Epigenetics: Above and Beyond DNA
Shelley Berger, Ph.D., leads Penn Medicine’s wholehearted venture into a rapidly expanding field of science.
By Lisa J. Bain
From Penn Medicine, Spring 2012
“Shelley, what the heck is epigenetics?”
The question was posed by Glen Gaulton, Ph.D., executive vice dean and chief scientific officer of the Perelman School of Medicine. All eyes turned to Shelley Berger, Ph.D., sitting among other faculty panelists at the official opening of the Translational Research Center last May. Colleagues, administrators, students, and other interested parties in the center’s large and modern auditorium waited for her answer. “Frankly,” Gaulton continued, “I don’t think there’s an area that epigenetics does not touch nowadays.”
But he was not putting Berger on the spot. After all, he knew she was director of the Penn Epigenetics Program, established only a couple of years ago to make sure the school did not lag behind in what the popular press had begun calling “the new science.”
Taking Gaulton’s bait, Berger explained that, until recently, scientists believed that gene mutations were the only source of human diseases – but it turns out to be more complicated. “Epigenetics is a layer of regulation over our genes that is key to how genes are turned on and off.” Identical twins, Berger noted, have identical genomes – but as they age, they become different because of epigenetic changes. “One of the areas that’s fascinating to study with respect to epigenetics,” she continued, “is aging.” Currently, her laboratory is using a single-cell organism to try to understand how changes in the epigenome underlie aging – and are relevant even to human aging. As another example, Berger cited the work of Ted Abel, Ph.D., the Brush Family Professor of Biology in Penn’s School of Arts & Sciences, who is studying how “a single change in the epigenome” can impair memory function in mice.
It’s clear, then, that this “new science” has a tremendous reach. It’s also clear that Penn Medicine was determined not to be left behind. And that is where Shelley Berger comes in.
On the Trail of Gene Regulation
Weekends are a good time to get work done with few distractions. So it was not surprising that even though it was the Saturday after Christmas in 1995, Berger was working as usual in her office at The Wistar Institute when she got a call from Jerry Workman, Ph.D., a scientist and collaborator from Penn State University. Berger and Workman had been trying to understand the biochemistry of Gcn5, a yeast protein that Berger had found to play a role in the process of activating genes. “We figured this gene was going to be doing something interesting, we just didn’t know what,” said Berger.
“Are you sitting down?” asked Workman.
“No,” replied Berger.
“Well, sit down,” he said and then proceeded to tell her that another scientist, David Allis, Ph.D., then at the University of Rochester, had discovered a mechanism by which the Gcn5 protein regulates gene activity. Gcn5, it turns out, is a histone acetyltransferase (HAT), which means it’s an enzyme that adds chemical tags called acetyl groups to histone proteins that package DNA, relaxing the tightly compacted structure so it can be more easily copied.
“I was depressed all weekend,” recalled Berger. “I thought, ‘oh man, the biggest thing that happened about this gene I was studying – and I didn’t discover it!’ But by Monday I thought, ‘Wow, this is amazing.’ I could just understand how important this was going to be.” Moreover, she realized that she was in a great position to ask important questions about whether the enzymatic activity Allis had discovered was really essential for gene regulation. “So we immediately set out to do that. We started collaborating with David Allis, and within six months or less my group had some very nice papers on the subject.”
Today, the study of how the inherent DNA structure can be modified by adding acetyl groups and numerous other chemical groups that influence how genes are expressed has coalesced into one of the hottest areas of biomedical science – epigenetics.
“Shelley and her colleagues have played a seminal role,” said Allis. “It started with her postdoctoral work with the genetics of these proteins. . . . And now 15 years removed, it’s really a huge enterprise of people.”
In January, 2010, Time magazine brought epigenetics to the mainstream by featuring the topic as its cover story, “When Your DNA Isn’t Your Destiny.” A few years earlier, at the urging of Arthur H. Rubenstein, M.B., B.Ch., then the dean, Penn’s medical school had begun to build an epigenetics program. Looking around for someone to lead the program, Rubenstein said that he and his Penn colleagues found that “the very best person in the country was actually at The Wistar Institute, and that was Shelley Berger. She had done extremely innovative and creative work in a variety of areas of epigenetics and was already viewed as an international leader in this area.” A program was crafted with Berger as the leader, and she was recruited as director of the Penn Epigenetics Program and the Daniel S. Och University Professor in the departments of Cell & Developmental Biology and of Genetics. She was also named the 10th Penn Integrates Knowledge (PIK) University Professor, a University-wide initiative that recruits faculty whose research and teaching cross multiple disciplines and at least two schools at Penn. Berger’s other appointment is in the Department of Biology of the School of Arts & Sciences (SAS).
Shortly after her arrival at Penn, Kenneth Zaret, Ph.D., was recruited from the Fox Chase Cancer Center to serve as co-director of the Penn Epigenetics Program as well as associate director of the Penn Institute of Regenerative Medicine. Scientists on the program’s executive board come from departments ranging from Biochemistry and Biophysics to Pediatrics, and members of the program are drawn from Penn as well as The Children’s Hospital of Philadelphia, Wistar, Fox Chase, Drexel University, Thomas Jefferson University, and Temple University. According to Rubenstein, Berger and Zaret have built a program that “has become among the leading epigenetics programs in the country in a very short time.”
The sequencing of the human genome was announced with much fanfare in 2003, but the Human Genome Project raised as many questions as it answered, if not more. Scientists were hoping that by mapping all the genes, they would be able to identify mutations in those genes that caused disease. But what they found was that simple disease-causing mutations, such as the mutations in the BRCA 1 or 2 genes that cause breast cancer, are relatively rare. Most diseases, even those that run in families and thus are thought to be inherited, arise through much more complicated mechanisms. What had become apparent, even before the Human Genome Project started, is that gene activity depends not just on the sequence of the gene but on whether and when that gene is expressed. And gene expression is a complicated process controlled by proteins that package, spool, and compact the DNA in the nucleus of cells; and by chemical groups that sit on top of the package like switches, turning the genes on and off. The spool (chromatin) comprises proteins called histones, and the switches are called epigenetic marks. The prefix “epi-” means “above.”
Epigenetics can help explain some of the perplexing biomedical questions that simple Mendelian genetics cannot. Why, for example, might one child have autism while his identical twin is unaffected? Or what could explain the observation that pregnant women who experienced starvation during the Dutch famine of 1944, when the Nazis blockaded food and fuel shipments to the Netherlands, gave birth to children who were more susceptible to a variety of health problems, including diabetes? The answer, it seems, lies in the fact that environmental conditions can alter the epigenome, resulting in heritable changes that can be passed to offspring, even though there has been no change in the genes themselves.
Epigenetics is also thought to play a critical role in the development of cancer, for example, by turning off a tumor-suppressor gene. “Cancer is one disease that is unequivocally linked to epigenetic dysfunction,” said Allis. Last fall, Mariusz A. Wasik, M.D., professor of pathology and laboratory medicine, Qian Zhang, M.D., Ph.D., research assistant professor, and colleagues in the Perelman School of Medicine found that a cancer-causing fusion protein works by silencing the tumor suppressor gene IL-2R common gamma-chain (IL-2Rγ). In Proceedings of the National Academy of Sciences, they reported that the IL-2Rγ gene promoter is silenced by a chemical change to the DNA itself – in this case, the adding of a methyl group to DNA’s molecular backbone. Describing his study, Wasik wondered whether this form of epigenetic silencing could be made more generally applicable. “Can we overcome the tumor-suppressor gene silencing using inhibitors of DNA methylation – which are already approved to treat some forms of blood cancer – to inhibit the expression of NPM-ALK and possibly other cancer-causing proteins in patients?”
Epigenetic dysfunction has also been linked to problems in the brain affecting learning and memory, addiction, alcoholism, and complex psychiatric disorders, and to chronic inflammation and other immunologic disorders.
At a more basic level, epigenetic changes underlie many aspects of cell differentiation and cellular memory. How, for example, does a single fertilized egg that contains all the DNA for an organism give rise to separate populations of liver cells and brain cells with distinctly different patterns of gene expression? And how does a liver cell, when it divides, remember that it is a liver cell and not a brain cell? The answers to these questions are not known, but the study of epigenetics is likely to provide some clarity.
Epigenetics on the rise
The publication of Allis’s landmark paper resulted in a sea change in scientists’ understanding of gene regulation. Almost immediately, Berger converted her entire research focus to the study of chromatin mechanisms, particularly how modifications to histone proteins regulate not just gene transcription, but also replication and DNA damage. “Everything that has to be done on the genome is regulated by these enzymes that add and take off these little chemical groups,” she said. “It was lucky for me that I was in the beginning of this change, because I had been studying this gene and we had all the reagents in hand.”
Moreover, the excitement about epigenetics extends into almost every area of both basic and translational science. In Berger’s lab alone, research projects include studies related to aging, infertility, immunological memory, cellular metabolism, and the response to stress, and even a study on how epigenetic differences between different castes of female ants (queens vs. workers) might explain the extreme differences in both physical and behavioral characteristics. The ant study was published in Science (August 2010) with colleagues Danny Reinberg, Ph.D., of New York University, and Juergen Liebig, Ph.D., of Arizona State University. Despite the extreme differences, all females within the ant colony appear to be genetically identical. The researchers believe that epigenetic mechanisms are critical in establishing the variations. “Think of the workers and the queen as different tissues in our bodies,” said Berger. “It’s an epigenetic marking system on the scale of a whole organism.”
Indeed, epigenetics is the link that ties these different research programs as well as different organisms together. Berger said that after studying chromatin mechanisms in yeast for many years, she wanted to place it into the context of physiological pathways, so she started to cast around for some pathways in yeast that could be used as models for what is going on in mammalian cells. She chose spermatogenesis, the making of sperm, which is very similar to the process of sporulation (the making of spores) in yeast. Since then, her lab has found a number of important modifications in chromatin histone that occur during sporulation that are relevant to spermatogenesis.
Now, funded with a grant from the National Institutes of Health, Penn recently launched the Penn Center for the Study of Epigenetics in Reproduction. Marisa Bartolomei, Ph.D., professor of cell and developmental biology, is the principal investigator. The center also includes the Berger lab as well as Ralph Meyer, Ph.D., assistant professor of developmental biology at the School of Veterinary Medicine, and Richard M. Schultz, Ph.D., the Charles and William L. Day Distinguished Professor of Biology in SAS. “The really cool part in my opinion is that we reach all the way to a human in vitro fertilization clinic,” said Berger. Collaborators Christos Coutifaris, M.D., Ph.D., the Nancy and Richard Wolfson Professor of Obstetrics and Gynecology professor of obstetrics, and Carmen Sapienza, Ph.D., at Temple University, will be collecting samples from humans who are having fertility problems. They will try to determine whether epigenetics plays a role in increasing the risk of complications among babies conceived through in vitro fertilization.
Other scientists in the Epigenetics Program are going in equally diverse directions, across departments and schools. Cancer is a major area of epigenetics research, and many cancer researchers have hopped on the epigenetics bandwagon. For example, Roger Greenberg, M.D., Ph.D., assistant professor of cancer biology, is studying how, in cancer, epigenetic changes in proteins affect the ability of cells to repair damaged DNA. Meanwhile, Ted Abel at the School of Arts & Sciences is trying to understand learning and memory in terms of epigenetic marking. Indeed, a whole field is emerging that tries to understand how neurons become differentiated based on epigenetic changes.
Penn has tremendous strength in the area of neuroscience and neurodegenerative disease, and combining that with epigenetics has given rise to a new project recently funded by the N.I.H. (with scores almost unheard of in the peer-review process). Berger is a co-principal, along with Nancy Bonini, Ph.D., in the Department of Biology at SAS and an investigator of the Howard Hughes Medical Institute, and Brad Johnson, M.D., Ph.D., a physician at the Hospital of the University of Pennsylvania and associate professor of pathology and laboratory medicine, on a study aimed at understanding epigenetic changes that may underlie neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s dementia, frontotemporal lobar degeneration (FTLD), and amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig’s disease).
The project takes advantage of brain tissue collected by Penn’s Center for Neurodegenerative Disease Research under the supervision of collaborator John Trojanowski, M.D., Ph.D., director of Penn’s Institute on Aging, as well as Bonini’s fruit fly model of neurodegeneration; a cultured human astrocyte model developed by Claudio Torres at Drexel; second-generation sequencing technologies; and bioinformatics expertise available at Penn. “There have been broad hints in the scientific community that epigenetics plays an important role in aging,” said Johnson, “but neurodegeneration is kind of an unexplored area.”
Modifying the epigenome: a promising translational path
One of the aspects of epigenetics that has the biomedical community so excited is that translating it to the clinic appears to be the next logical step. In fact, there are already cancer drugs on the market that act by inhibiting the chemical groups that are epigenetic marks. As Johnson puts it, “It’s druggable. These aren’t necessarily irreversible changes. If you get a mutation in the DNA or a deletion in the DNA, it’s hard to change that. But if it’s just the chromatin state, you might be able to reset it – and that’s exciting.”
Epigenetics also holds great promise in the area of personalized medicine, according to Garret FitzGerald, M.D., the Robert L. McNeil Jr. Professor in Translational Medicine and Therapeutics and director of Penn’s Institute for Translational Medicine and Therapeutics. “There is a remarkable variability in how people respond to drugs, and obviously getting an understanding of that leads us progressively to a more personalized approach to medicine. So clearly one of the marks of environmental influences on the genome is on the epigenome, and that’s where Shelley’s expertise and the group of people she’s built around her is so vital.”
Another area in which epigenetics offers great translational potential is in regeneration. Understanding how worker ants and queens, or liver cells and pancreas cells, can develop along different pathways despite having the same DNA could eventually lead to techniques that would enable the regeneration of damaged or diseased organs. For example, Kenneth Zaret’s laboratory studies how stem cells, which have the potential to develop into multiple cell types, make the choice for one cell fate or another.
“We’re asking very basic questions about how cell fate choices are made in a mammalian embryo,” said Zaret, who is also the Joseph Leidy Professor of Cell and Developmental Biology. “We think if we understand how cells get programmed, then we can understand how they can get reprogrammed as well.” His lab has already deciphered much of the wiring that leads a precursor cell to become a liver cell or pancreas cell. “Now we are interested in using the same approaches to understand the wiring of how a pancreas progenitor would make the choice to become a beta cell, which is important for diabetes.” Reprogramming pancreas cells to become insulin-producing beta cells could potentially lead to a treatment for diabetes.
Diabetes, infertility, neurodegenerative disease, cancer. As Glen Gaulton remarked, epigenetics touches just about every area of medicine. In similar fashion, FitzGerald said, “I think it’s fundamental to everything. But our understanding of the biology is still very much at an evolutionary stage, and particularly our understanding of the implications of blocking changes in the epigenome are at an extraordinarily early stage.” At a recent symposium, Berger showed graphically the correlation between aging and changes in the epigenome. “That’s very provocative,” said FitzGerald, while noting that Berger was careful to ask, “are these a consequence or a cause of aging?” As FitzGerald sees it, “There are huge questions that we haven’t even begun to ask, never mind answer.”
Berger faces these daunting questions with seemingly limitless energy and enthusiasm – and, as noted by Arthur Rubenstein, with charisma. Said Allis: “She’s smart and high energy. There’s no moss growing on Shelley. She’s a go-getter. She can synthesize the big picture.”
The big picture, that is, as it pertains all the way to the smallest units in human biology.
Using Epigenetics to Predict the Fate of Personalized Cells
Discovering the step-by-step details of the path embryonic cells take to develop into their final tissue type is the clinical goal of many stem cell biologists.
To that end, Kenneth S. Zaret, Ph.D., the Joseph Leidy Professor of Cell and Developmental Biology, and Cheng-Ran Xu, Ph.D., a postdoctoral research associate in the Zaret laboratory, looked at immature cells called progenitors and found a potential way to predict their fate. The study appeared last spring in Science.
In the past, researchers grew progenitor cells and waited to see what they differentiated into. Now, they aim to use this epigenetic marker system, outside of a cell’s DNA and genes, to predict the cell’s eventual fate.
“We were surprised that there’s a difference in the epigenetic marks in the process for liver versus pancreas before the cell-fate ‘decision’ is made,” said Zaret, who also serves as co-director of the Penn Epigenetics Program and associate director of the Penn Institute for Regenerative Medicine. “This suggests that we could manipulate the marks to influence fate or look at marks to better guess the fate of cells early in the differentiation process.”
How the developing embryo starts to apportion different functions to different cell types is a fundamental question for developmental biology and regenerative medicine. Guidance along the correct path is provided by regulatory proteins that attach to chromosomes, marking part of the genome to be turned on or off.
Chemical signals from neighboring cells in the embryo tell early progenitor cells to activate genes encoding proteins. These proteins, in turn, guide the cells to become liver or pancreas cells, which have been found to originate from a common progenitor cell. Over several years, Zaret’s lab has unveiled a network of the common signals in the mouse embryo that govern development of these specific cell types.
“By better understanding how a cell is normally programmed we will eventually be able to properly reprogram other cells,” noted Zaret. In the near term, the team also aims to generate liver and pancreas cells for research and to screen drugs that repair defects or facilitate cell growth.
With regenerated cells, researchers hope to one day fill the acute shortage in pancreatic and liver tissue that is available for transplantation in cases of type I diabetes and acute liver failure.
– Karen Kreeger
At the Crossroads of Chromosomes: Revealing More of the Epigenetic Structure of Cell Division
On average, one hundred billion cells in the human body divide over the course of a day. Most of the time, the body gets it right but sometimes problems in cell replication can lead to abnormalities in chromosomes. Many types of disorders, from cancer to Down syndrome, can result.
In 2010, researchers at the Perelman School of Medicine defined the structure of a molecule that plays a central role in how DNA is duplicated and then moved correctly and equally into two daughter cells to produce two exact copies of the mother cell.
Ben E. Black, Ph.D., assistant professor of biochemistry and biophysics, and Nikolina Sekulic, Ph.D., a postdoctoral fellow in the Black lab, described the structure of the CENP-A molecule, which defines a part of the chromosome called the centromere. The centromere is a constricted area to which specialized molecules called spindle fibers attach and help pull daughter cells apart during cell division.
“Our work gives us the first high-resolution view of the molecules that control genetic inheritance at cell division,” said Black. “This is a big step forward in a puzzle that biologists have been chipping away at for over 150 years.”
Investigators have known for more than 15 years that part of cell division is controlled by epigenetic processes rather than encoded in the DNA sequence itself. The tightly bound DNA spools are built of histone proteins, and chemical changes to these spool proteins can either loosen or tighten their interaction with DNA. Epigenetics alter the readout of the genetic code, in some cases ramping a gene’s expression up or down. In the case of the centromere, it marks the site where spindle fibers attach, independently of the underlying DNA sequence. Researchers have suspected that CENP-A is the crucial epigenetic marker protein.
What hasn’t been known, however, is how CENP-A epigenetically marks the centromere to direct inheritance. The Black team found the structural features that confer CENP-A the ability to mark centromere location on each chromosome. Without CENP-A or the centromere mark it creates, the entire chromosome — and all of the genes it houses — are lost when the cell divides.
The work by Black and Sekulic is a major advance in the understanding of the molecules that drive human inheritance. But it also raised the exciting prospect that the crucial epigenetic components are now in hand to engineer clinically useful artificial chromosomes that will be inherited alongside our own natural chromosomes — and, says Black, with the same high fidelity.
– Karen Kreeger
A Recipe for Heart Disease: High-Fat Diet and Lack of an Epigenetic Enzyme
It’s no secret that a high-fat diet is not healthy. Now Penn Medicine researchers have discovered a molecular clue as to precisely why that is.
Writing last fall in the Journal of Biological Chemistry, Mitchell Lazar, M.D., Ph.D., Zheng Sun, Ph.D., a postdoctoral fellow in Lazar’s laboratory, and their colleagues revealed that when mice lacking a particular enzyme that controls gene expression are fed a high-fat diet, they experience rapid thickening of the heart muscle and heart failure. This molecular link between fat intake and an enzyme tasked with regulating gene expression – at least in mice – has implications for people on so-called Western diets and for combating heart disease. Modulating the enzyme’s activity could be a new pharmaceutical target.
The team found that the mice engineered to lack the enzyme HDAC3 tended to underexpress genes important in metabolizing fat and producing energy. Essentially, when fed a high-fat diet, the hearts of these animals cannot generate enough energy and thus cannot pump blood efficiently.
These same mice tolerate a normal diet as well as non-mutant, normal animals. “HDAC3 is an intermediary that normally protects mice from the ravages of a high-fat diet,” says Lazar, the Sylvan Eisman Professor of Medicine and director of the Institute for Diabetes, Obesity, and Metabolism.
HDAC enzymes control gene expression by regulating the accessibility of chromatin – the DNA and protein structure in which genes reside. Within chromatin, DNA is wound around proteins called histones.
When an animal eats, its metabolism changes, but food doesn’t change a cell's genome. Instead, food modulates the “epigenome,” the molecular markers on the chromatin that influence gene expression by affecting how tightly DNA is wrapped around its protein scaffolding.
Previously, researchers at the University of Texas Southwestern Medical Center showed that if HDAC3 were deleted in heart tissue in the middle of embryonic development, the animals developed severe thickening of the heart walls (hypertrophic cardiomyopathy) that reduces the organ’s pumping efficiency. These animals usually died within months of birth.
Lazar and his team wanted to know what would happen if the gene was inactivated in heart tissue after birth. To their surprise, they found that these animals were essentially normal.
On a diet of regular chow, the engineered mice lived as long as their normal littermates, although they did tend to accumulate fat in their heart tissue. On a high-fat diet, however, these animals deteriorated rapidly and died within a few months of hypertrophic cardiomyopathy and heart failure.
To understand why, Lazar’s team compared the gene expression patterns of the young mutant mice to their normal siblings. They found that the mutant mice tended to underexpress genes important in fat metabolism and energy production.
According to Lazar, this study identifies an “interesting and dramatic example” of the link between diet and epigenetics. At present, his team is working to identify the molecular nature of that link. They are also investigating whether the same pathway and interaction occurs in humans because it may contribute to the increased heart disease associated with Western diets.
Whatever the outcome of those studies, says Lazar, there is one sure-fire intervention people can always use to stave off the ravages of over-nutrition: changing your diet.
– Jeffrey Perkel and Karen Kreeger