Zebrafish Circadian Clock Gene Clock1a Is Regulated At Transcriptional And Post-transcriptional Levels

In 1994,the Clock gene was first identified in mice,subsequently the Clock homologs have been isolated in fruit flies and zebrafish.Although they share many similarities,these Clock homologs also display many differences.For instance,in most tissues,clock oscillates in zebrafish and flies but not in the mouse SCN(Suprachiasmatic Nucleus).The striking differences in the pattern of Clock expression between mice and zebrafish implicate possible diverse modes of regulation of Clock in different species.While regulation of expression of circadian clock genes has long been studied at the transcriptional level,post-transcriptional mechanisms driving rhythmic protein expression have recently been paid attention.Our study is to explore whether zebrafish clock1 a is regulated at both transcriptional and post-transcriptional levels.At the transcriptional level,luciferase reporter assays demonstrated that the combination of Clock1 a and Bmal1 b enhances clock1 a expression and Cry1 aa represses this effect through E-box,Ror?a up-regulates clock1 a expression and Rev-erb? suppresses it through RORE,and Fox O3 a also can promote expression of clock1 a.At the post-transcriptional level,bioinformatics analysis revealed that clock1 a is a possible direct target of mi R-148.We found that mi R-148 exhibits rhythmic expression in zebrafish,luciferase reporter assay showed that mi R-148 can be activated by the Clock1a-Bmal1 b heterodimer,and mi R-148 also down-regulates clock1 a expression via binding to 3' UTR,and deletion of their binding site abolished this suppression.We also used CRISPR-Cas9,an effective genome-editing tool,to knock out mi R-148 in zebrafish.Behavioral assays conducted under light-dark(LD)condition showed that the total locomotor distance of mi R-148-/-mutant fish is significantly higher than that of wild types,the direct opposite of our previous observation of clock1a-/-mutant fish.Specifically,under constant dark(DD)and constant light(LL)conditions,the period of locomotor activities of mi R-148-/-mutant fish is shortened 0.5 h and 0.8 h,respectively;and quantitative RT-PCR showed that clock1 a and its downstream gene per2 is up-regulated in mi R-148 mutants,suggesting mi R148 is required for zebrafish circadian regulation.On the other hand,mi R-148 is known to regulate neurological development and function,and the expression of neuronal marker gene zic1 altered in mi R-148 morphant zebrafish.Meanwhile,fox O3 a is directly up-regulated by Zic1.Fox O3 is shown to be able up-regulate Clock1 expression in the mouse liver.We showed that Fox O3 a can enhance clock1 a expression in zebrafish,and fox O3 a is down-regulated in mi R-148 mutants.Hence we reason that mi R-148 also regulates clock1 a expression through the Zic1-Foxo3 a pathway.Take together,our results suggest that rhythmic expression of clock1 a ias regulated in both transcriptional level and post-transcriptional level in zebrafish.