Sixteen years after scientists found the genes that control the circadian clock in all cells, the lab of 黑料网鈥檚 Aziz Sancar, MD, PhD, discovered the mechanisms responsible for keeping the clock in sync.
Researchers at the 黑料网 have discovered how two genes 鈥� Period and Cryptochrome 鈥� keep the circadian clocks in all human cells in time and in proper rhythm with the 24-hour day, as well as the seasons. The finding, published September 16, 2014 in the journal Genes and Development, has implications for the development of drugs for various diseases such as cancers and diabetes, as well as conditions such as metabolic syndrome, insomnia, seasonal affective disorder, obesity, and even jetlag.
鈥淒iscovering how these circadian clock genes interact has been a long-time coming,鈥� said Aziz Sancar, MD, PhD, Sarah Graham Kenan Professor of Biochemistry and Biophysics and senior author of the Genes and Development paper. 鈥淲e鈥檝e known for a while that four proteins were involved in generating daily rhythmicity but not exactly what they did. Now we know how the clock is reset in all cells. So we have a better idea of what to expect if we target these proteins with therapeutics.鈥�
In all human cells, there are four genes 鈥� Cryptochrome, Period, CLOCK, and BMAL1 鈥� that work in unison to control the cyclical changes in human physiology, such as blood pressure, body temperature, and rest-sleep cycles. The way in which these genes control physiology helps prepare us for the day. This is called the circadian clock. It keeps us in proper physiological rhythm. When we try to fast-forward or rewind the natural 24-hour day, such as when we fly seven time zones away, our circadian clocks don鈥檛 let us off easy; the genes and proteins need time to adjust. Jetlag is the feeling of our cells 鈥渞ealigning鈥� to their new environment and the new starting point of a solar day.
Previously, scientists found that CLOCK and BMAL1 work in tandem to kick start the circadian clock. These genes bind to many other genes and turn them on to express proteins. This allows cells, such as brain cells, to behave the way we need them to at the start of a day.
Specifically, CLOCK and BMAL1 bind to a pair of genes called Period and Cryptochrome and turn them on to express proteins, which 鈥� after several modifications 鈥� wind up suppressing CLOCK and BMAL1 activity. Then, the Period and Cryptochrome proteins are degraded, allowing for the circadian clock to begin again.
鈥淚t鈥檚 a feedback loop,鈥� said Sancar, . 鈥淭丑别 inhibition takes 24 hours. This is why we can see gene activity go up and then down throughout the day.鈥�
But scientists didn鈥檛 know exactly how that gene suppression and protein degradation happened at the back end. In fact, during experiments using one compound to stifle Cryptochrome and another drug to hinder Period, other researchers found inconsistent effects on the circadian clock, suggesting that Cryptochrome and Period did not have the same role. Sancar, a member of the 黑料网 Lineberger Comprehensive Cancer Center who studies DNA repair in addition to the circadian clock, thought the two genes might have complementary roles. His team conducted experiments to find out.
Chris Selby, PhD, a research instructor in Sancar鈥檚 lab, used two different kinds of genetics techniques to create the first-ever cell line that lacked both Cryptochrome and Period. (Each cell has two versions of each gene. Selby knocked out all four copies.)
Then Rui Ye, PhD, a postdoctoral fellow in Sancar鈥檚 lab and first author of the Genes and Development paper, put Period back into the new mutant cells. But Period by itself did not inhibit CLOCK-BMAL1; it actually had no active function inside the cells.
Next, Ye put Cryptochrome alone back into the cell line. He found that Cryptochrome not only suppressed CLOCK and BMAL1, but it squashed them indefinitely.
鈥淭丑别 Cryptochrome just sat there,鈥� Sancar said. 鈥淚t wasn鈥檛 degraded. The circadian clock couldn鈥檛 restart.鈥�
For the final experiment, Sancar鈥檚 team added Period to the cells with Cryptochrome. As 笔别谤颈辞诲鈥檚 protein accumulated inside cells, the scientists could see that it began to remove the Cryptochrome, as well as CLOCK and BMAL1. This led to the eventual degradation of Cryptochrome, and then the CLOCK-BMAL1 genes were free to restart the circadian clock anew to complete the 24-hour cycle.
鈥淲hat we鈥檝e done is show how the entire clock really works,鈥� Sancar said. 鈥淣ow, when we screen for drugs that target these proteins, we know to expect different outcomes and why we get those outcomes. Whether it鈥檚 for treatment of jetlag or seasonal affective disorder or for controlling and optimizing cancer treatments, we had to know exactly how this clock worked.鈥�
Previous to this research, in 2010, Sancar鈥檚 lab found that the level of an enzyme called XPA increased and decreased in synchrony with the circadian clock鈥檚 natural oscillations throughout the day. Sancar鈥檚 team proposed that chemotherapy would be most effective when XPA is at its lowest level. For humans, that鈥檚 late in the afternoon.
鈥淭his means that DNA repair is controlled by the circadian clock,鈥� Sancar said. 鈥淚t also means that the circadian clocks in cancer cells could become targets for cancer drugs in order to make other therapeutics more effective.鈥�
This research was funded by the National Institutes of Health and the Science Research Council and Academia Sinica in Taiwan.
Other authors of the Genes and Development paper are 黑料网 postdoctoral fellows Yi-Ying Chiou, PhD, and Shobhan Gaddameedhi, PhD, and 黑料网 graduate student Irem Ozkan-Dagliyan.
Written by : Mark Derewicz, 919-923-0959, mark.derewicz@unchealth.unc.edu