field of research will be critical to understanding the impacts from environmental toxins on human genes and the origin of disease, infertility, and birth defects. Epigenetics will show us the mechanisms through which toxins cause harm and alter us as a species.

I have discovered the transcripts!


Ghost in Your Genes – Transcripts

PBS Airdate: October 16, 2007
Go to the companion Web site

NARRATOR: Imagine sharing life with a person who seems to be you. Created from the same fertilized egg, you share exactly the same genes. So profound is their influence, everything about you appears the same: the spaces between your teeth, the way you laugh, your body language. You are, in a word, identical. Or are you?

SUSAN: As infants, they were very much alike. Their physical similarities are obvious. And all their physical milestones happened at the same time. But functioning today, for Jenna and Bridget…they’re completely different.

Jenna is enthusiastic, productive. Jenna’s going to college. She talks about it all the time now. Bridget is essentially non-verbal. She doesn’t have purposeful conversational speech. And there’s very unusual behavior. For example, she likes to spit on monitors and then rubs it in. I don’t know why, but that’s what she does.

How? How could these guys be identical and so, on such a different level, functioning-wise?

NARRATOR: So if genes don’t tell the whole story of who we are, then what does?

Scientists suspect the answer lies in a vast chemical network within our cells that controls our genes, turning them on and off.

ANDREW P. FEINBERG (Johns Hopkins University): It’s a little bit like the dark matter of the universe. I mean, we know it’s there, we know it’s terribly important, but we don’t really know all that much about how that symphony gets played out.

MARK MEHLER (Albert Einstein College of Medicine): We’re in the midst of probably the biggest revolution in biology that is going to forever transform the way we understand genetics, environment, the way the two interact, what causes disease. It’s another level of biology, which, for the first time, really, is up to the task of explaining the biological complexity of life.

NARRATOR: Ghost in Your Genes, up next on NOVA.

Major funding for NOVA is provided by the following:

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And by David H. Koch, and…

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And by the Corporation for Public Broadcasting, and by contributions to your PBS station from viewers like you. Thank you.

NARRATOR: In the early 1990s, the biggest project ever undertaken in biology captivated the world.

NEWS AUDIO: The Human Genome Project will be seen as the outstanding achievement in the history of mankind.

NARRATOR: The endeavor would reveal the chemical structure of each gene locked within our cells, the blueprint for life itself.

WOLF REIK (The Babraham Institute): The human genome is like a bible where everything was written down. The hope and the expectation was that once we had that book in front of us, and all the letters, we could just read down the pages and we would understand how the body was put together.

NARRATOR: Once the code was deciphered, scientists hoped to find the genetic cause and cure for every disease. They estimated that the human genome, the book of life, would contain around 100,000 genes.

MICHAEL SKINNER (Washington State University): And then when they started sequencing…and it popped down to 60. And then it popped down to 50. And, slowly, it went down to a much smaller number.

NARRATOR: Thirty thousand, twenty-five thousand…as the mapping drew to an end, it appeared that humans had about the same number of genes as fish and mice.

MICHAEL SKINNER: In fact, we found out that the human genome is probably not as complex and doesn’t have as many genes as plants do. So that, then, made us really question, “Well, if the genome has less genes in this species versus this species, and we’re more complex potentially, what’s going on here?”

NARRATOR: So few genes didn’t appear enough to explain human complexity. Even more startling, it turned out the same key genes that make a fruitfly, a worm or a mouse also make a human. Chimpanzees share 98.9 percent of our genome. So what accounts for the vast differences between species? Might genes not be the whole story?

Long before the genome was mapped, geneticists, like Marcus Pembrey, had caught hints of this possibility as they encountered baffling genetic conditions—Angelman syndrome, for instance.

MARCUS PEMBREY (University College London): …named after Harry Angelman, the pediatrician who first described Angelman syndrome. He referred to them as “happy puppet children,” because this described, to some extent, the features. They have a rather jerky sort of movement when they’re walking. These children have no speech; they are severely incapacitated in terms of learning but are uncharacteristically happy, and they’re smiling all the time.

NARRATION: The condition is caused by a genetic defect. A key sequence of DNA is deleted from chromosome 15.

MARCUS PEMBREY: Then we came across a paradox. At the same time, the same change, the same little deletion of chromosome 15, had been clearly associated with a quite different syndrome—much milder in terms of intellectual impairment—the Prader-Willi syndrome.

These children are characterized by being very floppy at birth, but once they started eating properly and so on, they then had an insatiable appetite and would get very, very large.

NARRATOR: Pembrey was stunned. Angelman syndrome and Prader-Willi syndrome, two completely different diseases, were caused by the same genetic abnormality.

MARCUS PEMBREY: So here we’re in a bizarre situation really. How could one propose that the same deletion could cause a different syndrome?

NARRATOR: As Pembrey looked at the inheritance pattern for the two conditions, he noticed something even stranger.

MARCUS PEMBREY: What really mattered was the origin of the chromosome 15 that had the deletion. If the deletion was on the chromosome 15 that the child had inherited from father, then you would have Prader-Willi syndrome, whereas if the deletion was inherited from the mother, you had the Angelman syndrome.

NARRATOR: It was a complete surprise that the same missing strip of DNA, depending upon its parental origin, could cause different diseases. It was as if the genes knew where they came from.

MARCUS PEMBREY: You’ve got a developing fetus manifesting this condition. How does the chromosome 15 know where it came from? There must have been a tag or an imprint placed on that chromosome, during either egg or sperm formation in the previous generation, to say, “Hi, I came from Mother.” “I came from Father, and we are functioning differently.” So that’s the key thing, that although the DNA sequence is the same, the different sets of genes were being silenced depending on whether it came from the mother or from the father.

NARRATOR: It was the first human evidence that something other than genes passed between generations. Something that could control genes directly, switch them on or off. But how exactly did these tags go about silencing a gene?

This odd strain of agouti mice provides a visual clue. Despite the difference in color and size, they’re twins, genetically identical. Both, therefore, have a particular gene, called agouti, but in the yellow mouse it’s switched on all the time.

RANDY JIRTLE (Duke University Medical Center): As a consequence, it inappropriately blocks a receptor in what’s called the satiation center of the brain, which tells mice and us when we’re full. So the yellow animals literally eat themselves into obesity, diabetes and cancer.

NARRATOR: So what switched the agouti gene off in the thin mouse? Exercise? Atkins? No, a chemical tag called a methyl molecule. Composed of carbon and hydrogen, it affixes near the agouti gene, shutting it down. Living creatures possess millions of tags like these. Some, like methyl molecules, attach to DNA directly. Other types grab the proteins called histones, around which DNA wraps, and tighten or loosen them to turn genes on or off.

JEAN-PIERRE ISSA (M.D. Anderson Cancer Center): And, in simple terms, this contact can be thought of as hugging the DNA. And if these proteins hug the DNA very tightly, then it is hidden from view for the cell. And a gene that is hidden cannot be utilized.

NARRATOR: These tags and others control gene expression through a vast network in the body called the epigenome.

RANDY JIRTLE: Epigenetics literally translates into just meaning above the genome. So if you would think, for example, of the genome as being like a computer, the hardware of a computer, the epigenome would be like the software that tells the computer when to work, how to work, and how much.

JEAN-PIERRE ISSA: Perhaps the best example of an epigenetic phenomenon…you’re actually looking at it. You see, skin and eyes and teeth and hair and organs all have exactly the same DNA. You cannot genetically tell my skin from my eyes or my teeth, yet you couldn’t really imagine that these are the same tissues.

NARRATOR: What distinguishes cells is not their genes, but how these genes are switched on or off by epigenetics.

WOLF REIK: And, as development unfolds, certain switches need to be thrown. And you can think of it as a light switch. Switch on the gene, the light is shining, the gene is active… makes the cell do a certain thing. Or the light switch is off, everything is dark. That gene is off.

And as the cells divide, the memory of whether it’s a liver cell or a brain cell, that’s brought about by these switches. And the switches are incredibly stable.

NARRATOR: But occasionally, some epigenetic switches can be flipped. To turn off the overactive agouti gene, researchers gave pregnant mothers foods rich in vitamins like B-12, or folic acid, from which they could make those methyl tags that silence genes.

The change was small, the effect huge. Fat yellow mothers gave birth to thin brown pups no longer prone to disease.

RANDY JIRTLE: This study, why it is so important is it opened the black box up and told us that this early stage of development—in the womb, basically—is linked to adult disease susceptibilities by, literally, tiny little changes in the epigenome.

NARRATOR: Agouti mice revealed the impact of an epigenetic change, one that occurred without altering a single chemical letter in the agouti gene.

It was increasingly clear that genes needed instructions for what to do, when and where. If the thousands of genes identified by the Human Genome Project symbolized the words in the book of life, it was the epigenome that determined how that book got read.

MARK MEHLER: We thought that by understanding the genetic code, we would understand life, disease, and then we’d all go home and be fine. But, in fact, the human genome project was just the beginning. What it did was it opened us up to this new world, getting us to the point where we’re understanding another level of biology which, for the first time, is up to the challenge of the biological complexity of life.

NARRATOR: If the epigenome controls the expression of our genes, could it solve the mystery of identical twins?

These rare individuals are living illustrations of the boundary point between nature and nurture. For, since their DNA is 100 percent the same, any difference should reveal the influence of the outside world.

Most identical twins appear so similar they seem the product of genes alone. Consider Javier and Carlos. Their every gesture seems the same. Or take Ana Mari and Clotilde, who show up in nearly the same red dress when, in fact, neither had a clue what the other was going to wear. They moved through life in symmetry.

CLOTILDE: When I see my sister, I see myself. If she looks good, I think, “I look pretty today.” But if she’s not wearing makeup, I say, “My god, I look horrible.”

NARRATOR: But, five years ago, symmetry appeared to break. Ana Marie was diagnosed with cancer and Clotilde was not.

CLOTILDE: I’ve been told that I am a high risk for cancer. Damocles’ sword hangs over me.

NARRATOR: In fact, it’s not unusual for one twin to get a dread disease while the other does not. But how? How can two people so alike, be so different?

Intrigued by the mystery, Spanish geneticist Manel Esteller set out, in 2005, to find the answer to that question.

MANEL ESTELLER (Spanish National Cancer Center): One of the questions of twins is, “If my twin has this disease, I will have the same disease?” And genetics tell us that there is a high risk of developing the same disease. But it’s not really sure they are going to have it, because our genes are just part of the story.

NARRATOR: Esteller suspected epigenetics was the rest. To find out, he and his team collected cells from 40 pairs of identical twins, age three to 74.

Then began the meticulous process of dissolving the cells until all that was left were the wispy strands of DNA, the master molecule that contains our genes. Next, researchers amplified fragments of the DNA, revealing both the genes and their epigenetic tags.

Those that had been turned off appear as dark pink marks on the gel. Now, notice what happens when these genes are cut out and overlapped. The epigenetic effects stand out, especially when you contrast the genes of two sets of twins who differ in age.

Here, on the left, is the overlapped DNA of six-year-old Javier and Carlos. The yellow indicates where their genes are functioning identically.

On the right, is the DNA of 66-year-old Ana Mari and Clotilde. In contrast to the younger twins’, hardly any yellow shines through. Their genome may be the same, their epigenome clearly is not.

Identical genes active in one twin maybe shut down in the other. Thus, as the years pass, epigenetic changes accumulate in twins, as in the rest of us.

MANEL ESTELLER: One of the main findings of our research is that epigenomes can change in function of what we eat, of what we smoke or what we drink. And this is one of the key differences between epigenetics and genetics.

NARRATOR: But why does the epigenome change, when the genome does not?

In Montreal, scientists Michael Meaney and Moshe Szyf believe the question contains its own answer.

MOSHE SZYF (McGill University): We have this very, very static genome, very hard to change. It could be only changed by really dramatic things like nuclear explosions or, you know, hundreds of thousands of years of evolution. On the other hand, we have the dynamic environment that changes all the time. And so what there is here is an interface between the highly dynamic world around us and the highly static genome that we have. Epigenome is an in-between creature, built in a way, to respond to changes around us.

NARRATOR: Szyf and Meaney believe that experience itself changes the epigenome. To reach this startling conclusion they studied two kinds of rats: those born to nurturing mothers who licked and groomed them intensely after birth, and those born to mothers who took a more paws-off approach.

MICHAEL MEANEY (Douglas Institute/McGill University): What we were particularly interested in is the way in which these animals might respond to stressful events. And we found the offspring of low-licking mothers, during periods of stress, show greater increases in blood pressure and greater increases in stress hormone production.

MOSHE SZYF: They will scream. They will try to bite you. Just walking into their cage, those rats will respond differently.

NARRATOR: To rule out a genetic cause, high-licking mothers were given the babies of low-licking ones and vice versa. Once again, the less-nurtured pups grew up markedly different, and not only on blood tests.

MOSHE SZYF: So the conclusion from that is, it’s not the genes that the mother brings into the game. It is the behavior of the mother that has an impact on the offspring years after the mother is already gone. And the basic question was, “How does the rat remember what kind of care it received from its mother, so that it now has better or worse health conditions?”

And we reasoned that there must be some mark in genes that marks that memory.

NARRATOR: But could such a mark, capturing memory, be found? The researchers focused on a gene which lowers the levels of stress hormones in the blood. It’s active in a part of the rat’s brain called the hippocampus. By extracting and analyzing the gene, they could compare how its activity varied between low- and high-licked rats.

The difference was striking. Less nurtured rats had multiple epigenetic marks silencing the gene.

The result? With the gene less active, stress levels in neglected rats soared. In stark contrast, nurtured rats could better handle stress because they had nothing dimming the genes’ activity.

MOSHE SZYF: The maternal behavior essentially sculpted the genome of their babies. We looked at one gene; we know hundreds of genes were changed. But for me, it was a fantastic thing that just a behavior of one subject can change the gene expression in a different subject.

NARRATOR: The most surprising phase of the experiment, however, was yet to come. Szyf and Meaney injected anxious rats with a drug known to remove epigenetic marks.

MOSHE SZYF: And as we injected the drug, the gene turned on. And when it turned on, the entire behavior of the rat changed. It became less anxious. Also, it responded to stress like a normally-reared rat. And we looked at the way that gene was marked in the brain, and we saw that we actually changed the epigenetic marking of that gene.

NARRATOR: Although the work has yet to be replicated, it appears that Szyf and Meaney have linked personality traits, albeit in a rat, to the epigenome.

Could this have implications for humans? We will not know until the completion of a 10-year study, now underway, that will look at children from both nurturing and neglected backgrounds.

But even now, says Meaney, we have clues that our own upbringings produce the same effects.

MICHAEL MEANEY: If you grow up in a family that involves abuse, neglect, harsh and inconsistent discipline, then you are statistically more likely to develop depression, anxiety, drug abuse. And I don’t think that surprises anyone. But what is interesting is that you are also more likely to develop diabetes, heart disease and obesity. And the stress hormones actively promote the development of these individual diseases.

MOSHE SZYF: So, one day, we’ll be able, perhaps, to chart the pathway from child abuse to changes in the way certain genes are epigenetically marked in the brain that unfortunately affect our health years later in life.

NARRATOR: This work is controversial. Still, many scientists now believe that epigenetic changes in gene expression may underlie human diseases.

Take a disorder like M.D.S., cancer of the blood and bone marrow. It’s not a diagnosis you would ever want to hear.

SANDRA SHELBY (Medical patient): When I went in, he started patting my hand and he was going, “Your blood work does not look very good at all,” and that I had M.D.S. leukemia, and that there was not a cure for it, and, basically, I had six months to live.

NARRATOR: With no viable treatment, Sandra entered a clinical trial experimenting with epigenetic therapy. It was the result of a radical new way of thinking about the causes of diseases like cancer.

JEAN-PIERRE ISSA: If one has a genetic basis of cancer in mind, then one is simply asking, “What causes genetic damage?” Cigarette smoking, certain types of environmental exposures and radiation causes genetic damage. But, now if I come in and say, “Well, wait a minute, epigenetic damage can also cause cancer,” then you’ve got to ask “Well, why does this come about?”

NARRATOR: The trouble begins, believes Issa, when our stem cells, the master cells that create and replace our tissues, overwork.

JEAN-PIERRE ISSA: Every time a stem cell has to repair injury, it is aging a little more. And because each time a stem cell divides there is a finite chance of some sort of epigenetic damage, what we find is that in older people there’s been an accumulation of these epigenetic events that is easily measurable in DNA.

Now where does the cancer angle come from? Well, if you count age as how many times a stem cell has divided, then cancers, which copy themselves tirelessly, are awfully old tissues.

NARRATOR: As epigenetic errors pile up, the switches that turn genes on and off can go awry, creating havoc within the cell.

ANDREW FEINBERG: There are genes that help to prevent tumors that are normally active that epigenetically become silenced. Those are called tumor suppressor genes. And there are other genes, called oncogenes, that stimulate the growth of tumors. And then the tags, such as the methylation tags, come off those genes, and those genes become activated. So both ways, turning on and turning off, is a way of getting epigenetic disease.

NARRATOR: But could misplaced tags be rearranged? In 2004, Sandra and other patients began taking a drug to remove methyl tags silencing their tumor suppressor genes.

ROY CANTWELL (Medical patient): Your number one thing is, “Okay, is it going work?” And when you know that before this there was nothing, then yeah, it makes you pretty happy that there is a chance to go forward in your life.

NARRATOR: Ironically, the drug, decitabine, was tried in conventional chemotherapy in the 1970s and deemed too toxic. Today, Issa is giving his patients a dose 20 to 30 times lower.

JEAN-PIERRE ISSA: The idea of epigenetic therapy is to stay away from killing the cell. Rather, what we are trying to do is diplomacy, trying to change the instructions of the cancer cells, reminding the cell, “Hey, you’re a human cell. You shouldn’t be behaving this way.” And we try to do that by reactivating genes.

SANDRA SHELBY: The results have been incredible. And I didn’t have, really, any horrible side effects.

ROY CANTWELL: I am in remission, and going in the plus direction is a whole lot better than the minus direction.

NARRATOR: Roy has not been cured, but he has been cancer-free for two years. And he is not alone.

JEAN-PIERRE ISSA: Spectacular results—complete disappearance of the disease—can be seen in almost half of the patients that receive this drug. And 20 years ago we wouldn’t have dreamed that a drug that affects DNA methylation could have such a profound effect on patients.

NARRATOR: As epigenetic therapy takes off, so do the expectations for this new science. Many believe that a multitude of complex diseases, from Alzheimer’s to autism, may have epigenetic triggers.

Consider autism, a mysterious disorder characterized by social withdrawal. This is Bridget. She passes her day running her fingers across her computer screen. Locked in her own world, she has spent the past 13 years drifting apart from her identical twin sister, Jenna.

SUSAN : As infants, they were very much alike. Their physical similarities are obvious. And all their physical milestones happened at the same time. And then, at their first birthday party, we had a big party at the house, lots of balloons, lots of people. And I remember watching Bridget maneuver around the house as if there were nobody there. She was fixated on a balloon, which a lot of babies would be, but something struck me that she was not in tune with everybody that was there.

NARRATOR: Bridget was eventually diagnosed with severe autism. As the girls developed, so did their differences.

SUSAN : Functioning today for Jenna and Bridget…they’re completely different. Jenna is enthusiastic, productive, you know? Jenna’s going to college, talks about it all the time now. Bridget is essentially non-verbal. She doesn’t have purposeful conversational speech, so everything she does say is very memorized because she was taught over and over again.

Do you want grilled cheese?

BRIDGET: Grilled cheese?

SUSAN: Yes or no?

BRIDGET: Yes or no? No.

SUSAN: No?

BRIDGET: Yes, grilled cheese. Yes.

SUSAN: You want grilled cheese, yes?

BRIDGET: Yes.

SUSAN: Good.

There’s no prescription that you get when your child’s diagnosed with autism.

And new things come that weren’t there before, new behaviors that are very problematic, that interfere with her ability to learn anything. So we don’t know, really, what the prognosis is.

NARRATOR: And, for a long time, doctors couldn’t really help. Despite millions of dollars and years of searching, no single definitive autism gene had been found.

But about a decade ago, scientists at the Kennedy Krieger Institute, in Baltimore, turned their high power imagers on the problem. They scanned the brains of both healthy and autistic children, searching for a biological cause of the disorder. One of the researchers involved was Walter Kaufmann.*

WALTER KAUFMANN (Kennedy Krieger Institute): For a long time, people questioned whether autism was a real entity, because the ways to diagnose autism had been behavior…behavioral abnormalities. And those sometimes are difficult to identify in a consistent and reliable way.

NARRATOR: But, in comparing the brain scans of identical twins discordant for autism, Kaufman finally saw the definitive data he was searching for: an area in the brain linked to learning, memory and emotions—called the hippocampus—was smaller in the twin with severe autism. But how could the same genes create different brain structures? Kaufmann asked Andy Feinberg at Johns Hopkins University.

ANDREW FEINBERG: And suddenly we were able to form an epigenetic hypothesis. And that hypothesis is that they have the same genome, but one of them maybe has an epigenetic change that’s leading to a difference in their brain that you don’t see in the other twin.

NARRATOR: Kaufmann and Feinberg are now searching for methyl marks in the DNA of identical twins discordant for autism. The work has just begun, but the hope is that by finding identical genes that differ in their expression, some causes of autism may emerge.

WALTER KAUFMANN: We know environmental stimulation, sensory stimulation, auditory, visual stimulation, have an impact on brain development and brain function. And this impact we know now is mediated, at least in part, by epigenetic mechanisms.

ANDREW FEINBERG: Epigenetic changes…generally they stand at the cornerstone between our genome—in other words, all of our genes, the development of the cells of our body—and the environment that we live in.

NARRATOR: So the environment molds our epigenomes. But might it do more?

At the far speculative edge of this new science, some are seeing evidence of an astonishing possibility, that genes may not be all that passes from generation to generation.

The evidence comes from this Swedish village huddled on the Arctic Circle. Overkalix stands out for one reason, its archives.

Olov Bygren, a Swedish public health expert, has been studying them from more than 20 years. What makes these records unique is their detail. They track births and deaths over centuries—and harvests. This is significant because, in years past, Overkalix’s location left it particularly vulnerable to crop failures and famines.

LARS OLOV BYGREN (The University of Umeå): In the 19th century this was a very isolated area. They could not have help from outside. As it was so poor, they really had a hard time when there was a famine, and they really had a good, good time when the harvests were good.

NARRATOR: Bygren was studying the connection between poor nutrition and health when he stumbled on something curious.

It appeared that a famine might affect people almost a century later, even if they had never experienced a famine themselves. If so, past and future generations might be linked in ways no one had imagined.

Wondering if epigenetics might explain the phenomenon, Bygren sent his research to geneticist Marcus Pembrey.

MARCUS PEMBREY: I was terribly excited to get this, completely out of the blue. And for the first time it seemed that there was some data that we could then start to explore, so that was the beginning of our collaboration.

NARRATOR: Overkalix offered Pembrey a unique opportunity to see if the events that happened in one generation could affect another decades later.

MARCUS PEMBREY: Olly first reported that the food supply of the ancestors was affecting the longevity or mortality rate of the grandchildren, so I was very excited. I responded immediately.

NARRATOR: Pembrey suspected the incidence of one disease, diabetes, might show that the environment and epigenetics were involved. So Olov trawled the records for any deaths due to diabetes and then looked back to see if there was anything unusual about the diet of their grandparents.

MARCUS PEMBREY: A few months later he emailed me to say that indeed they had shown a strong association between the food supply of the father’s father and the chance of diabetes being mentioned on the death certificate of the grandchild.

NARRATOR: In fact, a grandson was four times more likely to die from an illness related to diabetes if his grandfather had plenty of food to eat in late childhood.

This was one of the first indications that an environmental exposure in a man, one that did not cause a genetic mutation, could directly affect his male offspring.

MARCUS PEMBREY: It really did look as if there was some new mechanism transmitting environmental exposure information from one generation to the next.

NARRATOR: Because these ideas were so heretical, Pembrey knew the results could be dismissed as nothing more than a curiosity. To bolster the research, he needed to find out how a trans-generational effect impacted each sex and if it was linked to a specific period of development.

MARCUS PEMBREY: We wanted to tease out when you could trigger, in the ancestor, a trans-generational response.

NARRATOR: So he and Bygren went back to the data. The more they looked, the more patterns started to appear.

MARCUS PEMBREY: We were able to look at the food supply every year, in the grandfather and the grandmother, from the moment they were conceived right through to the age of 20. We found that there are only certain periods in the ancestors’ development when they can trigger this trans-generational response. They’re what one might call sensitive periods of development.

NARRATOR: They discovered that when a famine was able to trigger an effect was different for the grandmother than the grandfather. The grandmother appeared susceptible while she, herself, was still in the womb, while the grandfather was affected in late childhood.

MARCUS PEMBREY: And the timing of these sensitive periods was telling us that it was tied in with the formation of the eggs and the sperm.

NARRATOR: This suggested what might be happening. Perhaps environmental information was being imprinted on the egg and sperm at the time of their formation.

At last a sharper picture was beginning to emerge. The next step was to compile their findings. Bygren drew up a rough diagram and sent it to Pembrey.

MARCUS PEMBREY: Hand-drawn…this is what Olly sent me, you know he was too excited to wait for the thing to be drawn out properly. You know, he sent me the data, and, in fact, I was recovering from having something done on my heart, so he sent it saying, you know, “I hope this helps you get better quickly,” you know? Because it was so exciting.

NARRATOR: When Pembrey looked at the diagram, he was immediately struck by seemingly bizarre connections between gender, diet and health, connections that were most pronounced two generations later. Men, for example, who experienced famine at around age 10, had paternal grandsons who lived much longer than those whose grandfathers experienced plenty. Yet, women who experienced famine while in the womb, had paternal granddaughters who died on average far earlier.

MARCUS PEMBREY: Once I had plotted out the full extent of those results, it was so beautiful and such a clear pattern, I knew then, quite definitely, that we were dealing with a trans-generational response. It was so coherent—and that’s important in science, that the effect was coherent in some way—was tying in when eggs and sperm were being formed.

NARRATOR: The diagram showed a significant link between generations, between the diet in one and the life expectancy of another.

OLOV BYGREN: When you think that you have found something important for the understanding of the seasons itself, you can imagine that this is something really special.

MARCUS PEMBREY: This is going to become a famous diagram, I’m convinced about that. I get so excited every time I see it. It’s just amazing. Every time I look at it, I find it really exciting. It’s fantastic.

NARRATOR: Much about these findings puzzles researchers. Why, for instance, does this effect only appear in the paternal line of inheritance? And why should famine be both harmful and beneficial, depending on the sex and age of the grandparent who experiences it?

Nonetheless, it raises a tantalizing prospect: that the impact of famine can be captured by the genes, in the egg and sperm, and that the memory of this event could be carried forward to affect grandchildren two generations later.

MARCUS PEMBREY: We are changing the view of what inheritance is. You can’t, in life, in ordinary development and living, separate out the gene from the environmental effect. They’re so intertwined.

NARRATOR: Pembrey and Bygren’s work suggests that our grandparents’ experiences effect our health. But is the effect epigenetic? With no DNA yet analyzed, Pembrey can only speculate. But in Washington state, Michael Skinner seems to have found compelling additional evidence by triggering a similar effect with commonly used pesticides. Skinner wanted to see how these chemicals would affect pregnant rats and their offspring.

MICHAEL SKINNER: And so I treated the animals, the pregnant mother, with these compounds, and then we started seeing, between six months to a year, a whole host of other diseases that we didn’t expect. And this ranged between tumors, such as breast and skin tumors, prostate disease, kidney disease and immune dysfunction.

NARRATOR: He checked that there were no genetic mutations and then proceeded to breed the rats.

MICHAEL SKINNER: The next step was for us to go to the next generation. And the same disease state occurs. So after we did several repeats, and got the third generation showing it and then a fourth generation, we sat back and realized that the phenomenon was real. We started seeing these major diseases occur in approximately 85 percent of all the animals of every single generation.

NARRATOR: His discoveries were a revelation.

MICHAEL SKINNER: We knew that if an individual was exposed to an environmental toxin that they can get a disease state, potentially. The new phenomenon is that environmental toxin no longer affects just the individual exposed but two or three generations down the line.

I knew that epigenetics existed. I knew that it was a controlling factor for DNA activity—whether genes are silenced or not—but to say that epigenetics would have a major role in disease development, so…I had no concept for that. The fact that this could have such a huge impact and could explain a whole host of things we couldn’t explain before took a while to actually sink in.

NARRATOR: Further work has revealed changed epigenetic marks in 25 segments of the affected rat’s DNA. The implications, if they apply to humans, are sobering.

MICHAEL SKINNER: What this means, then, is what your grandmother was exposed to when she was pregnant could cause a disease in you—even though you’ve had no exposure—and you’re going to pass it on to your great-grandchildren.

NARRATOR: And if a pesticide can generate such effects, what about stress, smoking, drinking? To some, the epigenetic evidence is compelling enough already to warrant a public note of caution.

RANDY JIRTLE: We’ve got to get people thinking more about what they do. They have a responsibility for their epigenome. Their genome they inherit. But their epigenome, they potentially can alter, and particularly that of their children. And that brings in responsibility, but it also brings in hope. You’re not necessarily stuck with this. You can alter this.

NARRATOR: Might our lifestyle choices resonate down the ages, effecting people yet unborn? Such ideas remain, to say the least, controversial. But one thing many in the field can agree upon is the need to take a cue from the Human Genome Project and launch a similar effort, this time to decipher the epigenome.

ANDREW FEINBERG: Mapping the human epigenome is the most important thing that we could do right now, as a big project in science, because it will tell us some very important things about why organisms function the way they do, why cells have the behavior they do.

JEAN-PIERRE ISSA: We now know how many genes we have, but we really don’t know how they are regulated inside the cells. And mapping the epigenome will give a much better understanding of this particular process.

NARRATOR: The hurdle is that, unlike the genome, which is the same in every cell, the epigenome varies from tissue to tissue, between individuals and over time.

MARK MEHLER: If you thought sequencing the human genome took years and was difficult, you’re talking about levels of complexity that will dwarf anything we knew about the human genome. But it’s crucial, it’s essential. That’s the way that the future is going to unfold. So in a sense, the Human Genome Project was just the beginning.

NARRATOR: The end may be the realization that the code of life is more complex and interactive than we ever imagined.

MARCUS PEMBREY: I’ve thought of nothing else really for the last five years.

It is said, the first time, you know, one had a photograph of the Earth, you know this sort of delicate thing sailing through the universe, you know, it had a huge effect on the sort of “save-the-planet” type of feeling, you know? I’m sure that’s part of why the future generation think in a planetary way, because they’ve actually seen that picture, you know? And this might be the same. It may get to a point where they realize that you live your life as a sort of guardian of your genome. It seems to me you’ve got to be careful of it because it’s not just you. You can’t be selfish because you can’t say, “Well I’ll smoke,” or “I’ll do whatever it is because I’m prepared to die early.” You’re also looking after it for your children and grandchildren. It is changing the way we think about inheritance forever.

*This study was led by the Kennedy Krieger Institute’s Dr. Wendy Kates, who is now (late 2007) at the State University of New York at Upstate Medical University. NOVA would like to thank Dr. Kates for providing access to the autistic twins featured in the program.

On the Ghost in Your Genes Web site, an expert answers viewer questions about the growing field of epigenetics and its potential. Find it on PBS.org.

Educators and other educational institutions can order this or other NOVA programs for $19.95 plus shipping and handling. Call WGBH Boston Video at 1-800-255-9424.

NOVA is a production of WGBH Boston.

Major funding for NOVA is provided by the following:

For each of us, there is a moment of discovery. We understand that all of life is elemental, and as we marvel at element bonding with element, we soon realize that when you add the human element to the equation, everything changes. Suddenly all of chemistry illuminates humanity, and all of humanity illuminates chemistry. The human element: nothing is more fundamental, nothing more elemental.

And by David H. Koch, and…

Discover new knowledge: HHMI.

And by the Corporation for Public Broadcasting, and by contributions to your PBS station from viewers like you. Thank you.

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Autism & Alzheimer’s

I watched the Nova Program called, “Ghost in Your Genes,” from HHMI – well four times. I keep thinking about the studies on identical twins where one twin has severe autism and the other does not have any symptoms (Identical twins discordant for autism). This means that it is not genetics itself or both twins would have developed autism. I was reading how twins are more likely to both have autism but that would make sense because most twins have the same environmental exposures. The studies on twins discordant for autism are critically important. Walter Kaufmann was the researcher. I wanted to review his findings.

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