Sumoylation Of AhR Regulates Its Transcriptional Activity And Enhances Its Stability

AhR (Aryl hydrocarbon receptor, AhR) is a basic helix-loop-helix (bHLH)-PAS protein, which plays a very important role in the regulation of gene expression through modulating the activity of a transcription factor. AhR is involved in many signaling pathways, such as those mediated by nuclear receptor. HIF (Hypoxia-inducible Factor, HIF), p53and the cell cyclins. Moreover, AhR also regulates the processes of cell growth, cell proliferation and cell differentiation. AhR has been unconventionally implicated in many cancers,in which it is thought to play a role in their development.AhR is a nuclear receptor that is synthesized in the cytoplasm,where it then binds to ARNT (AhR nuclear transportor, ARNT) and subsequently transported into the nucleus upon binding with its ligand TCDD (tetrachlorodibenzo-p-dioxin, TCDD).The heterodimer of AhR/ARNT could promote expression of some downstream target genes, such as CyplAl/Cyp1B1(cytochrome P4501A1/cytochrome P4501B1, CyplAl/Cyp1B1) and regulate the relevant cell process. TCDD is an agonist for AhR and it has an important role in the regulation of AhR signaling pathway.It has been known that post-translational modification plays a very important role in regulating gene expression. SUMOylation is an important posttranslational modification that can affect many functions of a protein, as well as its localization and interaction with other proteins.Our research has focused on the function of SUMOylation that is concerned with the modulation of AhR protein.The following results were obtained.(1)Endengous AhR can be modified by SUMO1in MCF-7cells and the level of SUMOylated AhR can be reduced by the agonist TCDD. SUMOylation of AhR was also increased in MCF-7cells that were co-transfected with AhR and the SUMO E2enzyme (Ubc9) expressing plasmids.Through analysis of the amino acid sequence of AhR, two potential SUMOylation sites were identified;one in the N-terminal domain (K63) and one in the C-terminal domain (K510). Both sites were shown to be SUMOylated,as demonstrated by Western blotting.(2) SENP1was found to be a major deSUMOylation enzyme that mediated the deSUMOylation of AhR. and such process had the effect of promoting AhR-mediated gene expression. (3) Breast cancer cells (MCF-7) treated with10nM TCDD for different times showed that the levels of AhR SUMOylation was down-regulated, but no obvious change occurred with the protein level of AhR.Besides, separation of cytoplasmic and nuclear proteins in MCF-7cells showed that TCDD mainly reduced the levels of nuclear AhR-SUMO,without affecting the localization of AhR.(4) The half life of wild-type AhR was shown to be much longer than that of mutant AhR deficient in SUMOylation. SUMOylation is often linked to enhancement in protein stability. Further study showed that SUMOylation of AhR increased its stability through reducing its interaction with ubiquitin.(5) SUMOylation of AhR increased its transcriptional activity, but did not affect the TCDD-activated AhR signal pathway. SUMOylaiton of AhR also did not affect the growth of MCF-7cells, but had some antagonistic effects on TCDD-mediated cell apoptosis.In conclusion, this study mainly demonstrated identified SUMOylation as an important post-translational modification of AhR, and showed that SUMOylation can improve the stability of AhR by inhibiting its ubiquitination.Functionally, SUMOylation repressed the transcriptional activity of AhR,and TCDD reversed this effect through suppressing AhR SUMOylation.Two major SUMOylation sites were identified for AhR and a SENP-1was revealed as a specific deSUMOylation enzyme of AhR. The results of this study may important theoretic foundation for further study seeking to enhance our understanding of the factors that are involved in the development of breast cancer.