The ChIP assays were performed in terms of A20, IκBα, or GAPDH promoters using antibodies and corresponding primers as described.
To substantiate this finding, we took advantage of the chromatin immunoprecipitation (ChIP) assay to examine whether binding of NF-κB to its endogenous promoter was influenced in vivo when the expression of UXT was diminished. Consistently, deficiency of the endogenous UXT level resulted in considerable decreases in the amount of p65 associated with its endogenous cognate promoters upon TNF-α stimulation.
Given that UXT interacted with p65 and was essential to maintain the presence of NF-κB inside the nucleus, we wondered whether UXT was an integral component of the NF-κB transcriptional enhanceosome in vivo. To address this possibility, we transfected HA-UXT into 293T cells and performed systematic ChIP assays on the promoters of A20 and IκBα as described in Materials and methods. It turned out that UXT was indeed present within the NF-κB transcriptional enhanceosome. Notably, its presence became much more prominent upon stimulation, which suggested that UXT was dynamically recruited onto the enhanceosome. In addition, UXT had nothing to do with the transcription complex on the GAPDH promoter, indicating the selectivity of UXT action (Fig. 7 A). We also stimulated 293T cells and performed similar ChIP assays to confirm again that endogenous UXT was recruited onto the NF-κB enhanceosome in response to stimulation (Fig. 7 B).
After 10 ng/ml TNF-α stimulation, ChIP assays were performed on A20, IκBα, or GAPDH promoters as described.Chromatin immunoprecipitation indicated that DDX3X is recruited to the IFN promoter upon infection with Listeria monocytogenes, suggesting a transcriptional mechanism of action. DDX3X was found to be a TBK1 substrate in vitro and in vivo. Phosphorylation-deficient mutants of DDX3X failed to synergize with TBK1 in their ability to stimulate the IFN promoter. Overall, our data imply that DDX3X is a critical effector of TBK1 that is necessary for type I IFN induction.
To address this question, we used chromatin immunoprecipitation (ChIP) and amplified the enhanceosome-binding region of the IFN-β promoter by quantitative PCR (Figure 5A). IRF3, absent under non-stimulated conditions, was recruited to the IFN promoter upon infection with L. monocytogenes (Figure 5B). The specificity of this signal was ascertained using a control serum for ChIP.
ChIP suggests that DDX3X can be recruited to the IFN promoter, positioning DDX3X downstream of TBK1 at the level of IRF3.Cells were cultured for 24h and prepared using a ChIP assay kit from Upstate Biotechnology, Inc. (Lake Placid, NY) according to the manufacturer's recommendations and preformed as previously describe 28. Primers for the FOXO3a and SCO2 promoter are shown (Supplemental Methods) and their location is shown in supplemental Fig. S3. IP and transient transfection IP westerns were done as previously described 28. Bands for all IPs were detected using an ECL protocol (Santa Cruz Biotechnology, Santa Cruz, CA) and visualized with Fuji Las-3000 intelligent darkbox (FujiFilm Medical Systems, Stamford, CT).
ChIP analysis showed that wt-SIRT3 cells have an increase in FOXO3a binding to upstream regulatory regions of gene promoters that each contain two canonical FOXO3a binding sites roughly 1 kb upstream of the transcription start site (supplemental Fig. S3). FOXO3a binding to both the MnSOD (Fig.3D, upper panel) and SCO2 (lower panel) promoters is increased in the wt-SIRT3, as compared to mt-SIRT3 cells. These experiments imply that SIRT3 may increase FOXO3a DNA-binding.
(D) ChIP analysis of FOXO3a finding to the MnSOD and SCO2 promoters in HCT116 cells that overexpress either a wild-type or deacetylation null SIRT3 gene.