Effect Of Transcription Co-Regulator TLE1 On Clock Gene Expression

Objectives:Circadian rhythm is an internal periodic rhythm of life activities that has evolved over time under about 24-hour cycles.Disturbance of circadian rhythm will increase the incidence of diseases such as diabetes,obesity,neurological dysfunction and cancer.During evolution,with the periodic changes in the external light and dark environment,the circadian rhythm system of organisms continuously adjusts the expression of clock genes and clock control genes to maintain the internal physiological functions of the body consistent with changes in the external environment.TLE protein family is a transcription co-regulator that do not bind directly to DNA,but are recruited into gene sequences through interactions with many DNA binding proteins to play a transcriptional regulatory role.Currently,studies have shown that TLE1 can bind to a variety of transcription factors.play a role in inhibiting neuronal differentiation,promoting neuronal survival,inhibiting inflammation,and affecting intestinal epithelial cell differentiation,thereby affecting the process of various activities.Previous work of our laboratory found that knockout of Tle1 in mouse normal hepatocyte AML 12 cell lines can affect the normal expression of clock genes,suggesting that there may be some subtle links between TLE 1 and circadian rhythm.On this basis,this study aims to further explore the relationship between TLE1 and circadian rhythm.Methods:In this study,Rhythmic AML 12 WT and Tle1 KO cell lines were performed serum starvation and synchronized with horse serum to detect changes in clock gene expression through real-time fluorescent quantitative PCR;At the same time,The rhythmic human osteosarcoma cell lines U2OS-BMAL1/PER2:LUC containing luciferase was transfected with siRNA to knock down TLE1,the expression of clock genes was observed after synchronization with dexamethasone.Secondly,in order to further verify the regulatory effect of TLE1 on the expression of clock genes,we overexpress mouse TLE1 protein(mTLE1)in AML 12 Tle1 KO cell lines with lentiviruses by four plasmid packaging system.After synchronization for 24 hours,RNA was collected for sequencing analysis,and real-time fluorescence quantitative PCR was used to further verify whether clock gene expression were rescued;In U2OS-BMAL1:LUC cell lines,human TLE1 protein(hTLE1)was overexpressed through liposome transfection to detect whether the expression of the clock genes had changed.Then,the effects of knockdown of TLE1 on the expression period and amplitude of ARNTL(encoding BMAL1 protein)and PER2 were observed by Lumicycle biorhythm photometer,and the expression changes of clock genes were detected by collecting samples every four hours for a total of 24 hours.Next,in order to further study how TLE1 regulates the expression of clock genes,we detected whether TLE1 can either directly regulates expression of clock genes by combining with clock proteins through co-immunoprecipitation experiment,or binding to the promoter of clock genes through Chromatin immunoprecipitation experiment.Finally,in order to verify whether there are similar changes in gene expression and lead to changes in the intrinsic rhythm of the mice,the Tle1+/-mice were run on autonomous wheels to observe the activity cycle,and the expression of clock genes in liver was detected.Results:1.The deletion of mTLE1 in AML 12 cell lines will downregulation of clock gene expression significantly,mainly including Per1,Per2,Arntl,Nr1d1,Dbp,and Rorc;After knock down TLE1 in U2OS-BMAL1:LUC cell lines by siRNA,the expression of clock genes were also downregulated significantly.2.Overexpressing mTLE1 in AML 12 Tle1 KO cell lines,we found that 97 genes were downregulated and 38 genes were upregulated significantly through RNA-seq;GO biological process analysis of upregulated genes revealed that they could be enriched in circadian rhythm regulation,with the most significant changes were Nr1d1 and Dbp.KEGG analysis also enriched in circadian rhythm pathway.At the same time,overexpression of mTLE1 in AML 12 Tle1 KO cells can rescue the expression of clock genes downregulated after Tle1 knockout;After overexpression of hTLE1 in U2OSBMAL1:LUC cell lines,the expression of the clock genes were upregulated.3.Knockdown of TLE1 in U2OS-BMAL1/PER2:LUC cell lines can affect the period and amplitude of ARNTL and PER2,and change the expression pattern and amplitude of PER1 and DBP.4.In the study of regulatory mechanisms,we found that TLE1 may not directly bind to clock proteins.but TLE1 can bind to the promoter regions of transcription factors TEF and HES1,indicating that TLE1 may affect the expression of clock genes through TEF and HES1,and overexpression of hTLE1 and hHES1 simultaneously can reduce expression of clock genes significantly.5.In vivo experiments in mice,the autonomous running wheel activity of Tle1+/mice was not affected,and the expression of clock genes in the liver did not change significantly.Conclusions:In this study,knockdown and overexpression of TLE1 in AML12 cells and U2OS cells have proved that TLE1 can regulate the expression of clock genes and may affect the rhythm of U2OS cells.High-throughput expression analysis suggested that TLE1 might regulate the expression of clock genes through TEF and HES1 in U2OS cells.However,the circadian activity rhythm of Tle1+/-mice will not be affected,and the effect of mTLEl on clock genes in vivo needs further exploration.