he sequence composition of the ISREI and -II present in the wt promoter is optimal for ZNF395 to activate. To test whether ZNF395 is located at the endogenous ISG56 promoter, a ChIP assay with RTS3b-TR-FLAG-ZNF395 cells was performed. The amount of the fragment containing the ISG56-specific ISREs which was pulled-down by the FLAG-antibody from the extracts was determined by qPCR. With extracts from cells grown in the presence of Dox to induce the expression of FLAG-ZNF395, the FLAG antibody precipitated 288% of the input, representing a 2.3-fold increase compared to the IgG control which recovered 127% of the input. To confirm that ZNF395 can directly bind to the ISREs, we performed gel shifts. His-tagged purified ZNF395 shifted a radiolabeled oligonucleotide encoding the two ISREs of the ISG56 promoter. Surprisingly, the oligonucleotide encoding these two ISREs with the mutations T-G in pos. 114 and 101 was equally 2181489 well bound and was able to compete for the binding when added in excess. The direct binding of ZNF395 was confirmed with nuclear extracts prepared from the RTS3b-TR-FLAG-ZNF395 cells either grown with or without Dox and polyI:C, a mimic of dsRNA, respectively. One prominent band was visible with all extracts, independent of polyI:C and Dox. The homologues ISG56-ISRE-wt oligonucleotide, but not the mutated ISRE-mut oligonucleotide was able to compete this complex. Upon induction of ZNF395 expression by Dox, an additional faint band migrating just above the major complex appeared. This complex disappeared in the presence of an excess of unlabeled ISRE-wt as well as ISRE-mut oligonucleotide. Since this band only was detected with extracts of cells overexpressing recombinant ZNF395 and behaved like purified ZNF395 regarding the sequence specificity, it might represent ZNF395 bound to the ISREs. These observations imply that ZNF395 is directly located at the ISREs. However, the sequence specificity 10884437 observed with the transient transfections may not rely on ZNF395 binding. IKK marks ZNF395 for proteasomal degradation The results described so far show that ZNF395 is required for the full induction of ISG56, IFI44 and IFI16. These IFNstimulated factors are known to be 
part of an antiviral innate immune response. In addition, ISG56 can be directly activated by IRF3 upon TLR3 signaling in response to dsRNA. TLR3 is expressed in cells of the immune system and epithelial cells, as are RTS3b. We wanted to analyze the fate of ZNF395 within this pathway by transient transfections. Incubating transfected cells with IFN- increased the ISG56 promoter in the ISG56-Luc construct by 6-fold, which was not affected by the coexpression of ZNF395. When we incubated transiently transfected cells with polyI:C, to stimulate signaling by TLR3, we observed a 3-fold induction of the ISG56 promoter in average. However, polyI:C reduced activation obtained by overexpressed ZNF395 by 50%. This was even more obvious with ZNF395mtNES, whose 8.3-fold activation MedChemExpress AIC316 decreased to 2.3-fold due to polyI:C, representing a 70% repression. An IB with extracts from transiently transfected cells revealed that the amount of recombinant ZNF395 was markedly reduced upon incubating the cells with polyI:C. This was not observed when IFN- was added to the cell culture medium. Similar to ligand-bound TLR3, the proinflammatory cytokine TNF results in the activation of the canonical IKK pathway. Treatment of transfected cells with TNF decreased the protein level of recombinant ZNF395