Study Reveals Mechanism for Maintaining Circadian Rhythms in Mammals

By Eric Sauter

Scientists from The Scripps Research Institute and RIKEN, a Japanese research institute, have validated a key step in the mechanism of the circadian clock in mammals. Many critical human activities including sleeping, eating, and even hormonal activity are determined by circadian rhythms, fundamental functions that adapt to the cycle of light and dark and are controlled at the genetic level. Along with some accessory factors, clock genes work in concert to generate circadian rhythms over a 24-hour period.

The study, published in the journal Nature Genetics, provides for the first time direct evidence that proteins expressed by clock genes join together to inhibit their own transcription in the cell nucleus, bringing the active clock to a halt. This inhibitory process creates a self-regulating feedback loop that drives the gears of the mammalian clock and starts it running again according to the 24-hour rhythm. Transcription is the coding of DNA information to an RNA molecule to produce proteins.

The study’s findings could have a significant impact on the development of novel treatments for sleep disorders as well as new therapeutic approaches to other serious diseases, including cancer.

According to John Hogenesch, who initiated the research at the Genomics Institute of the Novartis Research Foundation and continued it as a professor of biochemistry at The Scripps Research Institute’s new Florida campus, evidence for the inhibition or repression of the gene transcription process has been difficult to obtain.

“Until our study,” Hogenesch said, “proof of the need for transcriptional inhibition to maintain the 24-hour circadian rhythm was elusive. To prove it, we developed a novel molecular genetic screen in mammalian cells to identify mutants of two key transcriptional activator proteins that were immune to inhibition and maintained normal activity. After screening a library of 6,000 mutant forms of activator proteins, we found five clones that were not affected by repression activity. When tested in vitro we found they completely disrupted the circadian rhythm. These data and other supporting information provide direct evidence that feedback repression is a requirement for proper mammalian clock function.”

The work of characterizing the cloned proteins in mammalian cells was led by co-corresponding author Hiroki Ueda, from the RIKEN Center for Developmental Biology in Kobe, Japan, Hogenesch said. Founded in 1917 as a private foundation, RIKEN was reorganized in 2003 as an independent administrative institution under the Japanese Ministry of Education, Culture, Sports, Science, and Technology. The RIKEN laboratory focused on the use of high throughput imaging of the clock mutant genes to provide the critical data demonstrating requirement of feedback repression.

Formal proof of this fundamental clock regulating mechanism, Ueda said, has implications that go beyond the value of the initial discovery: “If we could find a way to manipulate this particular element in the circadian rhythm process, it could one day provide new therapeutic opportunities in areas like sleep disorders, especially for those people whose lives are disrupted because of shift work.”

The study, Hogendesch said, took two years to complete, and used an innovative technology to determine the underlying basis of the feedback repression loop.

“While our discovery solves a major issue in mammalian clock biology,” Hogenesch said, “one of the most important aspects of the study was the method we developed to uncover the affects of mutagenesis in mammalian cells. While we utilized the same screening techniques used by bacterial researchers for decades—mutating a single gene and then screening for particular functions—this has not been done before in simple assays with mammalian cells. But with this study, we have moved well beyond the single gene approach—we did it in multiple genes and created easier assays to uncover their functions. Ultimately, we believe that this new cellular genetics technology will have a significant impact on the study of mammalian biology.”

Other authors of the study include Trey K. Sato, Genomics Institute of the Novartis Research Foundation and Scripps Research Department of Genome Technology; Tetsuya J. Kobayashi, Hiroki R. Ueda, Hideki Ukai and Rikuhiro G. Yamada of the RIKEN Laboratory for Systems Biology and Center for Development Biology; Julie E. Baggs, Scripps Research Department of Biochemistry; Loren J. Miraglia, Genomics Institute of the Novartis Research Foundation; David K. Welsh, Scripps Research Department of Cell Biology, Department of Psychiatry, University of California, San Diego, and The Veterans Administration San Diego Healthcare System; and Steve A. Kay, Scripps Research Departments of Cell Biology and Biochemistry.

The research was supported by the Novartis Research Foundation; a Rena and Victor Damone Postdoctoral Fellowship from the American Cancer Society; the National Institutes of Health; Scripps Florida; the RIKEN Center for Developmental Biology; RIKEN Strategic Programs; and a New Energy and Industrial Technology Organization (NEDO) Scientific Research grant and Scientific Research and Genome Network Project grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology.

Send comments to: mikaono[at]scripps.edu

"Until our study, proof of the need for transcriptional inhibition to maintain the 24-hour circadian rhythm was elusive."

—John Hogenesch