The Role Of Pgc-1α In The Zebrafish Circadian Clock

The circadian clock exits in almost all living things, from single-celled organisms to higher animals and plants on the Earth. The circadian clock broadly refers to the time-keeping mechanisms evolved from adaptation of life to the cyclic environment of the Earth. Life processes from gene expression, all kinds of metabolic activities and behaviors of life are subject to the regulation of the circadian clock. In addition, clinical studies reveal that disorders of circadian clock malfunctions can lead to various physical diseases, such as metabolic disorders, sleep disorders, cancer and early aging. Therefore,elucidating the circadian regulatory mechanisms is of great significance to human health.The circadian clock is composed of three pathways, the input pathway, the output pathway and the core oscillator. The molecular mechanism of the fish circadian clock is also a transcription/translation feedback loop regulated by multiple genes at the single-cell level, similar to that of the mammal circadian clock. First, the Clock:Bmal complex, as a positive regulatory e element, binds to the E-box or E’-box in the promoters of the negative regulatory element such as per and cry, and other clock genes,to activate their transcription in the nucleus. Then, a heterodimer Per: Cry formed in the cytoplasm enters the nucleus to turn off the activation of the Clock:Bmal complex,thereby completing the feedback loop.The peroxisome proliferator-activated receptor gamma coactivator 1(PGC-1)family possesses three members, PGC-1α, PGC-1β and PRC. The PGC-1 family is involved primarily in energy metabolism, mitochondrial proliferation and lipid metabolism, body temperature, sugar metabolism and so on. PGC-1 can bind to the nuclear receptors to recruit other regulatory factors, and together they form the complex to drives or enhances the transcription of target genes.PGC-1α as one of the three-member PGC-1 family is shown to contribute to energy metabolism and mitochondrial proliferation, and in particular mice PGC-1α is known to serves as PGC-1α works as an important link between the circadian clock and energy metabolism. However, the functions of zebrafish pgc-1α have not beendetermined thus far.In this study, qRT-PCR analysis show that pgc-1α oscillates robustly at mRNA levels under both DD and LD conditions, suggesting that pgc-1α is controlled by the circadian clock in zebrafish. Analysis of the promoter sequence shows that the pgc-1αpromoter harbors D-box and E-like box. Luciferase reportor assays show that that the co-transfection of Bmal1 b and Clock1 a can activate the transcription of pgc-1α, which can be suppressed by Cry1aa; and that Tefa can also promote the expression of pgc-1α,which can be inhibited by E4bp4-4. We also showed that pgc-1α is significantly down-regulated in clock1 a mutant fish. These results suggest that pgc-1α is regulated directly by the circadian clock through both E like box and D-box in zebrafish.To further study the functions of Pgc-1α, we generated pgc-1α mutant zebrafish with TALEN. qRT-PCR result showed circadian clock genes bmal1 a, bmal1 b and bmal2 are significantly down-regulated; and clock1 a, cry1 aa, per1 b, per2, per3 and rorαa are also changed in pgc-1α mutant fish. Cell transfection luciferase asaays indicate that pgc-1α can also enhance the activation of Rorα on bmal1 b, which can be inhibited by Rev-erbα. Behavioral experiments show that pgc-1α mutant fish display significantly higher amplitude of locomotor activities than wild types under both LD and DD conditions. Moreover, we also revealed that Pgc1α appears to have tissue-specific regulatory role in different adult fish organs. These results suggest that Pgc-1α play an impotant role in the zebrafish circadian clock. Metabolism-related genes pepck,ucp1,pparα, srebf1 are down-regulated.Taken together, our findings indicate that Pgc-1αplays an important roles in the circadian clock and metabolism in zebrafish.