A Preliminary Study On The Molecular Mechanism Of CHRONO Participating In Biological Clock Regulation Network

The biological clock is a kind of endogenous biological oscillator,which can oscillate and maintain itself in the body,and produce a rhythm with a period of approximately 24 hours.This periodic rhythm can not only make the activity of life coincide with the rotation of the earth,but also make many physiological activities of the body change with the changes of the external environment,which is a very important physiological system in organisms.In the core system of the biological clock,the combination of BMAL1 and CLOCK activates the expression of PER and CRY.To a certain extent,PER and CRY enter the nucleus to inhibit the activity of the BMAL1/CLOCK complex,forming a negative feedback regulation loop.In2014,the study found the existence of a new biological clock transcriptional inhibitor CHRONO.However,the role of CHRONO in the whole negative feedback regulatory loop and the specific molecular mechanism are not clear,and the effect of CHRONO on the biological clock and its downstream genes is not clear.In order to better understand the mechanism of CHRONO participating in biological clock regulation network,CHRONO was studied and analyzed.In this paper,the cell lines overexpressing U2OS-C26 were obtained by infecting CHRONO with lentivirus.In order to obtain more stable experimental results,a single positive cell line was sorted according to the GFP fluorescence signal by sorting flow cytometry.When the cells overexpressed CHRONO protein,it was found that the expression of BMAL1 increased continuously,and reached the highest level at about 72 h,and then maintained the highest level,and the biological clock rhythm of the cells disappeared.Through immunofluorescence and nuclear-cytoplasmic separation experiments,it was found that CHRONO all existed in the nucleus and performed its biological function in the nucleus.The effect of CHRONO on the core clock was detected at the transcriptional level,and it was found that CHRONO protein could significantly activate the expression of RORa,thus further up-regulating the downstream genes of BMAL1.Finally,mass spectrometric analysis of stable CHRONO cells showed that CHRONO protein was mainly related to ribosome function,transcription and translation and signal transduction.Through the basic research of CHRONO,it is helpful to understand the new biological clock element CHRONO and further understand the molecular mechanism of CHRONO participating in the biological clock regulatory network.