A Lasting Legacy

Over the past half-century, advances in the life sciences have been profound, and epigenetics is one of the most exciting new frontiers. The “epi” prefix is from the Greek for “above” – or, in this case, “in addition to.” It was once thought that the genetic code with which we were born was a static blueprint for all that would happen in our cells throughout our lives. Epigenetics describes the revelation that, in fact, that blueprint can be affected to a large degree by the environment. Changes in gene expression dictated by the environment can even be passed down to the next generation and beyond. Many of us would have received a failing grade in high school biology if we had said that (information about) someone’s life experiences could be passed down to their children. Now, epigenetics is recognized as a basic biological phenomenon.

One of the first documented manifestations of epigenetics in humans arose from dire circumstances: war and famine. During World War II, France was liberated after D-Day, but the Netherlands remained occupied by the Germans. To liberate the Dutch and hasten the end of the war, British Field Marshall Montgomery developed a plan that became known as Operation Market Garden. The Allies would drop 20,000 paratroopers into the Netherlands to seize a series of bridges along a highway leading to Germany, allowing subsequent Allied troops to move swiftly into the country.

If you have seen the movie “A Bridge Too Far,” you will already know that the plan was a failure. The Allies encountered heavy resistance from German forces at Arnhem, the bridge over the Rhine River just before the German border. The Allied paratroopers were surrounded by German defenses and sustained heavy casualties – about 15,000 killed and many more wounded. Even before Operation Market Garden, food was in short supply and, with no reinforcements coming, the surviving Allied paratroopers were soon starving along with the Dutch.

One soldier's story

One of the paratroopers of the US 101st Airborne, a sharpshooter, flew out of England on September 17, 1944, and his division was dropped over Veghel under enemy fire. His pack was so loaded with supplies that his combat helmet popped off when his parachute opened. But it was easily replaced; so many of his fellow paratroopers were shot as they descended that the ground below was littered with their helmets. His division captured the bridge at Veghel and then marched to Nijmegen, where they helped take control of the bridge over the Waal River. Then came the order to march onward to Arnhem. With German panzer divisions defending Arnhem and no supplies coming, the Allies ran out of food. They had been told that the Dutch would feed them; some did, but they had very little food to share, and those who were caught helping the Allies were executed by German soldiers.

One day, the hungry sharpshooter and his best buddy, Bob Sherwood, were out scouting for enemy soldiers when they saw an apple tree laden with fruit. Delighted, they raced to the tree. Bob climbed and started shaking the branches to drop apples down to his friend below.

A single shot rang out.

Bob fell from the tree, killed instantly by a German sniper. Afterwards, the sharpshooter lay on the ground motionless for hours until he was able to escape under the cover of darkness.

The sharpshooter was not so lucky on October 5, 1944, when he ran in to replace a gunner who was killed by heavy German fire. Although he knew he would be the next high-value target, he quickly wiped out five gunners with five shots. But a sixth, hidden in the woods, fired a mortar shell that gravely wounded the sharpshooter with shrapnel. He lay bleeding in a wine cellar for days before being loaded into a truck overflowing with casualties. A week later, the sharpshooter was transported to the US by hospital ship – and, 10 months later, was finally released. For his efforts, he earned a Bronze Star for bravery. I am particularly thankful that he survived, because he is my father: Robert “Red” Wirth. Now 93 years old, he still tells stories of his harrowing wartime service.

The thrifty gene

During and after the battle at Arnhem, the Germans destroyed the transportation infrastructure to secure their hold on the Netherlands, cutting off the food supply just as an unusually hard winter approached. The result was widespread famine. The winter of 1944–1945 is known as the Hongerwinter –“the hunger winter.” It was an inadvertent and tragic experiment on the long-term effects of famine. From December 1944 until Germany surrendered in May 1945, food was rationed to 1,000 dietary calories per day, and from February to May rations were cut again to just 580 calories per day. More than 20,000 people died of starvation.

Dutch babies born early during the famine were born underweight but, after the war, many grew to normal weight. Babies who spent only the first trimester in utero during the famine, born after the war, were born at a normal weight. Initially, it appeared that this latter group had escaped the worst impact of the famine. However, in the long term, people in this group were in fact some of the hardest hit. Far from being cushioned from the effects of the famine, their early exposure to starvation in the womb led to increased rates of obesity and a host of metabolic diseases affecting cardiac health once they reached middle age. They had somehow acquired what is popularly called the “thrifty gene.” A half-century after World War II, Dutch people born just after the famine found that their bodies behaved as though they were still living under conditions of starvation. We now know that the malnutrition suffered by parents caused epigenetic changes in offspring conceived during the famine.

Not quite a clean slate

The conventional understanding of molecular biology – its central dogma – was once simple: genes are transcribed to make mRNA, which is translated to proteins. In reality, though, things are not so simple. Transcription can be blocked by environmental effects, and genes that are supposed to be blocked can be unblocked. Two main types of gene silencing occur in cells: modification of the DNA and modification of histones. DNA is wrapped around histones like pearls on a necklace; histones are opposite in charge to DNA, yielding a strong electrostatic attraction. Modifications to either component can affect how tightly the DNA is held by histones.

In reproduction, the egg and sperm typically lose their DNA methylation, erasing any memory of the parental environment. The DNA is then re-methylated during development. Folic acid is a source of methyl groups for this critical process, which is why the mother’s nutrition in the first trimester is critical. For the Dutch Hongerwinter Cohort, as researchers call them, maternal malnutrition meant that methylation was not completely erased, and the epigenetic changes caused by the famine were passed down to the children and, in some cases, even the grandchildren – imprinting them for life with the traits needed to survive conditions of starvation.

Scientists in the Netherlands and the USA recently pinpointed the mechanism behind the higher body mass index and elevated serum triglycerides seen in the Dutch Hongerwinter Cohort: DNA methylation at CpG sites in genes mediating energy metabolism (1).

Experiments on animals shed light on the epigenetics of starvation. The two mice pictured in Figure 1 are genetically identical; the only difference is in the diets of the mothers during pregnancy. Both mice were engineered to be predisposed to obesity, but the mother of the smaller mouse was fed a much more nutritious diet. Although the genetic blueprint of each mouse is identical, the expression of genes relating to metabolism is radically different, reflected in their disparate appearance. In other words, they have the same genotype, but a different phenotype. DNA hypomethylation even affects the coat color.

In this case, the mother’s diet was responsible for passing on unfortunate epigenetic traits, but a study on extreme exercise (mimicking starvation) in male mice showed that the father can also pass down the so-called “thrifty” gene or genes through epigenetic changes in sperm, leading to obese offspring (2). But that doesn’t mean that mothers are better off choosing a man who lies on the couch watching TV all day to father their children; too little exercise in male mice causes other epigenetic problems (3).

DNA methylation, once presumed to be a persistent gene silencer, is now appreciated to be a reversible, dynamic process; an individual’s genome undergoes significant change over their lifespan (4). The average centenarian has significantly less cytosine methylation than a baby, with de-silencing of certain genes responsible for some age-related conditions (5). Even identical twins have virtually identical epigenomes at three years old but, by age 50, their epigenomes significantly differ (6), which may help to explain why identical twins usually die of different diseases. For example, the famous American twin-sister advice columnists, Abigail Van Buren and Ann Landers, were amazingly identical when they were young, and even pursued the same career path. However, they died of completely unrelated diseases – Ann at age 83 from multiple myeloma and Abby at age 94 from complications of Alzheimer’s disease. As well as this drift, epigenetic changes also lead to predictable effects. The epigenetic clock, for example, is based on a steady rate of DNA demethylation for a specific set of genes as people age – and it is even used in forensics to estimate the age of crime suspects (7).

Overcoming destiny?

Can we do anything about our aging epigenomes or must we make do with what we have inherited and put up with whatever changes occur over our lifespan? It appears that we can, in fact, manage epigenetic changes to some extent. A quick online search for “DNA methylation and exercise,” for example, brings up a wealth of studies. Heart disease is the most common cause of death in industrialized countries, and we know that exercise helps prevent heart disease. Now we know why: because our epigenetics change with exercise to help lower our risk of heart disease (8). Even if your genetics predispose you to heart disease, your epigenetics can offset some of the risk.

The breadth of studies on epigenetics is vast. Though we have focused on metabolism thus far, DNA methylation plays a role in a huge assortment of diseases (9). The epigenome of a cancerous cell is very different from that of a healthy cell, and this fact is being exploited for new therapies (10). In other illnesses of old age – everything from Parkinson’s disease to chronic obstructive pulmonary disease – you will again find epigenetic factors at work. The diseases that the famous advice column twins succumbed to are now both known to be associated with epigenetic changes.

How can science help us to understand – and maybe even control – our epigenome? The key ingredients for a major (or minor) scientific advance are, first, asking the right questions and, second, having the ability to answer them. We can use analytical chemistry to measure DNA methylation and histone modification to answer a vast array of interesting biological questions. The method we presently use for detecting cytosine methylation was invented in 1992 by Frommer and colleagues, who demonstrated that bisulfite deaminates cytosine, converting it to uracil (11). Methylated cytosine (see Figure 2) is much slower to react with bisulfite; therefore, they are still visible as cytosines in DNA sequencing after bisulfite treatment.

Analyses in epigenetics began with single-mode measurements; for example, detecting average methylation across all DNA or average modifications across all histones. Once scientists began to understand that DNA modifications and histone modifications work together, new methods were needed (12). The overlapping nature of epigenetic changes poses a measurement challenge, because there is just one copy of each nucleosome per cell. PCR does not amplify methyl modifications, and, of course, protein concentrations cannot be amplified.

As a result of the high sensitivity needed, single-molecule techniques are now widely used for these analyses. In a technique called ChIP-seq, a highly specific antibody for a histone modification (for example, lysine acetylation) selects the nucleosomes with this modification by chromatin immunoprecipitation (ChIP) (13), and single-molecule DNA sequencing using nanopore technology (14) identifies which cytosines in the chromatin are methylated. The nanopore technology sequences DNA and detects modifications without the need for PCR and does not require bisulfite treatment. Future measurement technology will need to address an even greater challenge: there are multiple modifications of histones and other modifications of DNA besides methylation of cytosine at CpG sites. The same histone can have multiple modifications, all of which work in concert with one another and with the DNA modifications. These comprise a complex epigenetic code that describes how our cells operate, how they respond to the environment, and how diseases arise.

The limitations of current technology are clear when considering the vast complexity in analytical measurements required to unravel this code (15).

Beyond biomedical research

Epigenetics has captured the interest of social scientists, who are concerned about the epigenetics of social status. After all, human epigenetics came to our attention in part because of a social issue in the Netherlands: war and famine. It is striking that an environment that we have no control over can cause deleterious biological changes, and that this effect can be passed down to the next generation and beyond. The Hongerwinter demonstrated that there can be a critical window, such as the first trimester of gestation, that impacts one’s entire life and sometimes the lives of one’s children. Beyond heart disease, folic acid deficiency during gestation in the Hongerwinter has been associated with a higher level of schizophrenia (16). But malnutrition is just one factor affecting the epigenome – in mice, maternal care in the first months of life has been demonstrated to epigenetically affect stress response in later life (17). Plus, pollution, drug addiction, family dysfunction and stress may all play a part in our individual epigenetic code. These issues and more are discussed in a recent review in the sociology literature (15).

Epigenetics is a rapidly growing and expanding science, encompassing nutrition, exercise, disease, substance abuse, family life and socioeconomic status. There is much we still don’t know, but one thing is certain: progress in all areas would be accelerated with better, faster laboratory tools and techniques.