Roles Of Dec2 In Zebrafish Circadian Regulation And Sleep Homeostasis

The circadian clock is an autonomous timekeeping system in organisms to be adapted to the 24-hour earth rotational cycle.This system is regulated by a negative feedback loop of the transcription-translation level,and related circadian clock genes and proteins constitute these negative feedback loops.The circadian clock system controls human metabolism,eating/fasting rhythms,etc.,among which the sleep-wake cycle is the most typical one controlled by the circadian clock.Sleep is the basic process of almost all animal life activities.It is highly conserved in animals.Insufficient sleep seriously affects people’s physical and mental health.However,there are still many unclear mechanisms for regulating sleep homeostasis,especially how the circadian clock regulates sleep.The circadian clock protein DEC2(Differentiated embryo-chondrocyte expressed gene 2)/BHLHe41/Sharp1 is an important element involved in the negative feedback loop of the circadian clock,and also plays an important role in sleep regulation.In humans,the variant DEC2 P385R,wherein proline is mutated to arginine at the 385th position,significantly reduces the sleep duration of the carriers.However,the results of the mouse Dec2 knockout mice display significantly more sleep at night,i.e.,human DEC2 mutation(P385R)’s sleep phenotype is opposite to Dec2 knockout mouse’s.Therefore,there is an urgent need for novel model animals to study the functions of DEC2 in the circadian clock and sleep regulation.70%of zebrafish genes have human homologs,and zebrafish and human share core circadian clock genes with similar circadian mechanisms.Further,like humans,zebrafish are diurnal animals and sleep at night.Therefore,zebrafish are an ideal model for studying the circadian clock and sleep.In this study,we first investigated the role of dec2 in the zebrafish circadian clock.qRT-PCR experiments showed that dec2 is rhythmically expressed in zebrafish.Cell transfection experiments showed that dec2 is regulated by the circadian clock through E-box and participated in the negative feedback loop of the circadian clock.We then used CRISPR-Cas9 to generate zebrafish dec2 nonsense mutants.Behavioral experiments showed that under the conditions of LD(light:dark=14 h:10h),DD(constant dark)and LL(constant light),dec2-/-display significant changes in the amount of activity has undergone but does not affect the period and phase of zebrafish locomotor activities.Among them,under LD conditions,the activity level of dec2-/during the day was significantly lower than WT.While qRT-PCR experiments showed that the expression of most core circadian clock genes was down-regulated in dec2-/-,per1b is up-regulated in dec2-/-.Luciferase reporter assays showed that Dec2 inhibits perlb via E-box.Moreover,sequence analysis revealed that Dec2 has an LXXLL motif,which is known as the nuclear receptor cassette that can directly bind to the C-terminal ligand of the nuclear receptor to promote the transcription of downstream genes.Cell transfection experiments showed that zebrafish Dec2 and human DEC2 can significantly increase the expression of bmallb via binding to Roraa,as shown by the Co-Immunoprecipitation(Co-IP)assays.These results indicate that Dec2 has a positive regulatory role in the zebrafish circadian clock.Together,our data showed that Dec2 regulates the zebrafish circadian clock in two ways:one is to play a negative role via the E-box-mediated inhibition of the expression of circadian clock genes,and the other is to play a positive role through the RORE-mediated enhancement of the expression of circadian clock genes via binding to Roraa.We also conducted RNA sequencing-based transcriptome analysis of the adult brains of dec2-/-and WT,and found that hcrt,the orexin precursor gene that maintains wakefulness,was down-regulated in dec2-/-at ZTO,and melatonin biosynthesis ratelimiting gene aanat2 was significantly up-regulated in dec2-/-at ZTO.In addition,loss of dec2 also affected the expression of genes involved in zebrafish rhythmic process,DNA sequence-specific binding,response to mechanical stimulation,phototransduction,forebrain development,DNA methylation or demethylation,PPAR signaling pathway,and neuropeptide activity implicating that Dec2 plays an important role in the normal growth and development processes.In particular,both hcrt and aanat2 play important roles in the regulation of the sleep/wake cycle.The significant changes in these two genes in the dec2-/-mutant suggest that Dec2 may affect zebrafish sleep/wake cycle.Therefore,after further analysis of the behavioral results,we found that compared with WT,dec2-/-has a phenotype similar to Excessive Daytime Sleepiness(EDS)in human type 2 narcolepsy,and the sleep latency of dec2-/-is significantly lower compared to WT.In addition,dec2-/-is not sensitive to sleep disturbance.At the molecular level,independent qRT-PCR experiments showed that the expression of hcrt gene in dec2-/-was significantly down-regulated during the day,and the expression of aanat2 was significantly up-regulated in dec2-/-,which corroborate the RNA-seq data.ELISA experiments showed that hcrt expression was down-regulated in zebrafish dec2/-mutants,and melatonin was up-regulated in dec2-/-during the day.The above results suggest that in zebrafish,Dec2 may activate hcrt expression.Further analysis showed that the hcrt gene promoter has RORE elements.Cell transfection experiments showed that zebrafish Dec2 can promote hcrt expression.Chromatin immunoprecipitation(ChIP)experiments showed that Roraa binds to the RORE element of the hcrt promoter.Cell transfection experiments showed that Dec2 significantly inhibits the expression of aanat2.In order to further verify the function of dec2 gene in zebrafish sleep,we constructed Tg(hsp70l:dec2;CG2)and Tg(dec2:ChR2;CG2)transgenic fish.We observed that following heatshock at 37℃,sleep time is reduced,aanat2 gene expression is down-regulated,and hcrt gene expression is up-regulated in Tg(hsp70l:dec2;CG2).Moreover,after blue light illumination,we observed reduced sleep time,down-regulation of aanat2 gene and up-regulation of hcrt gene in Tg(dec2:ChR2;CG2),indicating that Dec2 can activate hcrt expression by regulating RORE elements,and inhibit the expression of aanat2 gene through E-box elements.Together,our data showed that Dec2 plays roles in regulating sleep in two ways,one is to inhibit sleep via E-box-mediated repression of the expression of aanat2 gene,and the other is to promote wake via RORE-mediated enhancement of the expression of hcrt.In summary,our studies demonstrate that Dec2 plays a positive role through the RORE-mediated enhancement of the expression of circadian clock genes via binding to Rorαa in circadian regulation and sleep homeostasis,shedding light on our understanding of circadian roles in sleep.dec2-/-zebrafish mutant may serve as an animal resource model for screening narcolepsy-related drugs.