Unwinding the clock
Carrie Partch breaks circadian rhythms
When the circadian clock stops, most healthy cells wither and cancer cells thrive. The clock is our body’s 24-hour timekeeping system that helps regulate everything from insulin levels to cell division. For years, scientists wondered how cancer cells dodged the circadian cycle. Then, Carrie Partch, UC Santa Cruz assistant professor of biochemistry, discovered a protein with the unwieldly name of PER-ARNT-SIM domain containing 1 (or PASD1) and began to understand what made that clock tick.
Partch was the first to show that PASD1 can stop circadian rhythms. Her study findings, published in Molecular Cell, strengthen the link between disrupted clock function and cancer. Her research also points toward a new target for cancer therapies.
The relationship between cancer and the clock is just one of the questions driving Partch’s research. Her lab creates high-resolution “snapshots” to see how globs of proteins interact with each other and the cell’s DNA to keep the clock running. The health implications are big; many chronic illnesses, including diabetes and heart disease, arise from irregular circadian rhythms. Uncovering the underpinnings of the clock could improve medications that target disrupted circadian-controlled genes.
“There are so many important questions to answer that haven’t been dealt with yet,” Partch said of the potential discoveries in the circadian field. “It’s not low-hanging fruit; the fruit is so ripe it’s falling off!”
For billions of years, life on Earth has abided by a strict solar schedule. In animals, plants, and even single-celled bacteria, the circadian clock ticks tirelessly over 24 hours, driving daily cellular rhythms to match the planet’s light and dark cycles.
In people, a “master clock” contained in the hypothalamus of the brain takes light cues from the Sun to send a ripple of chemical and temperature signals that tell the body’s tissues when to turn on, or off, the right genes. These circadian-controlled genes regulate metabolism, blood pressure, hormone levels, and most other aspects of our physiology.
“The clock has evolved, I think, as a mechanism to help us prepare for the profound transitions from being upright and active to horizontal and sleeping,” said John Hogenesch, professor of systems pharmacology and translational therapeutics at the University of Pennsylvania, in Philadelphia. Hogenesch discovered two of the four core proteins that regulate the circadian cycle in cells.
The 24-hour feedback loop starts when the proteins CLOCK and BMAL1 bind together to activate the production of two more proteins: period (PER) and cryptochrome (CRY). After accumulating in the cell, at just the right time PER and CRY bind together. This new PER:CRY complex migrates into the cell’s nucleus to block the CLOCK:BMAL1 complex; this binding stalls further transcription until the proteins degrade and the cycle starts anew.
Studies have shown that a dysfunctional circadian clock leads to health problems, including diabetes, neuro-degenerative diseases, and cancer. Disruption usually occurs when something disturbs the complex interactions of proteins driving the clock.
The only human cells without circadian rhythm are embryonic stem cells and some cancer cells. They share one thing in common: the PASD1 protein. For years, scientists assumed there was no built-in way to shut down these vital circadian rhythms, but Partch had a hunch that this protein was the key.
Proteins are made up of multiple units called domains that fit together like Legos, said Partch. The shape of the domain determines which molecules the protein can interact with. PASD1 is structurally similar to the clock protein except it’s missing the domain that binds to DNA. This made Partch think that it could block the CLOCK protein’s ability to activate genes. When she started her lab at UC Santa Cruz, Partch began a series of experiments to test her hypothesis.
In a healthy cell, scientists can see the clock “ticking” by fusing a bioluminescent tag to one of the core clock proteins and then tracking it through the course of one day. “We can watch the light being emitted in these beautiful waves of the clock,” said Partch. She traced an imaginary sine wave. Each peak represented a burst of gene expression every 24 hours, she explained.
In a tumor cell, the waves are irregular and weak, a clear sign that circadian rhythms are not functioning at full steam.
The success of this project drew Alicia Michael, one of Partch’s first graduate students, to the lab. “It got me really excited that we could basically change the Wikipedia page if we figured out what it does,” said Michael, lead author of the paper in Molecular Cell.
Partch and Michael altered the PASD1 gene on tumor cells to either overproduce the PASD1 protein or prevent its expression. The cancer cells without the PASD1 protein began to emit more regular, glowing waves, demonstrating the protein’s disrupting effect on the circadian cycle. They think that PASD1 binds to the CLOCK and BMAL1 proteins, blocking circadian gene expression.
Cancerous cells have an advantage by shutting off the clock, said Hogenesch, who was not involved in the study. “If the cell wants to keep dividing and dividing and dividing, maybe it doesn’t want to be told it has to divide at one time of the day.”
Germline stem cells are the only healthy cells that produce PASD1, so when the protein shows up outside those cells, the immune system takes notice. That reaction means that targeting PASD1 could lead to cancer treatments, said Hogenesch.
Partch first learned the power of protein structures as a graduate student in the lab of biochemist Aziz Sancar at the University of North Carolina School of Medicine, in Chapel Hill.
Sancar may be best known for his Nobel Prize–winning work on a bacterial protein that uses blue light to repair UV-damaged DNA. But, years ago, he was interested in cryptochrome because of its similarity to the DNA repair enzyme. He discovered cryptochrome didn’t repair DNA; instead, it was one of the core clock proteins. Moreover, unlike most proteins, which stacked neatly together, cryptochrome had a strange, wiggly tail. Sancar wanted to know what the heck it did, Partch recalled.
With Sancar’s encouragement, she collaborated with a structural biology lab that made high-resolution images of the oddly built protein. They showed that in plants, exposure to light made the cryptochrome release an intrinsically disordered tail; a dynamic, flexible domain that tells the plant it’s time to rise and shine.
“That has been really key to understanding how cryptochrome works, so she gets full credit for it,” said Sancar. This insight into biochemical problems keeps Sancar sending his research papers to Partch for her opinion before he submits them to journals for publication.
Structural biologists approach the clock by taking high-resolution pictures to try to tease out the details of protein interaction at an atomic level, said Ning Zheng, professor of pharmacology at the University of Washington, in Seattle.
Zheng was the first to solve the overall structure of cryptochrome in mammals, but was missing key information about the mobile element. It was difficult to find an imaging technique that could capture precisely how the flexible tail region engaged in protein interactions.
“Why do we want to know the details? The clock is super important to all living systems. In humans, it’s highly related to health,” said Zheng.
Partch nailed down cryptochrome’s movement—solving how the protein works as the “pause button” of the clock—using nuclear magnetic resonance (NMR) spectroscopy. The technique picks up radio signals emitted in a magnetic field from every amino acid, the molecular building blocks that make up a protein. The signals build a “fingerprint” of the protein; throw another protein into the mix and the fingerprint will change. The method tells you where important interactions are happening on the protein, an essential tool to guide experiments, said Partch.
To spotlight these key places, Partch made mutations at the site where BMAL1 and cryptochrome interacted. When they strengthened the connection of the two proteins, the circadian clock extended another hour. When they weakened the interaction, the circadian clock got as short as 19 hours, or fell apart altogether. This revealed that the binding site was crucial to holding off transcription until just the right time. The study was published last year in Nature Structural and Molecular Biology.
Our understanding of the circadian clock is transforming the way we treat disease, said Hogenesch. For instance, cholesterol is on a circadian schedule and its production peaks while we sleep. Since many cholesterol-lowering medications are only active for a few hours at a time, taking the drugs at bedtime works best to prevent heart disease. In a 2014 study, Hogenesch found that hundreds of FDA-approved drugs targeted circadian genes, suggesting that many other medications could improve with dosing timed to the body’s clock instead of the wall clock.
Partch’s lab is now developing direct drug-screening approaches to find new therapies aimed at the molecular underpinnings of the clock. For example, scientists know that high-fat diets can weaken the circadian rhythms’ controlling metabolism. By pinpointing how two proteins come together to regulate that circadian-controlled gene, perhaps a molecule could be found to strengthen those clock rhythms and override the effects of the high-fat diet.
“It’s not just lip service,” said Partch. “Direct screening would save us years of asking what this amazing molecule does in the black box of the cell.”
Beyond understanding how pieces of clock proteins interact, Partch and her graduate students are asking a more fundamental question: “How do these different protein complexes change over time to give rise to the timekeeping itself?”