The circadian clock coordinates an animal’s entire body with the 24-hour cycle of the day using a circuit of transcription factors. But different tissues show distinct patterns of circadian gene expression, and it has not been clear how broadly expressed transcription factors interact with local networks of transcription factors to control tissue-specific circadian rhythms.
In a study published in Current Biology, Alexander Stark of the Research Institute of Molecular Pathology in Vienna, Austria and his team analyzed how globally expressed transcription factors known as CLOCK and CYCLE interact with tissue-specific transcription factors in the fruit fly (Drosophila melanogaster) (1). “This sort of study has been long overdue,” said Krish Krishnan of Mississippi State University, who was not involved in the study. “Circadian biologists using the Drosophila model system have already reported differential rhythmic expression of genes in heads versus bodies, but this study provides the answer to how and why there is differential tissue-specific regulation.”
Stark and his team used ChIP-seq—a method that combines chromatin immunoprecipitation with massively parallel DNA sequencing—to identify the CLOCK and CYCLE genome-wide binding sites in fly heads and bodies. They found that CLOCK and CYCLE bind to distinct targets in these different tissues. Using gene ontology analysis, the researchers observed that the CLOCK and CYCLE binding targets common to both tissues were located next to genes that play a role in regulating transcription. However, genes next to head-specific binding sites play a role in behavior and vision, whereas genes near body-specific binding sites are involved in metabolism.
These findings suggest that CLOCK and CYCLE are master regulators at the top of a transcription factor hierarchy, but they also drive tissue-specific functions. “Since the key circadian activators in Drosophila have mammalian orthologs, tissue-specific clock outputs are likely regulated the same way as in Drosophila,” said Paul Hardin of Texas A&M University, who wrote an accompanying Dispatch article in Current Biology (2).
Using computational analyses, Stark and his team identified sequence motifs that differentiate CLOCK and CYCLE binding sites in the head versus the body. They found that the opa motif was enriched in head-specific binding sites, whereas the GATA motif was enriched in body-specific binding sites. In fruit fly cells, they found that Serpent—a transcription factor that binds to the GATA motif—activated a CLOCK and CYCLE binding site specific to the body. On the other hand, the presence of OPA—a transcription factor that binds to the opa motif—was crucial for the ability of CLOCK to activate its head-specific binding sites. These findings suggest that CLOCK and CYCLE act synergistically with local transcription factors to regulate gene expression in specific tissues.
According to the authors, transcription factors such as Serpent and OPA could partner with CLOCK and CYCLE to control specific clock functions in different tissues. But experts say that more experiments are necessary to flesh out the relationship between this transcription factor network and circadian rhythms. “One prime drawback would probably be that the authors chose only one time point of maximal binding of CLOCK,” Krishnan said. “As a circadian biologist, I would have liked to see a full circadian profile of CLOCK binding.”
Moving forward, improvements in the techniques could allow scientists to take a closer look at how CLOCK and CYCLE regulate circadian rhythms. “As the technology advances, it will become easier to perform these genome-wide sequencing techniques using very small amounts of biological material,” said Jennifer Mohawk of the University of Texas Southwestern Medical Center, who was not involved in the study. “This should allow for even finer resolution in terms of cell-type specificity—perhaps allowing us to look at clocks within single cells.”
In the end, these types of studies could have an impact beyond revealing transcriptional circuits in cells and tissues. “Looking ahead, this research may lead to new ways to manipulate circadian clocks at the level of a single organ or tissue type,” Mohawk said. “This could have important implications for many physiological processes, including sleep, hormone release, metabolic function, and disease states—such as depression, Alzheimer’s disease, and some types of cancer—that have a circadian component.”
1. Meireles-Filho AC, Bardet AF, Yáñez-Cuna JO, Stampfel G, and Stark A. 2014. cis-Regulatory Requirements for Tissue-Specific Programs of the Circadian Clock.
Curr Biol. 24(1):1-10. doi: 10.1016/j.cub.2013.11.017.
2. Menet JS and Hardin PE. Circadian clocks: the tissue is the issue. Curr Biol. 24(1):R25-7. doi: 10.1016/j.cub.2013.11.016.