The Interaction Between CARM1 And CLOCK And Relative Functional Research In PC12 Cell

CARM1 (Coactivator- Associated arginine(R) Methyltransferase 1) is a ubiquitous co-regulator with various functions like HMT activity. It can be recruited by SRC-3 (Steroid Receptor Coactivator-3) to the target gene to methylate special Arg of histone residue and then regulate gene transcription. While similar to SRC-3 in structure, CLOCK (Orcadian Locomotor Output Cycles Kaput) is a key regulator in biological rhythm, where it forms heterodimmer with BMAL1 to combine onto the E-box of target gene and activate transcription by acetylating lysines of histone through its C terminal HAT activity. However, it is still unknown whether there are still other co-regulators participating in this process. So our research is to find the interaction between CARM1 and CLOCK in vivo, and the potential regulation of CLOCK by CARM1 to prove the participant status of CARM1 in the biological rhythm regulation. These findings will definitely support the crossover theory between steroid signal transduction and biological rhythm regulation. And the methods and results are as follows:The endogenous CARM1 complex is immunoprecipitated by antibody anti-CARM1 from lysates of PC12 cell in Immunoprecipitation, and proteins were blotted with antibody anti-CLOCK. We found that CARM1 interacts with CLOCK in vivo. Moreover, this interaction is dependent on serum and different in serum hungry and serum stimulation. Then plasmids expressing HA-CARM1 and Flag-CLOCK were transfected into COS-7 cell to detect the interaction of these two proteins in vitro with the same method, and identical result was found.In the following step, we detect the distribution of CARM1 and CLOCK in PC12 cellwith Immunofiuorescence assay, in which the results showed that CARM1 and CLOCK colocates well in the whole cell with preparences in nuclear. When treated with serum, the distribution of CARM1 was unchanged but CLOCK translocated from cytoplasm to nucleas.To investigate the functional effects of the interaction between CARM1 and CLOCK, Luciferase Assay was conducted, in which we found the luciferase activity was highly improved when Gal4DBD-CLOCK was overexpressed, but co-transfection of CARM1 plasmid simultaneously reduced the activity obviously. These results indicated that CARM1 can inhibit the transcriptional activity of CLOCK. Through the following cycle analysis of the interaction between CARM1 and CLOCK while treated with serum, we found the interaction strenthened first and then weakened, and the maximum combination happened at about 20hrs.In conclusion, we found the interaction between CARM1 and CLOCK, which belongs to steroid receptor pathway and biological circadian pathway respectively. Our finding made a connection between these two independent regulation systems and proved that the interaction shows some sort of periodicity under the inducement of serum. Moreover, CARM1 was found to inhibit the transcriptional activity of CLOCK, which indicates CARM1 may be a potential circadian regulator.