How Genes Express Themselves

Robert Roeder was starting to worry in early 1969. A graduate student at the University of Washington, he’d been working long hours in the lab and filling notebook after notebook with data, but he hadn’t made any exciting finds. Meanwhile, his peers were publishing papers and finishing their degrees. “I was a fifth-year graduate student and I didn’t have any papers,” says Roeder, now a biochemist at Rockefeller University.

His feeling left behind partly explains why he was in the lab at 3 a.m. on Valentine’s Day, plotting data on graph paper from his latest experiment. To produce a protein, cells first make an RNA version of the protein’s gene through a process called transcription. Roeder was trying to identify the enzyme, known as RNA polymerase, that transcribes genes in sea urchin cells. The graph of his results threw him for a loop. Bacteria get by with one version of RNA polymerase, but the sea urchin cells sported three. “I was mesmerized,” says Roeder, who called his supervisor to share the news. Despite the early hour, “he was as excited as I was.”

Cells use the information carried in a gene to produce a protein or an RNA molecule in a process known as gene expression. Roeder’s identification of a triumvirate of RNA polymerases, earning him that long-sought first paper, “was a major event in the study of gene expression in animals,” says molecular biologist James Kadonaga of the University of California, San Diego, who wasn’t connected to the work. “And he didn’t stop there,” Kadonaga says. In the years since, Roeder has meticulously dissected the elaborate mechanism that allows cells in eukaryotes such as sea urchins and humans to transcribe genes. For his contributions, he won the 2003 Albert Lasker Award for Basic Medical Research.

Organisms carefully control the activity of genes, turning them on and off at specific times and in specific locations. Vital functions such as cell division, neutralization of toxins, production of blood cells, and wound healing depend on the coordinated expression of genes. And gene activity patterns change in illnesses such as cancer, diabetes, and Alzheimer’s disease. Roeder is one of the researchers who received a Lasker award for discoveries that elucidated how cells activate or silence genes. Those scientists devised ingenious experimental methods, leveraged offbeat model organisms, and capitalized on underappreciated observations. Besides revealing fundamental cellular mechanisms, their findings served as the basis for new treatment approaches. Some of the results already have led to drugs for diseases such as muscular dystrophy, age-related macular degeneration, and cancer, and clinical trials are testing other potential therapies inspired by their work.

Unexpected Complexity
When Roeder began digging into the transcription mechanism in eukaryotes, “it was pretty much still the dark ages,” he says. After he identified the RNA polymerases, Roeder and his colleagues determined why eukaryotic cells need three varieties: Each transcribes a different type of RNA. The researchers also found that the enzymes were structurally elaborate, carrying up to 17 different subunits.

Delving deeper, Roeder and colleagues uncovered more proteins crucial for transcription. The group found that each type of RNA polymerase teams up with a distinct set of proteins called general transcription factors. The researchers showed that gene-specific transcription factors, which help ensure that RNA polymerase reads the right gene, are also necessary but need a hand from another group of proteins, called coactivators.

Scientists now recognize that more than 100 proteins congregate to enable RNA polymerase to transcribe a gene. “Nobody knew about the complexity that had evolved. Bob discovered this,” says University of Bordeaux molecular biologist Martin Teichmann, Roeder’s postdoc in the late 1990s and early aughts. “His impact on the field is enormous.”

Two Careers in One
Transcription yields a messenger RNA (mRNA) molecule that tells the cell what amino acids to string together to produce a protein. To carry out those instructions, however, the cell needs help from another type of RNA molecule, called transfer RNA (tRNA), that delivers the amino acids to the protein-synthesizing organelles.

Molecular biologist Paul Zamecnik of Massachusetts General Hospital earned the 1996 Albert Lasker Special Achievement Award in Medical Science in part for identifying tRNA. He received the award because he also devised a gene-blocking approach that spawned treatments for diseases such as age-related macular degeneration, hepatitis B, and muscular dystrophy. What makes Zamecnik’s achievements all the more impressive, says cell biologist Thoru Pederson of the UMass Chan Medical School, was that “he had no formal training as a biochemist. He was trained as a physician.”

An autopsy on one of Zamecnik’s former patients in 1938 sparked his interest in protein synthesis. The patient produced huge amounts of fat but little protein, and Zamecnik, then an intern, wondered why. He didn’t make much headway until after World War II, when he established his own lab. In 1956, he and a colleague developed the first procedure for replicating protein synthesis in the test tube. While studying that cell-free system, his team stumbled upon a small RNA essential for making proteins. It was tRNA, whose existence other researchers had predicted. “This was the discovery that opened the door to the molecular biology of protein synthesis,” Pederson says.

Soon Zamecnik switched to studying a virus that causes cancer in chickens. When the pathogen invades a cell, it forces its victim to produce viral RNAs. Zamecnik and colleagues reasoned that they could block that step by dosing cells with a small DNA molecule that was the mirror image of a specific RNA. The DNA would attach to the RNA molecule and thwart the virus’s replication. The approach worked in infected chicken cells, Zamecnik and a colleague revealed in 1978.

Zamecnik suspected that antisense oligonucleotides—short strings of nucleotides similar to the DNA he tested in chicken cells—could also treat diseases in humans, and in 1990 he co-founded a company to turn the technology into treatments. Although it never delivered, other companies did, and 17 approved antisense oligonucleotide therapies now exist. Zamecnik “was very careful in the laboratory,” Pederson says, but at the same time “he had an open mind.” The combination of those two qualities helped make him an excellent scientist, Pederson says.

Wrapped Up in Histones
Stretched out, the DNA in a human cell is about 2 meters long. The DNA fits inside such a small space because the molecule coils tightly around proteins called histones. As biological chemist Michael Grunstein of the University of California, Los Angeles, and molecular biologist C. David Allis of Rockefeller University helped reveal, histones perform another key job in cells: regulating gene expression. By attaching chemical groups to the tails of histones, cells adjust gene activity. Grunstein and Allis shared the 2018 Albert Lasker Basic Medical Research Award for their work.

In the 1960s, a group of scientists proposed that cells could tweak gene expression by adding acetyl groups to histones—a process known as acetylation—but the evidence for their hypothesis was slim. When Grunstein started studying histones in the 1970s, most researchers still thought that the proteins served only as spools for DNA. Grunstein “did a lot to bring to the forefront the importance of histones,” says Brian Strahl, a biochemist and molecular biologist at the University of North Carolina Medical School and Allis’s former postdoc.

Grunstein began investigating the proteins in sea urchins but soon switched to yeast, which proved to be a smart choice. He and colleagues could track the effects of mutating yeast genes, something they couldn’t do in sea urchins. That capability paid dividends in 1988, when the researchers showed that eliminating one histone from yeast cells boosted the activity of three genes, furnishing the first direct evidence observed in a living organism that histones help control gene expression. Replacing certain amino acids in the tails of histone molecules reduced the activity of two genes. The amino acids were attachment sites for acetyl groups, and the results bolstered the case that acetylation helps cells modulate gene expression.

In the early 1980s, Allis and his lab took up a project that had foiled Grunstein and colleagues: isolating the enzyme that affixed acetyl groups to histones. Like Grunstein, Allis and colleagues picked the right study organism, selecting Tetrahymena, a single-celled protist with two nuclei. One nucleus was rich in histones decorated with acetyl groups, and Allis reasoned that the enzyme was also plentiful there, which should make it easier to identify.

“What followed were long hours in the cold room looking for what seemed to be more challenging than a needle in a haystack,” Allis wrote. But in 1996, after analyzing 200 liters of Tetrahymena cells, Allis’s graduate student James Brownell pinpointed the enzyme. That Allis allowed a graduate student to take on the important task of isolating the enzyme illustrates one reason why he was so successful, Strahl says. “He energized people to do the hard experiments.”

Allis and Strahl went on to hypothesize that acetylation and other modifications to histones, such as adding methyl groups, create what they termed a histone code that tells cells how much gene expression to permit. Because of Allis’s experiments, “the whole field realized that there is a fundamental role for histone modification in regulating gene expression,” says cancer biologist G. Greg Wang of Duke University, also a former Allis postdoc. Some alterations cause DNA to wrap more tightly around histones, turning down gene expression, whereas others loosen the DNA, allowing gene activity to increase.

“We wouldn’t want to say that [gene regulation] is only via histone modification,” Wang says. “It’s one of several mechanisms.” Scientists are trying to work out how it meshes with those other mechanisms to keep genes under control. But histone research already has paid off with new drugs. The pattern of modifications is abnormal in diseases such as cancer, and the Food and Drug Administration (FDA) has approved five cancer treatments that target histone-modifying enzymes.

A (Hormone) Family Affair
Hormones induce their physiological effects by modifying the expression of many genes. More than 150 genes respond to insulin, for example. The 2004 Albert Lasker Basic Medical Research Award went to three researchers who discovered mechanisms by which hormones alter gene activity: molecular endocrinologist Elwood Jensen of the University of Chicago; molecular biologist Pierre Chambon of the Institute of Genetics and Molecular Cellular Biology in Strasbourg, France; and biologist Ronald Evans of the Salk Institute for Biological Studies.

In the 1950s, Jensen began investigating how estradiol, a form of estrogen, affects cells. The leading explanation then was that the hormone entered a cell and underwent chemical modifications that ultimately produced its physiological effects. Using a radioactively labeled form of estradiol and an extremely sensitive assay, Jensen’s lab tracked the hormone to its target tissues in rats. “To our surprise, nothing happens to it. It’s not changed chemically,” he told an interviewer. Jensen proposed that estradiol instead binds to a receptor in the cytoplasm of the cell. When that interaction occurs, he and colleagues found, the receptor moves to the nucleus, where it can manipulate gene expression.

Building on that work, in the mid-1980s Chambon and colleagues isolated the gene for one estrogen receptor. Around the same time, Evans and his team nabbed the gene for the glucocorticoid receptor, the target for hormones such as cortisol. The two receptors turned out to be similar to each other and to the receptor for thyroid hormone, which Evans and other researchers revealed shortly afterward. In 1987, Chambon’s and Evans’s groups almost simultaneously discovered another kindred receptor that responds to retinoic acid, a vitamin A derivative essential for vision, learning, and immunity.

Yet more similar receptors began turning up. Because the molecules that stimulated those receptors remained elusive, researchers dubbed them orphan receptors. Their roles have since been discovered to include managing fat storage, promoting calcium absorption, controlling cholesterol levels, and detoxifying drugs. “Through the discovery of orphan receptors, a ‘hidden’ wonderland of physiology has been unveiled,” Evans wrote.

The orphan receptors, along with the estrogen, glucocorticoid, retinoic acid, and thyroid receptors, belong to a family of nuclear receptors with 48 members. Nuclear receptors are central to many physiological processes and malfunction in many diseases, including diabetes, osteoporosis, heart disease, and cancer.

Turndown Service
It was, according to an over-the-top TV documentary from the mid-1960s, the key to understanding “why one cell creates a genius and another cell gives man cancer.” The key in question was a hypothetical molecule called a repressor that could regulate gene expression. Molecular biologist Mark Ptashne was one of two Harvard scientists trying to bag the molecule, a race that the documentary followed.

Ptashne’s rival identified a repressor in bacterial cells two weeks before Ptashne discovered a comparable protein for a type of virus known as lambda. But Ptashne stuck with the subject, and over the following decades he detailed how the viral repressor protein functions as part of a genetic switch to control gene expression. One scientist described that effort as “the greatest sustained experiment of the last century.” In 1997, Ptashne, who by then had moved to the Memorial Sloan Kettering Cancer Center, won the Albert Lasker Basic Medical Research Award for the work.

Ptashne is a larger-than-life character who once held a poolside chat with Fidel Castro and played violin with celebrated cellist Yo-Yo Ma. “I’ve never seen anyone who thought so clearly,” says University of Pennsylvania biophysicist Mitchell Lewis, Ptashne’s postdoc in the 1980s. That ability served Ptashne well during the chase for the repressor. Two future Nobel laureates, Jacques Monod and François Jacob, had posited the existence of the molecule in 1961, but researchers hadn’t been able to find it. Putting in 18-hour days, Ptashne and colleagues needed a year and a half to isolate the viral repressor protein.

The scientists then zeroed in on how the protein works once lambda invades a bacterium. The pathogen’s genome normally slips into its host’s chromosome and stays there, often for generations. During that latent period, the repressor silences 50 of the virus’s 51 genes. Ptashne and colleagues found that two molecules of the repressor pair up and attach to the DNA, forming a roadblock that prevents RNA polymerase from reading almost all the virus’s genes. However, the enzyme can still access the gene for the repressor, so levels of the protein stay high and keep the other genes quiet.

Some stimuli can cause the virus to emerge from hiding, and the switch flips. The researchers could trigger that event by exposing an infected bacterium to UV light, which destroys the repressor protein. As a result, RNA polymerase can start transcribing formerly silenced genes. The unfortunate bacterium then uses the proteins encoded by the viral genes to make a passel of new viruses.

Ptashne went on to investigate gene expression in eukaryotic cells, which rely on a different control system. However, his work on the lambda repressor protein “was the foundation for how people think about how genes are regulated,” Lewis says.

The Little RNAs That Could
The three researchers who shared the 2008 Albert Lasker Basic Medical Research Award—developmental biologist Victor Ambros of UMass Chan Medical School, molecular biologist Gary Ruvkun of Harvard Medical School, and plant scientist David Baulcombe of Cambridge University—showed that tiny RNA molecules can dial down gene expression. Ruvkun and Ambros also won the 2024 Nobel Prize in physiology or medicine. The discovery of those molecules, known as microRNAs and small interfering RNAs (siRNAs), “was quite astonishing,” says developmental biologist and molecular geneticist Eric Lai of Memorial Sloan Kettering. Their results “opened up a new universe of small RNAs.”

In the 1980s, Ambros and Ruvkun became intrigued by nematodes whose development was out of sync because of mutations in either of two genes, lin-14 and lin-4. After their initial data suggested that lin-4 inhibited lin-14, the duo investigated further. To their surprise, Ambros and colleagues learned that lin-4 encoded not a protein, but a miniature RNA. At the time, scientists usually ignored small RNAs that showed up in experiments, convinced that they were leftovers of larger molecules. Ambros and Ruvkun could have bailed out when they got that anomalous result, says molecular biologist Andrea Kasinski of Purdue University, who wasn’t connected to the research. “Instead of dropping it and saying, ‘this must be some kind of artifact,’ they followed up.”

Their persistence led to the discovery of the first microRNA. Ruvkun’s lab determined that a sequence at the tail end of lin-14’s mRNA was essential for controlling the gene’s expression. When he and Ambros exchanged results, they noticed that the control sequence and the sequence of the lin-4 RNA partially matched. In 1993, Ambros’s and Ruvkun’s teams proposed that lin-4’s RNA was latching onto the lin-14 mRNA and preventing the cell from using it to make a protein.

Most scientists were unimpressed, says molecular virologist Peter Sarnow of Stanford University. “They said, ‘OK, nice,’” because the phenomenon appeared to be exclusive to nematodes. In 2000, however, Ruvkun and colleagues isolated another microRNA and found it in the cells of many kinds of animals, including humans.

Working independently, Baulcombe discovered another small RNA variety, siRNA, in plants. Several lines of evidence spurred him and his colleagues to hypothesize that those RNAs were silencing genes. For example, when his group and other researchers introduced genes into plants, the activity of genes with corresponding nucleotide sequences fell. But no such RNAs had turned up until Baulcombe and his postdoc Andrew Hamilton identified the first batch in 1999.

Researchers have since uncovered much more about small RNAs. MicroRNAs play an important role, says RNA researcher George Calin of the University of Texas M.D. Anderson Cancer Center. Other regulatory mechanisms shut down genes, preventing them from generating mRNAs. By contrast, microRNAs target mRNA molecules that have already been made, spurring their destruction. In that way “they can fine-tune gene expression,” Calin says. Human cells also harbor siRNAs, whose main function appears to be fighting viruses.

But small RNAs also remain mysterious. The effects of microRNAs are complex and can depend on various factors, including what type of cell they are active in, says biochemist David Corey of the University of Texas Southwestern Medical Center. That complexity is one reason why, although microRNAs are involved in many diseases, no drugs to target them have received FDA approval, Sarnow says. “Even after three decades, there’s so much to learn. That is the mark of a great discovery,” Corey says.

Researchers have a lot to learn about other gene regulation mechanisms as well. Take the enigmatic DNA sequences called enhancers, which boost gene activity but are sometimes far from the genes they influence. “A big question is how enhancers actually work, especially if they are hundreds of kilobases away” from their target genes, Roeder says. With such challenging questions to answer, it’s no wonder that even at the age of 83, Roeder still works in the lab almost every day. And he’s just one of many researchers around the world who are trying to crack the remaining mysteries about gene expression.

By Mitchell Leslie