By Meagan Goldman ’16
Image at top: Professor Ben Carone with his students at this year’s biology thesis poster session. From left to right: Ronak Dave ’17, Emily Shea ’16, Ben Carone, Sierra McDonald ’16.
Ben Carone is a heretic. Part philosopher, part biologist, he stumbled as an undergraduate upon a branch of genetics that challenges one of biology’s most accepted dogmas. Once he found the field, there was no turning back. He used to think a lot about the meaning of life, he told me, but philosophy didn’t help him much with that. It was science – and belief in his research – that hooked him.
His blasphemy is this: Charles Darwin was wrong. At least, he was partly wrong. Across a bare desk in his basement office at Williams College, Carone explained to me that in the nineteenth century, two dueling theorists proposed their own versions of evolution. One was Jean-Baptiste Lamarck, the other Darwin.
“It really comes down to identity and trying to figure out what makes us who we are,” Carone said. He’s a short man in his thirties with a young face and an unassuming manner. When he starts talking about something that excites him, his lively gray eyes light up, and his words rush along and collide with other like dominos.
Lamarck believed we arrived at our current state through constant adaptation to the environment, Carone said. The classic example is the neck of the giraffe. According to Lamarck, if a short-necked giraffe wanted leaves from a tall tree, it would stretch its neck out to reach them, and it would pass this long-necked trait to its offspring. In other words, the physical changes in a parent’s lifetime affect subsequent generations, and that’s why giraffes have long necks today. Darwin disagreed with Lamarck, claiming that this kind of transgenerational inheritance doesn’t happen. Instead, Darwin wrote that long-necked giraffes would be more likely to survive than their short-necked counterparts, and they would pass the desirable neck length to their offspring. Darwin’s theory makes a lot more sense than Lamarck’s when you really think about it. If Lamarckian inheritance were true, then a mother who lost a finger during her lifetime would have children with nine fingers. We all know this doesn’t happen, don’t we?
But there’s a loophole, and that’s where Carone comes in. His field is called epigenetics, and it has proven that transgeneration inheritance does occur. Not in the extreme forms of the giraffe’s stretched neck or the mother’s missing finger, but rather in DNA expression. Experiences we have in our lifetimes can permanently affect which genes are turned on and off – not only in us, but also in our children.
Since leaving grad school, Carone has been chasing what he calls his pie in the sky: he wants to understand the mechanism behind these changes.
During the winter of 1944, when German troops were occupying the Netherlands, the Dutch government organized a railway strike to halt the transportation of enemy soldiers. In retaliation, the Germans cut off food supply. Famine swept through the country, with some people consuming only 30% of their normal daily calories. City dwellers went on “hunger expeditions” to farms, searching for food and trading their silverware, carpets, and jewelry in exchange for anything to eat. Others were so desperate they ate tulip bulbs. When the famine finally ended in May, over 20,000 people had died. In addition to marking one of many tragedies of the war, this period of starvation – now called the Dutch Hunger Winter – has acquired unexpected significance. For in the years following World War II, epidemiologists studying the effects of famine on the population came across an unusual discovery.
Children conceived at the beginning of the Hunger Winter were born small. That was to be expected, because their mothers were malnourished throughout most of their pregnancies. What was surprising was that these children remained small for life despite adequate nutrition, and even more astonishing, their own children were often small as well. On the other hand, children conceived near the end of the winter were average-sized at birth. The famine only affected the beginning of their gestation, while most growing occurs near the end of pregnancy. However, epidemiologists followed these children for years and found that they expressed higher obesity rates than average—and their own children did, too. A mother’s nutrition during pregnancy, it seemed, influenced not only her child’s lifetime health, but also her grandchildren’s health. Yet that could only occur if the effects of starvation had been incorporated into her – or her child’s – very genes.
This was the first inkling of evidence supporting transgenerational inheritance. Tragic though it was, the Hunger Winter served as a valuable experiment. Before the famine, the Dutch population was generally well fed and attended by doctors who kept meticulous public health records. The people experienced a relatively short period of hunger, after which they returned to prosperous lives. Thus the effects of starvation could be isolated from other factors normally associated with it, like poverty. Scientists could directly compare children conceived during the winter of 1944 with their siblings conceived before or after that time.
Sixty years later, geneticists studying the children of the Hunger Winter found a difference within their DNA that set them apart from siblings of the same gender. They were looking the gene for the hormone called Insulin-Like Growth Factor II, or IgF2, which promotes growth in fetuses and plays a role in adult reproductive and metabolic processes. What they discovered was a difference in gene activation between siblings.
Within a person’s genome – their full set of DNA – some genes are expressed more than others. A gene’s expression level corresponds with how easily the cell machinery can access it. DNA is typically stored wrapped around proteins called histones. Some stretches of DNA are loosely wrapped, while others are tightly wrapped. If the genome is a library, then genes expressed most frequently are the books near the front, spread out and easily browsed. The genes that are rarely used are located in condensed DNA, or the library’s compact shelving, requiring complicated maneuvering to read. And the ones that are never used are stashed in dusty boxes in a storage room, piled on top of each other, where no one could reach them even if they tried. The addition of chemical modifications such as methyl groups to histone proteins can change a gene’s accessibility.
The scientists found that even at age sixty, people conceived during the winter of 1944 had less methylation of their IgF2 gene than same-sex siblings conceived at different times. Although the biologists haven’t yet determined the effects of altered IgF2 methylation, it likely has lasting impacts. These kinds of chemical modifications – as well as modifications that occur during a person’s lifetime – are the basis of epigenetics. Born out of the Hunger Winter Studies, the idea that changes in DNA expression can be inherited between generations has revolutionized how scientists approach the human genome.
In Ben Carone’s lab, a colorful poster on the wall illustrates the characteristics of three distantly related species: yeast, tetrahymena, and lab mice. The day I visited, Carone led me to the poster and explained its relevance to his research, gesturing with his hands as he spoke. Across the room, two students used pipettes to fill small plastic centrifuge tubes, while 90’s rock music played in the background, and a paper sign wedged partway behind a fridge read: Beware of Dog. Do Not Enter. Dog May Attack and Eat You and That Might Make Him Sick.
After leaving grad school, Carone told me, he wanted to find the answer to his pie-in-the sky question: is there a causative relationship between histone modifications, like methylation, and the silencing of genes? Biologists had found a correlation, to be sure. Yet no one knew if methylation caused silencing or if it just marked silenced genes. Carone envisioned a simple experimental design. He wanted to use yeast, which are similar enough to animals to possess histone proteins, but are easy to manipulate because they’re single cellular.
“The most simple way you could do this is: I have a cell, I add the mark, I see what happens. Is the gene turned on or turned off? I have the mark in there, I take the mark out, I see if the gene turns on or off,” Carone said. “You have to do really fundamental binary questions. The tools that we build in yeast, if those tools work well, we can take them and potentially put them into a mouse or an animal model because their substrates are the same.”
It sounded straightforward. But in 2009, after landing a postdoc with Oliver Rando, one of the foremost epigeneticists in the country, Carone realized the technology didn’t yet exist for this kind of DNA modification. So he started helping Rando with his research examining how the diets of male mice affect their offspring’s metabolism. Though the Dutch Hunger Winter studies were groundbreaking in that they provided the first hard evidence of epigenetics, it’s not too difficult to accept that an embryo’s prenatal environment has lasting impacts on future health. Rando’s research, however, is shocking because it implicates fathers. What he’s discovered is that paternal diets may alter gene expression in sperm, linking their offspring to metabolic diseases like diabetes. Not only does a father’s present-day diet affect gene expression in sperm, but his lifetime diet – starting before puberty – also has significant impacts.
“Let’s say I was starved or really stressed for a period of my life,” Carone said to me. “My kids might actually inherit a metabolic program that will last their entire life based on my life experiences. It’s really cool to think of from a social responsibility perspective.”
After completing his postdoc with Rando two years ago, Carone moved to Williamstown with his wife – also a biology professor – and their two young kids. Since then, he’s been focusing on his own research. The work that took off the fastest was once again not directly related to his initial question. Rather, Carone and his students began examining histone protein spacing in Tetrahymena, single cellular creatures known to most people as pond scum. This research will help biologists understand the way DNA is condensed and packaged.
When he finished explaining the poster to me, Carone led me past a fridge in his lab. Like the other fridges nearby, he uses it to store yeast at the optimal temperature, but I later learned this one houses particularly special yeast. In a twist on his interest in these single cellular fungi, Carone brews beer for fun. He also maintains strains for the Berkshire Brewing Company, a popular New England craft brewery. If the strains for favorite beers ever diverge from their present state and start making different flavors, he told me, then they’ll be able to recover the old tastes. His own beer has been known to show up in kegs at the end-of-the-year Biology Department picnic.
Carone paused at the lab bench and asked one of his students, “Can I smell that real quick actually?”
The young man stopped pipetting and replied, “Go for it.”
Bending over the centrifuge tubes, which were full of pale liquid, Carone sniffed and said, “Smells pretty phenol-y to me. That was the top layer?”
The student was in the midst of pulling out DNA from yeast cells in a process called phenol-chloroform extraction. Afterwards, he would sequence the DNA and compare it to information on histone spacing that he previously obtained from Tetrahymena. This research, Carone said, is almost ready to be published.
Even more exciting is that now the advent of a new technology called CRISPR-Cas9 has finally made it possible for him to start chasing down the answer to the question he first posed years ago. Like a word-processor for the genome, CRISPR-Cas9 allows biologists to edit DNA. In Carone’s case, it lets him insert genetic marks at specific locations. The preliminary data suggest that adding a methylation mark can indeed silence a gene and keep it silenced for a period of time.
It’s only the beginning of a long road addressing the question. He will need to do two or three more experiments to confirm the data. Then one day he hopes to take the tools he’s building in yeast and use them in mice to turn on genes in mothers and see if the changes persist in future generations. If all goes according to planned, that will be the first formal proof of epigenetic inheritance caused by the addition of external factors to DNA.
When I met with Carone in his office one last time, I asked him about the implications of his research for medicine and public health. He explained that his work might play a role in stem cell research. Every cell in the body has the same DNA, but they’re all programmed differently to become, say, skin or muscle or liver.
“Programming patterns are stabilized by epigenetic patterns,” Carone said. “Understanding those patterns and actually being able to erase, create modify, whatever you want to do to them, is really the key to being able to unlock or reprogram cells.”
If scientists can reprogram cells, then they might be able to grow organs or replace cells post cancer treatment. In 2012, two scientists won the Nobel Prize for research showing that the introduction of only a handful of genes into mature cells could reprogram them into pluripotent stem cells. This reprograming is currently inefficient, with only one or two cells among thousands changing successfully. However, Carone said that knocking out additional factors that maintain epigenetic marks might improve the efficiency.
Carone’s philosophical bent has also led him to realize that research like his and Rando’s may have tremendous ethical implications.
“One of the things I’m always interested in is personal freedom versus social responsibility,” Carone said. “Our country is founded on personal freedoms. The idea of epigenetics and transgenerational inheritance actually adds another level to it, because what if I now said to you: if you smoke, your children have a higher incidence of cardiovascular disease, or diabetes, or lung cancer? So what you do in your life will permanently affect your children’s metabolism. It adds a new twist or a new dimension to what we should seriously consider as important. I think about that a lot.”