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RNA Screen IDs Pathways Influencing Human Circadian Clock

NEW YORK (GenomeWeb News) – The insulin metabolism pathway and others are not only regulated by — but also regulate — the circadian clock, according to a paper appearing in the advance, online edition of Cell today.

Researchers from the Universities of California at San Diego, Pennsylvania, and Memphis, as well as the Novartis Research Foundation did a genome-wide screen for circadian clock-related genes in a human cell line using a small interfering RNA library. In so doing, they identified hundreds of genes whose knockdown corresponded to changes in the circadian cycle.

Based on network and pathway analyses of the genes involved, the team concluded that the clock is linked to pathways controlling everything from insulin and folate metabolism to the cell cycle. And because genes in the insulin pathway seemed to regulate the activity of clock genes, the researchers argue that it may eventually be possible to treat conditions such as jet lag by tweaking the clock via related biological pathways.

"What came out very strongly was this close relationship between circadian regulation and insulin signaling," co-senior author Steve Kay, a cell and developmental biology researcher at UCSD, said in a statement. "There's a reciprocal relationship between circadian dysfunction and metabolic dysfunction."

The circadian clock, which governs sleeping and wake cycles over each 24-hour period, has been investigated in several animals. Studies in mammalian and Drosophila model organisms have unraveled complex genetic, biochemical, and cellular components of circadian rhythms, the researchers explained. But despite similarities in the circadian clocks found in humans and model organisms, differences remain.

"Although the genetic architecture of the clock is similar in human, mouse, and Drosophila … for a particular gene itself, [the clocks] are quite different," co-lead author Eric Zhang, a post-doctoral researcher in Kay's lab, who is also affiliated with the Novartis Research Foundation's Genomics Institute, told GenomeWeb Daily News.

For the current study, the team focused on a human osteosarcoma cell line called U2OS, which is known to have a robust circadian clock function, Zhang explained. They screened for circadian clock-related genes in the U2OS cells using a Qiagen siRNA library consisting of 89,872 siRNAs that targeted 17,631 known and 4,837 predicted genes.

Using a reporter assay involving a luciferase gene whose activity relied on two clock genes — the transcriptional activator Bmal1 and the repressor Per2 — the team was able to pick out siRNAs that altered the clock. To account for off-target siRNA effects, the researchers limited their analysis to genes targeted by at least two siRNAs detected in the study, Zhang noted.

Overall, they found 343 genes linked to changes in circadian phenotypes such as the length and amplitude of circadian rhythms, including most known circadian clock components.

After narrowing in the 200 or so most likely candidates, the researchers did network and pathway analyses aimed at understanding how the clock relates to other biological processes. In so doing, they found that changes in the clock were often linked to genes involved in insulin and hedgehog signaling, folate metabolism, and the cell cycle.

In particular, the team was intrigued by the finding that several insulin-related genes appear to regulate the clock. Past studies have identified links between insulin and clock pathways. But the new finding suggests that the pathways actually regulate one another.

"Clock biologists all appreciated that the communication went one direction — from the clock to biological processes — but I don't think anyone anticipated that there would be this level of integration with cell metabolism and the cell cycle, or all these other pathways impinging on clock function," co-senior author John Hogenesch, a pharmacology researcher at the University of Pennsylvania, said in a statement.

While some of these genes are likely tissue specific, co-lead author Tsuyoshi Hirota, a post-doc in Kay's lab who is also affiliated with Novartis Research Foundation, told GenomeWeb Daily News, others are expected to govern clock function in other tissues as well.

In the future, the team plans to do similar screens in other cell lines. They are also combining chemical and genomic screens aimed at pinpointing even more clock-related genes, Hirota explained.

Zhang also noted that the list of genes they detected includes potentially druggable targets. For instance, he explained, by targeting genes that shorten or lengthen circadian cycles, it may be possible to treat individuals with fast or slow clocks. On the other hand, targeting genes that affect clock amplitude could theoretically treat conditions such as sleep disorders or jet lag.

"Understanding this close relationship between circadian regulation and metabolic homeostasis should provide novel ways of identifying new therapies for metabolic disease," Kay said in a statement. Kay is currently on the advisory board for the San Francisco-based company, ReSet Therapeutics, which hopes to come up with new treatments for problems related to circadian rhythm disruptions.

Data from the current study, along with expression data and gene structure information, are available online through a searchable, open access database called BioGPS.

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