Constructing A Database Of Circadian Genes And Investigating The Mechanism Of LBR In Circadian Rhythm Regulation

Circadian rhythms exist in diverse organisms on the Earth.At the molecular level,a molecular clock composed of a series of transcriptional and post-transcriptional feedback loops drive circadian rhythms,resulting in rhythmic expression of a significant portion of the genome.In eukaryotes,approximately 1%-70%of genes exhibit cyclic expression pattern.The extensive existence of cyclic genes implicate the importance of the circadian clock.In depth understanding regarding the function of the clock and its regulatory mechanisms are important areas of research in the circadian field.In this study,we collected 1382 experimentally-characterized genes from the scientific literature which are oscillating at the mRNA level in eukaryotes.Then we performed homolog search for potential cycling genes in 148 eukaryotes.We also collected 26,582 osillating genes that were identified by microarray and/or RNA sequencing.All these genes were incorporated into a comprehensive database named CGDB(http://cgdb.biocuckoo.org).In total,the database contains 72,800 known cycling genes or potential cycling genes from 148 eukaryotes,along with relevant cycling information and known post-translational modifications.Moreover,we performed the Kyoto Encyclopedia of Genes and Genomes(KEGG)-based enrichment analyses on human genes,as well as genes from Mus musculus,Drosophila melanogaster,Neurospora crassa and Arabidopsis thaliana,four commonly used model organisms in circadian research.We found that metabolic pathways appear to be significantly enriched in all organisms analyzed.It suggested that the regulation of metabolism may be one of the basic functions of circadian clocks,thus cyclic regulation of these pathways are conserved throughout evolution.Recent studies found that the nuclear membrane may influence spacial conformation of the genome by wide-spread binding with chromatin.Moreover,some compoennts of the nuclear membrane may be involved in circadian-controlled transcription.Here we investigated the mechanism of how nuclear envelope protein Lamin B Receptor(LBR)regulates circadian clock in Drosophila and Mus musculus.First,we found that LBR is involved in regulating Drosophila locomotor rhythms using the upstream activating sequence(UAS)/GAL4 system and Drosophila activity monitoring system(DAMS).Over-expressing and knocking down LBR in Drosophila circadian neurons lengthened the period and reduced the amplitude of locomotor rhythms.LBR knockdown significantly decreased the level of core clock protein PERIOD(PER)in S2 cells co-transfected with LBR siRNA and pAC-per-v5 plasmid.Through the real-time reverse transcription PCR and Western blotting,we found that LBR affected core clock genes of Drosophila at the transcriptional level and post-transcriptional level.Knocking down LBR in Drosophila clock cells with timGAL4 resulted in significant reduction of PER protein.Over-expressing LBR in Drosophila circadian neurons using cryGAL4-16 delayed the phase of PER protein oscillation.Then we found a genetic interaction between LBR and mts or HP1c,which affected the circadian period.Knocking down LBR and reducing mts function simultaneously in circadian neurons with pdfGAL4 lengthened the period synergistically.Consistently,over-expressing LBR and mts simultaneously shortened the period synergistically.Over-expressing LBR and knocking down HP1c simultaneously in circadian neurons with cryGAL-39 lengthened the period synergistically.Finally,we also found that LBR affected the transcript level of some core clock genes in Mus musculus.In sum,our results indicate that LBR might cooperate with MTS to increase PER protein level,so as to affect the circadian period.All in all,this study shall help us further understand the function and regulatory mechanism of circadian rhythm,which may contribute to developing treatments for diseases and improving health.