HP1 staining also showed nuclear localization with over expressed FLAG TIF1 s. As a well known corepressor, TIF1 regulates gene expres sion through direct binding to HP1 proteins in numerous transcriptional repressed loci. Thus, the immunostaining data further indicate that TIF1 might control its co regu lator function in the nuclear microenvironment selleck products via Ser473 phosphorylation dephosphorylation. Phosphorylation of TIF1 Ser473 compromises interaction between HP1 and TIF1 The Ser473 is located close to the PXVXL containing HP1 box. To eluci date the role of TIF1 in regulating gene expression, we examined whether its interaction with HP1 was affected by the phosphorylation of S473. Immunoprecipitation and Western blotting experiments were performed using 293T cells that were co transfected with FLAG TIF1 , FLAG TIF1 S473A or FLAG TIF1 S473E and HA HP1 .
The interaction between FLAG TIF1 S473A and HP1 was stronger than that between FLAG TIF1 S473E or FLAG TIF1 and HP1 . To demonstrate that the reduced phosphor ylation level of TIF1 Inhibitors,Modulators,Libraries Ser473 was related to its stronger Inhibitors,Modulators,Libraries interaction with HP1 , a pull down assay was performed using purified GST HP1 from E. coli and FLAG TIF1 , FLAG TIF1 S473A or FLAG TIF1 S473E from 293T cells. GST HP1 interacted with non phosphorylated mimetic FLAG TIF1 S473A 1. 6 times more effectively than with phosphorylated mimetic FLAG TIF1 S473E. The similarity of the level of interaction between wild type FLAG TIF1 and GST HP1 and that of FLAG TIF1 S473A and GST HP1 may reflect a lower phosphorylation level of wild type FLAG TIF1 here.
To verify that TIF1 Ser473 phosphorylation compromises the interaction between TIF1 Inhibitors,Modulators,Libraries and HP1 , M2 or HA antibody pull down assays were conducted using in vitro translated HA HP1 and FLAG TIF1 , FLAG TIF1 S473A, or FLAG TIF1 S473E. The results of the M2 pull down and the HA antibody pull down assay indeed Inhibitors,Modulators,Libraries demonstrated that the interaction between HA HP1 and FLAG TIF1 S473A is much stronger than that between FLAG TIF1 S473E and FLAG TIF1 . Taken together, these data Inhibitors,Modulators,Libraries demonstrate that the phosphorylation state of TIF1 Ser473 influences the interaction between TIF1 and HP1 both in vivo and in vitro. When TIF1 Ser473 is phosphorylated, its interac tion with HP1 is compromised. Although the TIF1 co repressor function is known to be related to HP1 , few studies have addressed the specific gene targets of TIF1 . TIF1 S473 phosphorylation is up regulated during the S phase in HeLa cells and the interac tion between HP1 and TIF1 is compromised when Ser473 is phosphorylated. These www.selleckchem.com/products/BI6727-Volasertib.html observations suggest that the phosphorylation of TIF1 Ser473 may regulate gene expression by abolishing its interaction with HP1 .