How the renowned neuroscientist discovered the first mammalian circadian rhythm gene, and what it meant for human health.
Circadian rhythms, and the disruptions imposed on them, play an increasingly vital role in human health. This role is evident in sleep medicine, and in fields as far-flung as metabolic disorders, mental health, and even oncology. Awareness of the influence of circadian rhythms is rapidly rising, a significant development for a characteristic of the body that even today seems mysterious to many: the built-in 24-hour cycle that monitors everything from our sleep patterns to our digestion.
As with most medical advancements, there was a tipping point in the mystery factor surrounding circadian rhythms, when our vague knowledge of the body’s innate ability to regulate its own timing gave way to a precise understanding of where that ability resides and how it functions at the molecular and genetic levels. It happened in the mid-1990s, at Northwestern University’s department of neurobiology and physiology. There, a laboratory research team led by Joseph S. Takahashi, PhD, isolated and cloned the first mammalian circadian rhythm gene—also known as Clock.
Mapping with Mutant Mice
Now chair of the Department of Neuroscience and an investigator of the Howard Hughes Medical Institute at University of Texas (UT) Southwestern Medical Center, Takahashi was then an up-and-coming researcher who had been working to understand circadian rhythms through pharmacology and cell biology. But those efforts, largely pursued through work with laboratory birds, had “hit a brick wall,” Takahashi told the Proceedings of the National Academy of Sciences, “so it became apparent that we had to move to genetics and molecular biology to continue the search.”
In the 1970s, geneticists Seymour Benzer and Ron Konopka had published seminal circadian rhythm research involving Drosophila, or fruit flies. In Benzer’s lab at the California Institute of Technology, the duo had discovered what’s known as the period (or per) locus in the tiny insects, a gene that, as the name suggests, controls the period of their circadian rhythms. Their colossally important work was “the inspiration for why we went into mouse genetics to find clock genes in mice,” Takahashi tells Sleep Review. But even with a precedent set by two of the field’s greatest figures behind him, the way forward was fraught with challenges.
“Back when we did this, people thought we were crazy that you could actually look for mutants in mice for behavior,” Takahashi says. “At the time, people thought that would be impossible to do.” There were many reasons for his peers’ skepticism, beginning with an almost existential derision of the idea that human behavior, or at least one major facet of it, could somehow be dictated by a single gene. “People don’t like to think our behavior can be reduced to something so simple,” Takahashi says.
But the naysayers did not deter Takahashi and his colleagues Martha Vitaterna, Lawrence Pinto, Fred Turek at Northwestern, and William Dove at the University of Wisconsin, Madison. His team pushed forward, and in the early ’90s, began screening mutant laboratory mice, looking for altered circadian rhythms. Placed in perfect, constant darkness, the mice mostly proved to have very regular circadian rhythms, as evidenced by their penchant for running on a wheel at the same time every 23.7 hours on average, or very close to 24 hours. But one mouse behaved differently, running its wheel approximately every 25 hours, and this anomaly was significant enough to move to the next testing stage.
“At that point, you’re not sure whether it’s real or not—whether it’s genetic,” says Takahashi. “So the next test is: Will it transmit that altered circadian rhythm to its offspring when you test-cross it?” Ultimately, the mouse’s offspring did exhibit the 25-hour cycle, and “that was really very exciting because that confirmed it was a mutation that could be transmitted,” Takahashi says. But his work had only just begun. Confirming a genetic mutation exists is one thing; mapping its location on the genome is entirely another.
“Already at that stage we could tell that [the mutation] was likely to be caused by a single gene, just because of the way the phenotypes segregated in that very first cross,” says Takahashi. Pinpointing the location of that gene, however, was complicated by the fact that “the size of the genome of the mouse is the same as a human,” says Takahashi, “about 3 billion base pairs. And this mutation that causes the [altered circadian rhythms] is a single base-pair change. It’s one change out of 3 billion. That kind of gives you a feeling about what it’s like to try and find that: a needle in a haystack.”
Today’s scientists have access to the fully sequenced genomes of both a mouse and a human, a groundbreaking development that materialized in the early 2000s, years after Takahashi’s team succeeded in isolating the Clock gene. But “in the mid-’90s,” says Takahashi, “we couldn’t just go to the computer and try to look at the genome sequence where the mutation was. You had to actually isolate physically the DNA from that region. It’s kind of like a jigsaw puzzle, because all the DNA fragments that you isolate are short pieces, so you have to put together a mosaic of all those short clones or fragments of DNA to reconstruct the region.”
The process of piecing together those disparate sections of genome into a cohesive region of it is called “physical mapping,” and it is so labor-intensive it took Takahashi’s 10-person team 3 years of focused collective toil, from 1994 to 1997, to isolate and identify the area in which the Clock gene resides. Even then, the results were far from perfect. “We could have made a mistake,” Takahashi says, “and we could have been somewhere else than where we thought we were.”
Fortunately, another method was available of verifying that the section of DNA they had mapped contained the Clock gene. “We had some genetic evidence that the mutation [causing the altered circadian rhythms] could be reversed if we were able to put a normal copy of the gene into a mouse,” Takahashi says. By then, his team had created a strain of Clock mutant mice that carried two copies of the mutant gene and whose circadian periods were off by a whopping 4 hours—28 hours compared to the normal mouse’s 24 hours. Now, they took the region of the genome that they thought contained the gene, cloned it, and put it in the mutant mouse “to see if that would rescue the mutation,” Takahashi says. And sure enough, doing so “completely rescued that behavior. The mouse became completely normal….I can remember it. It was in August of 1996 that we got the first result where we could show that putting this big piece of DNA into a mouse actually repaired it.”
Partnering with Charles Weitz, MD, PhD, in Harvard’s Department of Neurobiology, Takahashi’s team followed up their breakthrough by locating Clock’s partner gene, called BMAL1, which paved the way to describing the molecular mechanism by which the two genes function together, as a unified protein. Acting as a “transcriptional activator” from their position inside the nucleus of cells, the protein CLOCK/BMAL1 directs other genes to transcribe and produce proteins in the cell outside the nucleus at the start of each day. At night, these proteins re-enter the nucleus to meet up again with their regulators and turn the tables, inhibiting CLOCK/BMAL1 and turning the protein off for the night. This transcriptional feedback loop is the “essence of how the clock works,” says Takahashi, and it happens every day, in nearly every cell in the body.
Expressions in Human Health
Before beginning work with Takahashi in 2000, Joseph Bass, MD, PhD, a professor at the Feinberg School of Medicine and chief of Northwestern’s Division of Endocrinology, Metabolism and Molecular Medicine, approached his metabolic research from an “insulin-centric universe,” Bass tells Sleep Review. But “pretty soon after we began systematically looking at the animals that [Takahashi] had characterized at a molecular level, it became apparent that there was this opportunity to bring these two [fields] together, and that was a hugely fortuitous opening for me, and my intellectual focus and excitement was redirected as a result of it, entirely.”
The merging of the disciplines of Bass, an endocrinologist by trade, and Takahashi (which included key contributions from Fred Turek, PhD, as well, director of Northwestern’s Center for Sleep & Circadian Biology) produced yet another extremely fruitful period in Takahashi’s career. Bass’ work in the pathology of glucose regulation led them to try putting the circadian-mutated mice on a Western, high-fat diet, which resulted in two observations that “were foundational for everything we did over the next 15 years,” says Bass. The first was a propensity the circadian-altered mice had for gaining weight when fed high concentrations of fat, which their normal counterparts did not possess, even while placed on the same diet. The second was even more surprising: Even though the mutated animals were susceptible to weight gain, “they never developed what is classically seen in humans with type 2 diabetes,” says Bass. “They always had insulin deficiency. So that told us that [clock] genes are of fundamental importance in the production of insulin.”
As a direct result of Takahashi’s influence, Bass has come to focus “on understanding how clock genes may give us an insight into the molecular effects of sleep loss. And one of our ideas is that the disruption of the clock may be an element that manifests as diabetes and obesity in conditions where people are restricted in their ability to sleep under shift work and so forth.”
Bass’ research is just one development of many to advance the notion that “one of the important functions of the clock is really to fine-tune metabolism in individual cells,” says Takahashi. “We think that may be one reason why the clock is in every cell: If you want to control the metabolism of every cell, then it’s better to do it locally than to have a brain clock, which it has to signal for the body, and do it in an indirect way.”
The implications of a regulatory mechanism that exists in every cell are as far-reaching in medicine as the Clock gene is in the body itself. At the University of California, San Francisco’s Department of Neurology, to cite one example, Louis Ptacek, MD, has directed the research toward familial advanced sleep phase disorder, a circadian condition that causes its victims to have to go to sleep extremely early, about 3 to 4 hours on average earlier than a normal person, and then wake up 3 to 4 hours earlier. “It turns out it’s genetically caused,” says Takahashi, “and the first gene that [Ptacek] found that caused that was a mutation in the PERIOD2 gene—a circadian clock gene. The work from [Ptacek] really showed that in humans, the same clock gene pathway is operating [as in mice] and that it has a very strong effect on the timing of sleep in this particular disorder.”
Additionally, Takahashi explains, common sleep disorders such as shift work disorder and jet lag are “disruptions to our circadian system caused by changing the phase or timing of the clock in an abnormal way. So as we understand the mechanism of the clock better, we’re going to have better ways of either resetting our clocks faster, or trying to correct some of the problems that are caused by misalignment….There are already some medications on the market for that.”
Takahashi also pointed to the work of Eva Schernhammer, MD, DrPH, MSc, MPH, as another particularly promising application of clock-based research to human health. Schernhammer, a professor at both UCLA and Harvard, focuses on linking circadian rhythms with chronic disease, and has uncovered substantial epidemiological evidence for increased cancer risk in people who undergo shift work.
Bass says, “The discovery of the mammalian clock gene…and the full mechanism that then emerged of how the clock works enabled us and positioned us to understand many different processors, ranging from behavioral disorders all the way to peripheral disorders and diseases….Now that we have that ability, which in part relates to [Takahashi’s] work, we can understand many different processes ranging from metabolic disease, inflammation, and even cancer, ultimately.”
One day, it may even help us better understand sleep itself, whose genetic underpinnings largely “remain a mystery,” says Takahashi, “much like the circadian clock field in the early ’90s….Finding ‘sleep genes’ that are analogous to ‘clock genes’ is an important goal for cracking open the molecular mechanism of sleep.”
Freelance journalist Justin W. Sanders wrote the article “Better Nutrition for Better Sleep During Menopause” for our July/August 2014 issue.