.clock/bmal1 Regulation Off The Atpase Nocturnin Transcription Mechanism And Clock Micrornas Identified

Nocturnin is one of the circadian genes which show day-night oscillation in transcription.The name 'nocturnin' came from the phenomenon of its high level expression at night and it is conservative from yeast to human.The certified function of nocturnin is still needed to be explored,although recent studies indicate that it may degrade mRNA as a deadenylase.Nocturnin knockout mice showed reductions in liver lipid accumulation(steatosis) compared with wild-type mice and,after high-fat feeding;they are resistant to obesity and steatosis.Nocturnin,a RNA deadenylase,may underlie the coregulation of circadian rhythms and metabolism.Obese and hyperlipemia influence human health around the world,and receive the widespread attention.The research of Nocturnin is important for people to understand and solve to the influence of obese and hyperlipemia.Nocturnin is involved in lipogenesis,lipid catabolism,bile acid synthesis,and gluconeogenesis,a series of studies should be undertaken to investigate whether Nocturnin is a clock-controlled gene.In Xenopus retina,high expression of nocturnin at night is regulated by phosphorylation of cAMP response element-binding protein through binding to the NE of the nocturnin promoter.Nocturnin transcript levels are greatly reduced in mice homozygous for a hypomorphic allele Clock.The night-time peak transcription of nocturnin in a mouse as well as the disruption of rhythmic expression of nocturnin in mouse homozygous for a hypomorphic allele Clock suggest nocturnin is controlled by CLOCK directly or indirectly.Based on our sequence analyses of E-box elements in the nocturnin promoter regions of human and the result from Oishi's laboratory,we suggest that the E-box could be involved in the regulation of nocturnin transcription in higher species.In Huh7 cells,we verified that CLOCK/BMAL1 bound to the canonical E-boxes in the promoter of human nocturnin in vivo.We demonstrated in this study that the transcription of human nocturnin displayed circadian oscillations in Huh7 cells(a human hepatoma cell line).The results indicate that hNoc and hPerl have similar oscillation in Huh7 cells.During the 72 hour observation period,hPerl and hNoc transcript levels showed three peaks at CT16-20,CT40 and CT68, respectively,and two toughs at CT24-32 and CT48-56.The results indicate that hNoc is regulated by molecular clock in Huh7 cells.To validate the activity hNoc E-boxes,a luciferase reporter system was used.The results suggested that the E-box2 of nocturnin might play a major role in circadian transcription driven by CLOCK/BMAL1 heterodimer.To further identify the relative importance of E-box 1 and E-box2 in the regulation of nocturnin transcription,the binding affinities of CLOCK/BMAL1 to the E-boxes 1 and 2 of nocturnin were examined by a series of DNA pull-down assays.Our results showed that human nocturnin transcription was regulated by CLOCK/BMAL1 via E-box.E-box2 was more efficient in binding CLOCK/BMAL1 than E-box1,suggesting that E-box2 may take a dominant role in mediating the regulation of CLOCK/BMAL1 on nocturnin transcription.In addition,base on these studies,we choosed 86 microRNAs which express in mammalian brain and searched the E-box elements in the regions around the microRNAs in genome sequences which come from the Ensembl database (http://www.ebi.ac.uk/ensembl).The results showed that E-box elements exist in regions around the genomic sequences of the 18 microRNAs.By blasting these genomic sequences in rat and human,we found those 12 E-boxes and their flanking sequences showed high identity.We then identified the binding between the predictive elements and transfactors(CLOCK and BMAL1) by useing chromatin immunoprecipition assay(CHIP) and transcription oscillation of these microRNAs by realtime PCR assay in running-wheel mice model.We found three clock-controlled microRNAs.In this study,we screened the clock-controlled genes and identified the transcription of nocturnin controlled by CLOCK/BMAL1.Base on the trascrptional mechnism of molecular clock,we identified the three microRNAs(miR140,miR329 and miR337).