Investigating The Mechanism Of TRRAP And Nipped-A Interacting Genes In Circadian Clock Regulation

Circadian rhythm is an internal rhythm formed by organisms to adapt to daily changes in the external environment.Circadian rhythm is precisely regulated by the internal circadian clock.Much evidence has shown that once the clock is disturbed,the risk of a series of diseases including metabolic diseases,cancer,and mental diseases will increase accordingly.Meanwhile,human patients with mental diseases(such as depression,schizophrenia and autism)have also shown circadian disruptions,but the mechanistic connection between these diseases and the circadian disruption is not clear.In our previous work,we found that Nipped-A,the Drosophila homolog of TRRAP which is associated with schizophrenia and autism spectrum disorder,is involved in regulating the period of fly locomotor rhythm.To further explore the mechanism of how Nipped-A regulates the circadian clock,I tested three genes X,Y,and Z,which were known to interact with Nipped-A.Using the UAS/GAL4 system,I knocked down these three genes in different cells of flies and monitored their locomotor rhythm.I found that the power of the rhythm was substantially reduced when either one of these three genes were knocked down.Knocking down Z also led to lengthened period.I also found that knocking down these three genes synergistically interacts with Nipped-ARNAi to enhance the long period phenotype,while knocking down Y and Z interacts with Nipped-ARNAi to reduce the power of the rhythm.At the molecular level,the m RNA levels of the core clock gene tim and the blue light receptor cryptochrome(cry)were significantly altered when these three genes were knocked down in all clock cells.To examine whether TRRAP is involved in the regulation of the circadian clock in mammals,I overexpressed or knocked down TRRAP in human osteosarcoma U2 OS cells and assessed their bioluminescence rhythm and clock gene expression using Lumi Cycle and/or q RT-PCR.The results showed that the circadian period and amplitude of U2 OS cells remain unchanged when TRRAP was disturbed.The m RNA levels of clock gene CLOCK,BMAL1,PERIOD1,and REV-ERB? were all significantly down-regulated when TRRAP was over-expressed,while the m RNA levels of clock gene PERIOD3 and ROR? were significantly up-regulated when TRRAP was knocked down.In conclusion,these findings provided basis and new thoughts for further research on the regulation of the circadian clock by TRRAP/Nipped-A.This work added to what is known regarding the molecular mechanism of the circadian clock in Drosophila,and to some extent,laid the foundation for elucidating the relationship between mental diseases and circadian disruptions.