Profile of Steve Kay

Blinking mustard plants and glowing fruit flies are just a few of the clever tricks that Steve Kay devised to explore the molecular genetic basis of circadian clocks in plants, flies, and mammals.

Kay, who was elected into the National Academy of Science in 2008, has spent two decades identifying the photoreceptors, genes, and complex networks that make these internal clocks tick.

Kay's work has revealed applications from agriculture to disease. For example, by tweaking one of the clock genes, researchers can engineer crops to alter their growth patterns, ultimately increasing yields and possibly expanding the geographical range where the plants can thrive. Circadian clocks are also important for how plants deal with stresses such as drought and salinity.

Steve Kay

Kay's work linking the clock to human health suggests that chemicals targeting the body's timing system may lead to new treatments for bipolar disorder, diabetes, and cardiovascular disease.

Although Kay, dean of biological sciences at the University of California, San Diego, is best known for his work on circadian rhythms, his inaugural article (1) is a divergence from his mainstream work on circadian networks.

The article is a tribute to his mother, who died >2 years ago of a progressive neurodegenerative disease. The article identifies a novel E3 ubiquitin ligase as the culprit behind some types of motor and sensory neuron degeneration in a mutant mouse model.

Seduced by Nature's Rhythms

Another of nature's rhythms inspired Steve Kay's interest in biology: the extraordinary tides that sculpted his birthplace and childhood home on Jersey, the southernmost of the Channel Islands located between England and France.

On those shores, the sea receded >3 miles at some low tides, he recalled, leaving beaches pockmarked with rock pools of the English Channel. In this environment, he discovered “amazing creatures” and other oddities of marine life, and his interest in science was born.

When he was ≈9 years old, his life changed when a teacher from mainland England brought a microscope to his small elementary school.

“That absolutely blew me away,” he recalled. “I'd never known what was in pond water or what the edges of a torn piece of paper looked like.” By his early teens he knew he wanted to study for a Ph.D.

Although his parents had not pursued college, they were phenomenally supportive of his interest in science.

“When you come from generations of fishermen,” Kay said, “they realize that there are better things to do than being frozen in the middle of the ocean. The North Sea is not the most inviting place in the world, however thrilling!”

Planted in the Beginning

Kay began his studies at the University of Bristol in the United Kingdom, where he earned a bachelor's degree in biochemistry. There he met a Welshman named Trevor Griffiths who would become his Ph.D. supervisor and introduce him to the world of plants.

Griffiths had observed that plants grow differently in the dark than they do in light. Previous studies had found that plants stop producing chlorophyll, the key to transforming light into energy, when grown in darkness. Kay's doctoral project was to identify and characterize the enzyme that catalyzes the light-dependent step of chlorophyll synthesis.

Kay noticed that the concentration of the enzyme seemed to rise and fall throughout a day–night cycle.

Using molecular biology techniques that were just being developed in England and the United States, he discovered, to his surprise, that light regulated the expression of the gene that produced the enzyme for chlorophyll synthesis (2).

To further pursue this research, Griffiths advised Kay to study in the United States. After completing his thesis, Kay secured a postdoctoral fellowship at The Rockefeller University (New York) lab of Nam-Hai Chua, who is a leader in the study of light-dependent gene expression in plants.

“We were collaborating closely with Monsanto on constructing some of the first vectors for making transgenic plants, mostly tobacco and petunias,” he said. “It was very, very exciting.”

Kay and another postdoc named Ferenc Nagy began studying the light-activated chlorophyll a/b binding (CAB) gene. They were trying to discover how plants convey light signals to the nucleus where they rapidly alter CAB expression level.

While recording CAB mRNA levels, the two noticed that their results conflicted. To resolve the discrepancy, they began conducting around-the-clock experiments for several days.

“We discovered that our results were different because I was doing my experiments in the morning, because I'm an early bird, and Ferenc was a night owl and doing his experiments in the evening,” said Kay.

The finding implied that a circadian clock regulated the CAB gene; some mechanism switched it on in the morning and off in the late afternoon.

“That [3] was my first clear glimpse of something called a circadian rhythm,” said Kay. It was 1985 and no one had described circadian rhythms acting at the molecular level in any organism. Researchers had only recently cloned the period (PER) gene in flies, which controlled the insects' 24-h body clock, and researchers had not yet identified such a gene in plants.

Blinking Mustard Plants

To examine the idea of a circadian clock in plants, Kay developed transgenic plants in which he fused a light-dependent promoter to the reporter gene LAC Z. This meant that the LAC Z gene was turned off and on in a circadian manner.

When Kay presented his work at the annual meeting of the Society for Research on Biological Rhythms in 1988 “everybody went absolutely nuts,” he said. “We showed we had a promoter element regulated by the clock.”

Kay's results were among the first observations to suggest that clocks were not just clusters of neurons in the brain that synchronized each other and regulated sleep/wake cycles. Instead, his findings showed that an individual cell could display circadian rhythms and that clocks could regulate intracellular processes such as gene expression.

The circadian field reacted to Kay's work with enthusiasm and excitement. At the time, he understood the importance of his findings and decided to shift his focus to plant circadian rhythms.

Later, while still at Rockefeller, Kay collaborated with geneticist Mike Young to use fly clock genes to search for homologues in plants. When that approach failed, he realized that they had to develop a method to “visualize the circadian rhythms in plants” and developed transgenic Arabidopsis plants.

Kay and graduate student Andrew Millar took photos of the Arabidopsis plants every few hours with a sensitive camera. “We were just shocked and elated to discover we could see the plants glowing on and off in a circadian fashion. That was definitely a ‘eureka!’ moment,” said Kay.

Because the CAB promoter is light-dependent, the plants glowed early in the morning. But when he mutated these glowing plants, he identified individuals that glowed out of sync, for example, once every 21, rather than 24, h.

The first gene Kay and Millar identified from this screen was TOC1, the first clock gene in plants (4). It took Kay another 12 years to figure out what the gene does; it is a transcriptional regulator in Arabidopsis and is recruited to the promoter of other clock genes (5).

The screening method and the discovery of TOC1 led to back-to-back papers in Science in 1995 (4, 6).

Kay examining transgenic Arabidopsis plants. Image courtesy of Kim McDonald, University of California, San Deigo.

“The luciferase tool became very, very powerful and, of course, it ended up being used by the whole circadian field,” said Kay.

Flashing Flies

In 1996, Kay moved to the Center for Biological Timing at the University of Virginia (Charlottesville, VA). Along with trying to identify all parts of the plant clock, he wanted to find more fruit fly clock genes. To this end, he engineered flies with the luciferase gene. When he fed these transgenic flies luciferin they started glowing.

“And so we had these Drosophila running around that were ‘fire-fruit’ flies,” he said.

In collaboration with Jeff Hall at Brandeis University (Waltham, MA), Kay used these flashing flies to search for clock genes. Kay and Hall discovered fly mutants that could not be entrained by light; these flies were glowing all over. This finding led the pair and their collaborators to discover a fly version of a gene called cryptochrome that Kay's lab had been working on in plants.

Kay recalled being perplexed by the glowing flies. Hall and Kay held a “cranio-centric view,” which suggested that that the circadian clock rested in the brain where it controlled behavior. However, when Kay dissected his glowing flies into body parts (heads, thoraxes, and abdomens), each part glowed independently. When he grew these insect body parts in culture, he could use light to reset the rhythmic flashing.

These findings showed that clocks were all over the body and predicted that there should be a widely distributed local photoreceptor. Kay and Hall identified that photoreceptor while screening their mutant flies (7), a discovery Science hailed as one of the top 10 breakthroughs of 1997.

Also in 1997, Joe Takahashi, then at Northwestern University (Evanston, IL) found the mouse circadian clock gene and wanted to find its counterpart in the fly. He and Kay teamed up.

Kay and Takahashi's teams worked together and identified not only the fly CLOCK gene, but the fly partner of CLOCK, a gene called BMAL1.

“It is a complete reversal of what normally happens in biology,” said Kay, meaning that in this case a mouse forward genetics screen led to the identification of new genes in flies.

Kay continued to tease apart to ultimately define a molecular feedback loop. The two then demonstrated that CLOCK and BMAL1 acted to turn on the PER gene and a gene called TIM. They later showed that PER and TIM proteins repressed their own expression by antagonizing CLOCK and BMAL1. This was the first demonstration of a molecular feedback loop defining the core mechanism of the circadian clock (8).

This work was part of an increasingly competitive circadian rhythms field.

“There was this amazing meeting in May in 1998, a clock meeting, where Michael Rosbash's lab, our lab, Takahashi's lab, and Chuck Weitz's lab all got up and described in various versions in mammals and flies this feedback loop at the molecular level, and people were just stunned,” Kay recalled. “That was incredibly satisfying to participate in.”

In 1998, Science again cited Kay's work as one of the breakthroughs of the year and the circadian rhythms field was working overtime, with discoveries about clock genes appearing almost weekly. By then, Kay had moved his lab from Virginia to the Scripps Research Institute (La Jolla, CA), and his team generated a steady stream of fundamental breakthroughs revealing the inner workings of clocks in plants and flies.

“It was really exciting,” he said. “You have to look back at it and say, wow, what a great mix of incredible colleagues, luck, and occasional good judgment.”

From Flies to Mice

In 1999, Kay turned his attention to mammals where understanding of the circadian clock was still relatively rudimentary. To facilitate this shift, he established a second laboratory at the Genomics Institute of the Novartis Research Foundation (GNF) (San Diego).

At GNF, Kay and postdoctoral fellow John Hogenesch took advantage of automation and large-scale genomics technology to identify new clock genes. He and his colleagues quickly realized that the mammalian clock was far more complex and made of multiple loops, not just one feedback loop. This discovery turned out to be universal in all organisms.

The technology at GNF also enabled Kay and his colleagues to characterize the novel photoreceptor melanopsin. In 2002, Kay's group showed that melanopsin in retinal ganglion cells detects light and signals the master circadian oscillator in the hypothalamus, which then signals the rest of the body. This photoreceptor can reset the clock (9).

Kay followed this work in a second collaborative article (10) where he and his colleagues showed that the visual photoreceptors and melanopsin work independently to entrain the clock gene. The article showed that even blind mice lacking rods and cones can detect light, which then sets their body clock. But when both the visual photoreceptors and melanopsin are eliminated, the animals become completely blind to light/dark cycles.

In 2002 Science, once again, cited Kay's melanopsin findings in the Top 10 breakthroughs.

“Those years are, I think, they are almost unbeatable, right?” said Kay. “There were just so many amazing discoveries and, it was a product of intense, but reasonably civil competition, as well as collaboration.”

Clock Disorders and Therapeutics

By 2002, his clock work in animals had shifted exclusively to mammals. In one study (a collaboration with Takahashi and the University of California, San Diego's David Welsh) Kay's team used the luciferase screen to study CLOCK gene expression in individual mammalian cells in culture.

They (11) found that the clock ticks along in liver cells, fibroblasts, and many other cell types. However, the circadian rhythm seems to “run down in whole tissue samples in culture,” said Kay. “Each individual cell expresses a clear rhythm, just slightly different in pace from its neighbors.”

Kay and his team extended those findings in 2007 when they showed that to appreciate the real impact of a clock mutant, researchers must not only look at behavior, but also at single cell phenotypes.

This is because when the cells in the suprachiasmatic nucleus (SCN), a section of the brain's hypothalamus known to contain a “master clock” that controls behavior, are coupled into a network, they are quite resistant to Clock gene mutations. However, if intercellular communication is disrupted, such as in dissociated neurons or cultured cells, the Clock gene knockouts generally show dramatic arrhythmic phenotypes (12).

“Understanding how circadian rhythms interact with other networks could provide new opportunities for treatments.”

Kay is applying this finding by using cell lines to screen for compounds that affect the pace or strength of the circadian clock. Those candidates that affect the circadian clock could be useful tools for probing the clock pathways and might serve as drug candidates for manipulating the clock for therapeutic purposes (13).

Throughout Kay's pursuit of the clock in the fly and mouse, he never abandoned his studies in his favorite model plant, Arabidopsis.

Kay said it is now very clear that the clock network regulates plant growth by issuing the wake-up call that triggers a growth spurt.

“I think it is going to reveal ways where, through a circadian rationale, we're going to be able to influence yield in the fields [5],” he said.

He has also described an “exquisite” mechanism whereby a 24-h clock is used as a seasonal timer. “It is [some of the most] satisfying work that I've ever done in my life,” Kay said.

A Tribute

When it comes to Kay's inaugural article, he notes, “It's one-off, opportunistic … essentially a nod to my mum.”

In 2006 his mother died of a very aggressive motor neuron disease called progressive bulbar palsy. The disease closely resembled a disease that Kay and a large team of collaborators from Phenomix identified and characterized in a mouse mutant.

So, with the mouse model, the team proceeded to clone the defective gene, an E3 ubiquitin ligase named Listerin that is involved in motor and sensory neuron degeneration when mutated.

“I had a strong personal motivation to follow up on this mouse mutant, inspired by the suffering that I saw my mum go through,” he said. “And, I think, she was very keen that I would contribute something in this field.”

Meanwhile, his mammalian clock research is yielding discoveries ripe for use in medicine. Agricultural biotechnology companies are keen to collaborate and translate Kay's discoveries in Arabidopsis to crop plants to improve yield.

Research has shown that circadian rhythms influence cancer treatment and Kay believes they also play a role in metabolic diseases. He has been investigating the clock's role in the liver and how it regulates hepatic glucose output, which may reveal a connection between the clock and diabetes.

In a recent study (14) Kay's laboratory together with John Hogenesch have performed a genomewide siRNA screen for clock modulators in human cells.

“This work has revealed that the clock is functionally interconnected with many fundamental signaling modules in cells” said Kay. Of relevance is their discovery of tight links between clock function and insulin signaling.

“Understanding how circadian rhythms interact with these other networks could provide new opportunities for treatments,” said Kay. The clock is ticking and he is not missing a beat.