Skiv2l2Positively Regulates The Circadian Clock Through Binding Clock And Bmal In Zebrafish

Most organisms on the Earth exhibit biological rhythms, i.e., circadian rhythmswith a period of approximately24-hour, which are generated and maintenaned by thecircadian clock. The circadian clock regulates a variety of fundamental life processesincluding body temperature, sleep-wake cycle, metabolism and hormone secretion. Thus,a full understanding of mechanisms and regulatory pathways of the circadian clock iscritical for accounting for the normal physiological and biochemical activities and alsoproviding insights into prevention and treatment of dysrhythmias.The zebrafish represents one of the most important model organisms for studyingkey aspects of the vertebrate circadian system. Like that in Drosophila and mouse， Acurrent zebrafish clockwork model is a transcription/translation-based negativefeedback loop composed of the Clock:Bmal heterodimer and Per:Cry heterodimer. Inaddition to these core circadian clock genes, numerous genes have recently beenrevealed to regulate the circadian clock.In Neurospora, the RNA helicase, FRH, is known to form FFC with FRQ andrepress the activation of WWC-mediated transcription. The specific role of FRH incircadian regulation, however, remains not fully understood. Here phylogenetic analysisshows that skiv2l2(superkiller viralicidic activity2-like2) is zebrafish orthologous geneof frh. A previous study shows that skiv2l2plays a role in larval melanocyteregeneration in zebrafish. However, whether skiv2l2is involved in the zebrafishcircadian clock is yet unknown. Quantitative real-time-PCR shows that skiv2l2mRNAis rhythmically expressed in both the larvae and liver in a robust manner, and it isup-regulated in per1b mutant but down-regulated in bmal1b mutant. We also observethat the skiv2l2promoter harbors E-box elements. Luciferase assays show that thetranscriptional activity of the skiv2l2promoter is enhanced by the Clock1a:Bmal1bheterodimer but repressed by Cry1.1a. Chromatin immunoprecipitation (ChIP) assaysshow that Bmal1b binds to the E-boxes in the skiv2l2promoter. These results suggest that the transcription of skiv2l2is directly regulated by the circadian clock.On the other hand, although the skiv2l2homozygous mutant fish cannot move withtheir malformed swimming bladders, the skiv2l2heterozygous mutant fish appearnormal. The locomoter activity assays show that the skiv2l2heterozygous mutant fishdisplay the abnormalities of swimming rhythms. Key circadian clock transcripts,including per1a, per1b, per2, per3, cry1.1a, cry1.1b, cry1.2a, cry1.2b, andclock-controlled genes, annat2and pck1, are all reduced in skiv2l2homozygous mutant,suggesting that skiv2l2plays an essential role in the zebrafish circadian clock.Furthermore, in vitro cell transfection experiments show that over-expression of skiv2l2activates the Bmal1b:Clock1a-mediated transcription. Furthermore, co-IP assays showthat Skiv2l2binds Clock1a and Bmal1b, rather than Per2or Cry1.1a. Taken together,our results show that skiv2l2is a new clock-controlled gene in zebrafish and Skiv2l2positively activates the transcription of Clock1a:Bmal1b. Thus, skiv2l2is a newessential element in the zebrafish circadian clock.