Identification Of The Role Of HMGB1in RNA Polymerase Ⅲ Sensation Of Cytosolic Poly(dA-dT)

The detection of microbial DNA and initiation of innate immune responses aredependent on cytosolic DNA sensors. The functional and regulational study of cytosolicDNA sensors are paid more attention in recent years.In our study, we explored the mechanism of HMGB1involved in RNA PolymeraseⅢ sensation of exogenous DNA. First, we determinated the effect of HMGB1in thepoly(dA-dT) stimulated cells, including293T cell line and Raw264.7cell line. It wasfound that HMGB1was robustly induced in poly(dA-dT) stimulated293T cells. ThemRNA level of HMGB1dramatically increaseed to forty folds of the control amount in293T cells, and it was similar with seven folds increasing in Raw264.7cells. Next, weused ML-60218, a specific chemical inhibitor of RNA polymerase Ⅲ, to confirm thecritical role of this enzyme in the detection of poly(dA-dT) and triggering of type Iinterferon production. The result showed that IFN-β RNA were reduced significantly bythe treatment with10μM ML-60218, indicating that Pol Ⅲ is critical for production ofIFN-β upon poly(dA-dT). Then in order to explore the role in activation of Pol Ⅲ withpoly(dA-dT) stimulation, we detected the production of IFN-β in HMGB1overexpression cells and HMGB1KDcells. Because there was large amount ofendogenous HMGB1expression, the transfection of pCGGS-HMGB1-HA didn’tchange the level of HMGB1significantly. When cellular HMGB1expression wasknocked down by lentivirus-based shRNA strategy, IFN-β production dramaticallydecreased to one eight of the original amount, indicating the key role of HMGB1inexogenous DNA induced type I interferon production.To further elucidate the intracellular interaction of HMGB1with Pol Ⅲ, here wepresent the application of immunoprecipitation(IP) and fluorescent co-localization. In IP,using HMGB1as a bait protein, we did not detected the expression of Pol Ⅲ subunitPLOR-3D-Flag in HMGB1interacting complex, suggesting that HMGB1might notinteract with Pol Ⅲ directly. However, our experiments further showed that Pol Ⅲ subunit POLR-3D and HMGB1were co-localized in both293T cell and Raw264.7cells.These findings suggested that HMGB1may regulate the detection of Pol Ⅲ toexogenous DNA and the pathway of type I interferon in an indirect way.Taken together, our work focuses on the regulatory mechanism of cellular DNAsensor Pol Ⅲ to exogenous DNA. This study would facilitate us to find the key pointactivated by cytosolic DNA downstream of innate immune response. Furthermore, it isalso helpful for us to develop novel strategies for SLE therapies.