©Mehau Kulyk/Photo Researchers, Inc
Toward the end of World War II, a German-imposed food embargo in western Holland – a densely populated area already suffering from scarce food supplies, ruined agricultural lands, and the onset of an unusually harsh winter – led to the death by starvation of some 30,000 people. Detailed birth records collected during that so-called Dutch Hunger Winter have provided scientists with useful data for analyzing the long-term health effects of prenatal exposure to famine. Not only have researchers linked such exposure to a range of developmental and adult disorders, including low birth weight, diabetes, obesity, coronary heart disease, breast and other cancers, but at least one group has also associated exposure with the birth of smaller-than-normal grandchildren.1 The finding is remarkable because it suggests that a pregnant mother's diet can affect her health in such a way that not only her children but her grandchildren (and possibly great-grandchildren, etc.) inherit the same health problems.
In another study, unrelated to the Hunger Winter, researchers correlated grandparents' prepubertal access to food with diabetes and heart disease.2 In other words, you are what your grandmother ate. But, wait, wouldn't that imply what every good biologist knows is practically scientific heresy: the Lamarckian inheritance of acquired characteristics?
If agouti mice are any indication, the answer could be yes. The multicolored rodents make for a fascinating epigenetics story, which Randy Jirtle and Robert Waterland of Duke University told last summer in a
This type of inheritance, the transmission of non-DNA sequence information through either meiosis or mitosis, is known as epigenetic inheritance. From the Greek prefix
Because a genome can pick up or shed a methyl group much more readily than it can change its DNA sequence, Jirtle says epigenetic inheritance provides a "rapid mechanism by which [an organism] can respond to the environment without having to change its hardware." Epigenetic patterns are so sensitive to environmental change that, in the case of the agouti mice, they can dramatically and heritably alter a phenotype in a single generation. If you liken the genome to the hardware of a computer, Jirtle explains, then "epigenetics is the software. It's the grey area. It's just so darn beautiful if you think about it."
Courtesy of Museum Online
Jean-Baptiste Lamarck (1744–1829) is best remembered for a discredited theory of heredity, the "inheritance of acquired traits." He proposed that environment changes caused changes in behavior which in turn led to the increase or decrease of particular structures. Lamarck had a colorful and distinguished career: in turns soldier, bank clerk, Professor of "insects and worms" he died a poor man and was buried in a rented grave.
The environmental lability of epigenetic inheritance may not necessarily bring to mind Lamarckian images of giraffes stretching their necks to reach the treetops (and then giving birth to progeny with similarly stretched necks), but it does give researchers reason to reconsider long-refuted notions about the inheritance of acquired characteristics. Eighteenth-century French naturalist Jean Baptiste de Lamarck proposed that environmental cues could cause phenotypic changes transmittable to offspring. "He had basically good idea but a bad example," says Rohl Oflsson, Uppsala University, Sweden.
Although the field of epigenetics as it is known today (that is, the study of heritable changes in gene expression and regulation that have little to do with DNA sequence) has been around for only 20 years or so, the term epigenetics has been in use since at least the early 1940s. Developmental biologist Conrad Waddington used it back then to refer to the study of processes by which genotypes give rise to phenotypes (in contrast to genetics, the study of genotypes). Some reports indicate that the term is even older than Waddington, dating back to the late 1800s. Either way, early use of the term was in reference to developmental phenomena.
In 2001, Joshua Lederberg proposed the use of more semantically, or historically, correct language.4 But it appears that today's use of the term is here to stay, at least for now, as are its derivatives: epiallele (genes with different degrees of methylation), epigenome (the genome-wide pattern of methyl and other epigenetic markers), epigenetic therapy (drugs that target epigenetic markers), and even epigender (the sexual identity of a genome based on its imprinting pattern).
Terminology aside, biologists have long entertained the notion that certain types of cellular information can be transmitted from one generation to the next, even as DNA sequences stay the same. Bruce Stillman, director of Cold Spring Harbor Laboratory (CSHL), NY, traces much of today's research in epigenetics back to Barbara McClintock's discovery of transposons in maize. Methyl-rich transposable elements, which constitute over 35% of the human genome, are considered a classical model for epigenetic inheritance. Indeed, the epigenetic lability of Jirtle's agouti mice is due to the presence of a transposon at the 5' end of the agouti gene. But only over the past two decades has the evidence become strong enough to convince and attract large numbers of epigenetics researchers. " [Epigenetics] has very deep roots in biology," says Stillman," but the last few years have been just an explosion in understanding."
METHYLATION AND MORE
One of the prominent features of DNA methylation is the faithful propagation of its genomic pattern from one cellular or organismal generation to the next. When a methylated DNA sequence replicates, only one strand of the next-generation double helix has all its methyl markers intact; the other strand needs to be remethylated. According to Massachusetts Institute of Technology biologist Rudy Jaenisch, the field of epigenetics took its first major step forward more than two decades ago when, upon discovering DNA methyltransferases (DMTs, the enzymes that bind methyl groups to cytosine nucleotides), researchers finally had a genetic handle on how epigenetic information was passed along. Now, it is generally believed that DMTs bind methyl groups to the naked cytosines based on the methylation template provided by the other strand. This is known as the maintenance methylase theory.
THE EPIGENOME IS REPROGRAMMED DURING DEVELOPMENT:
©The American Society of Human Genetics
Erasure of epigenetic marks, including DNA methylation and genomic imprinting, occurs as primordial germ cells migrate along the genital ridge. Reestablishment of mark takes place during gametogenesis, differentially in sperm (blue) and egg (pink). After fertilization another round of erasure occurs – apart from imprinted genes (dotted line), which are protected – followed by tissue-specific patterning. (Reprinted with permission,
But even a decade ago, says Wolf Reik of the Babraham Institute, Cambridge, UK, "a lot of epigenetics was phenomenology, and so people looked at it and said, well, this is all very interesting, but what's the molecular mechanism?" Reik points to recent evidence suggesting a critical link between the two main types of epigenetic regulation, DNA methylation and histone modification, as one of the most interesting recent developments in the field. Because of that link, researcher Eric Selker and colleagues at the University of Oregon, Portland, have proposed that there may be more to methylation propagation than maintenance, despite 25 years of evidence. In 2001, Selker and coauthor Hisashi Tamaru showed that
In a study by CSHL researchers Robert Martienssen, Shiv Grewal, and colleagues, evidence suggests that histone modifications are, in turn, guided by RNA interference (RNAi).6 Using the fission yeast
EPIGENETICS AND DISEASE
More than two decades ago, anyone who proposed that epigenetic regulation played a role in carcinogenesis was a "lone prophet in the desert," explains Jaenisch. Researchers didn't seriously entertain the notion until Andy Feinberg and Bert Vogelstein, both at Johns Hopkins University, reported a link between human cancer cells and aberrant DNA methylation patterns.7 Even then, Feinberg says "the initial reaction was disbelief. I think that people ignored it. Now, everyone accepts that epigenetics is important in cancer." The etiological link between epigenetic change and cancer has fueled both academic and pharmaceutical interest in the field.
Methylation usually silences gene expression. Normally, about 70% of all CpG dinucleotides in the mammalian genome are methylated. The remainder, clusters near the 5' end of genes known as CpG islands, are protected from it. Too little methylation across the genome or too much methylation in the CpG islands can cause problems, the former by activating nearby oncogenes, and the latter by silencing tumor suppressor genes. When Feinberg and Vogelstein linked cancer to epigenetics in the early 1980s, they linked it specifically to genome-wide hypomethylation. A few years later, German and US research teams discovered connections between cancer and tumor-suppressing silencing caused by hypermethylation. Both hypo- and hypermethylation can play significant regulatory roles even in the same tumor.
SAME GENOME, DIFFERENT EPIGENOME:
© 2003 American Society for Microbiology
Variability in CpG methylation at the agouti locus causes differences in coat color among genetically identical mice. Maternal nutrition affects the phenotype of offspring by influencing the degree of CpG methylation at the agouti locus. (Reprinted with permission,
It has taken more than correlations between methylation and cancer, however, to convince researchers that epigenetics is the cause, not consequence, of malignancy. Feinberg points to two pieces of evidence that have pushed epigenetics to the fore. First, several independent observations of epigenetic aberrations (specifically, impaired methylation patterns) in normal cells surrounding tumorous tissue suggest that epigenetic abnormalities are not simply an epiphenomenon of the cancer phenotype, as has been argued. But the real "smoking gun for epigenetics," says Feinberg, has been the detection of a clear causal link between Beckwith-Wiedemann syndrome (BWS) and a particular cluster of imprinted genes, which include the insulin-like growth factor II gene,
Imprinting is the differential methyl tagging and expression of genes depending on whether they came from the mother or father.
The etiological role of epigenetics in tumor formation has prompted efforts to create antitumor drugs that correct disrupted epigenetic inheritance. So far, says Jaenisch, no one has succeeded, although the Food and Drug Administration approved the epigenetic inhibitor azacitidine on May 19, for the treatment of the bone disorder myelodysplastic syndrome. The drug reportedly turns on genes that have been silenced by epigenetic methylation.
Epigenetic inheritance has been associated with a number of other human health conditions, including some whose incidence is higher among babies born with the aid of assisted reproduction technology (ART). As Reik explains, embryos normally develop in a protective environment, the womb. When they are put into the suboptimal environment of a culture dish, many things can go wrong. Methylation sites initially established in the oocyte may not be maintained properly, and imprinting patterns may be lost during development. Individuals conceived by ART techniques have a higher risk of being born with BWS, Angelman syndrome (AS), and retinoblastoma (a tumor of the retina). Like BWS, AS has been linked to imprinting errors. Typical features of babies born with AS include developmental delay, absent speech development, and seizures.
Epigenetic inheritance also may be the reason that human cloning is all but impossible. Indeed, Jaenisch considers cloning "the ultimate bioassay for epigenetic changes." When a differentiated somatic cell is put into an oocyte, its genome-wide epigenetic pattern must be reprogrammed in order to restore totipotency. The difficulties associated with reprogramming all the chromatin, histones, and methylation patterns along the entire length of the DNA sequence may explain why so many cloned embryos have so many developmental failures.
INHERITANCE OF METHYLATION STATES:
Replication of methylated DNA (a) results in hemimethylated progeny DNA(b). Maintenance methylation enzymes, such as DNA methyltransferases, methylate cytosine (c) based on the hemimethylated states of symmetric CG or CXG motifs. C and G represent cytosine and guanine, respectively, whereas X is any nucleotide. Other mechanisms of methylation inheritance are also known. (Reprinted with permission,
Normally, the fur of agouti mice is yellow, brown, or a calico-like mixture of the two, depending on the number of attached methyl groups. But when Duke University researchers Jirtle and Waterland fed folic acid and other methyl-rich supplements to pregnant mothers, despite the fact that all offspring inherited exactly the same agouti gene (i.e., with no nucleotide differences), mice who received supplements had offspring with mostly brown fur, whereas mice without supplements gave birth to mostly yellow pups with a higher susceptibility to obesity, diabetes, and cancer. The methyl groups bound to a transposon at the 5' end of the agouti locus, thereby shutting off expression of the agouti gene, not just in the murine recipient but in its offspring as well.
MORPHOLOGICAL EVOLUTION THROUGH AN EPIGENETIC MECHANISM:
Courtesy of Douglas Ruden
Light photomicrograph of the head of a fruitfly with epigenetically-induced ectopic bristles in the eyes.
Although the study demonstrates that, at least in mice, folic acid supplementation in pregnant mothers reduces the risk of their babies having certain health problems, Jirtle warns that the results cannot be extrapolated to humans. "Mice are not men," he emphasizes. But he doesn't downplay the proof of principle. The take-home message is not that folic acid supplements are a good thing. Rather, environmental factors such as nutritional supplementation can have a dramatic impact on inheritance, not by changing the DNA sequence of a gene or via single-nucleotide polymorphism, but by changing the methylation pattern of that gene. "It's a proof of concept," says Donata Vercelli, University of Arizona, Tucson. "That's why it's so important."
According to Vercelli, the environmental susceptibility of epigenetics probably explains why genetically identical organisms such as twins can have dramatically different phenotypes in different environments. She points to the agouti mice, as well as another recent cluster of studies on a heat shock protein, Hsp90, in
Hsp90 regulates developmental genes during times of stress by releasing previously hidden or buffered phenotypic variation. Douglas Ruden of the University of Alabama, Tuscaloosa, says he noticed some weird fruit fly phenotypes – things like appendage-like organs sticking out of their eyes – at about the same time that a paper appeared in
When the Lindquist paper came out, Ruden says he thought, "Ah, I'm probably seeing the same thing." He was doing some crosses, "and I started to see this weird phenotype." But Ruden and collaborators concluded that their strange eye phenotype was due to something other than, or in addition to, the sudden unleashing of hidden genetic variation.9 Indeed, the researchers used a strain of flies that had little genetic variation, and yet was still capable of responding to 13 generations of selection even after normal Hsp90 activity was restored. Because of the genomic homogeneity of their flies, combined with observations that mutations encoding chromatin-remodeling proteins induced the same abnormal eye phenotype, the investigators concluded that reduced levels of Hsp90 affected the phenotype by epigenetically altering the chromatin.
Although it is hard to imagine that an appendage-like structure sticking out of the eye would be adaptive in times of stress, Vercelli says that epigenetic change clearly can be environmentally induced in a heritable manner, in this case by alterations to Hsp90. The morphological variations in the eye were probably only the most obvious of many phenotypic differences caused by the chromatin changes.
In a written commentary, evolutionary biologist Massimo Pigliucci said that Ruden's experiment was "one of the most convincing pieces of evidence that epigenetic variation is far from being a curious nuisance to evolutionary biologists."10 Pigluicci doesn't go so far as to say that the heritable changes caused by Hsp90 alterations are Lamarckian, but Ruden does. "Epigenetics has always been Lamarckian. I really don't think there's any controversy," he says.
Not that Mendelian genetics is wrong; far from it. The increased understanding of epigenetic change and the recent evidence indicating its role in inheritance and development doesn't give epigenetics greater importance than DNA. Genetics and epigenetics go "hand in hand," says Ohlsson. In the case of disease, says Reik, "there are clearly genetic factors involved, but there are also other factors involved. My suspicion is that it will be a combination of genetic and epigenetic factors, as well as environmental factors, that determine all these diseases."
The Three Main Types Of Epigenetic Information
Cytosine DNA methylation is a covalent modification of DNA, in which a methyl group is transferred from S-adenosylmethionine to the C-5 position of cytosine by a family of cytosine (DNA-5)-methyltransferases. DNA methylation occurs almost exclusively at CpG nucleotides and has an important contributing role in the regulation of gene expression and the silencing of repeat elements in the genome.
Genomic imprinting is parent-of-origin-specific allele silencing, or relative silencing of one parental allele compared with the other parental allele. It is maintained, in part, by differentially methylated regions within or near imprinted genes, and it is normally reprogrammed in the germline.
Histone modifications – including acetylation, methylation and phosphorylation – are important in transcriptional regulation and many are stably maintained during cell division, although the mechanism for this epigenetic inheritance is not yet well understood. Proteins that mediate these modifications are often associated within the same complexes as those that regulate DNA methylation.