The Regulation Of Aryl Hydrocarbon Receptor(AhR) By SUMOylation

AhR (Aryl hydrocarbon receptor) is a transcription factor belonging to the basic helix-loop-helix (bHLH) PAS (Per-Arnt-Sim homology domain) family. When binding with the ligand 2,3,7,8-TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), AhR is activated and translocated from cytoplasm to nuclear, forming heterodimer with ARNT (AhR nuclear transportor), binding to DNA elements and leading to abnormal expression of the target genes such as CYP1A1/CYP1B1 (cytochrome P450 1A1/cytochrome P450 1B1), which finally causes several diseases including cancer.The changes occurred in the AhR activation includes many modifications of the protein, such as phosphorylation, ubiquitination, and so on. But another modification named SUMOylation, which regulates proteins' localization, activity and stability, is still unreported. Our research is to investigate the SUMOylation of AhR and the functions of this modification. These findings will benefit the prevention and therapy of the diseases especially cancer caused by TCDD. The methods and results are as follows:The endogenous AhR is immunoprecipitated by antibody anti-AhR from lysates of HeLa cells in Immunoprecipitation, and protein is blotted with antibody anti-SUMO-1. We found that AhR indeed is modified by SUMO-1 in vivo.We also constructed the plasmid expressing human AhR with Flag tag. The plasmid was transfected into HeLa, and then lysates was detected with antibody anti-Flag. Just as the endogenous AhR, Flag-AhR is modified by SUMO-1, and the modification is more strong when co-transfected with the plasmid expressing HA-SUMO-1. Besides, the co-transfection of GFP-SENP1 deletes the SUMOylation of AhR, indicating that AhR's deSUMOylation is mediated by SENP1.Since TCDD is the ligand of AhR, we then detected the effect of TCDD on AhR's SUMOylation. With the same method, we found that 10 nM TCDD decreases the SUMOylation AhR time-dependently, which occures after 20 min and has an obvious effect after 1h. But this decrease needs the existence of serum, because serum keeps the stability of AhR-SUMO-1, which makes the TCDD's effect easier to detect. Opposite to the time-dependence, TCDD's effect on AhR SUMOylation is not concentration-dependent.In order to make sure where the deSUMOylation of AhR occures, we separated the cytoplasmic and nuclear protein, and then detected with the same method. The result shows that TCDD decreases the AhR's SUMOylation in nuclear. And this is proved by immunofluorescence assay, which indicates that TCDD might activate SENP1 in nuclear, and then decrease the AhR's SUMOylation, leading to the activation and degradation.In conclusion, our research proved that AhR is modified by SUMO-1 in vivo and in vitro, and this modification is weakened by TCDD. The decrease occures in nuclear, because most of the modified AhR locates in nuclear, which may make AhR form heterodimer with ARNT and activate the target genes' expression to response to the stimulation quickly.