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. 2017 May 24;292(28):11873–11885. doi: 10.1074/jbc.M116.770412

Figure 6.

Figure 6.

The IIARE positively modulates transcription by increasing the recruitment of pol II, TFIIA, TAF4, and P300 at the promoter of AdML-IIARE. A, the scheme showing the generation of Flp-In 293 stable cell lines that integrate promoter-directed luciferase gene derivatives. B, mutation of the IIARE within the promoter of AdML–IIARE significantly reduced reporter gene expression in vivo. Flp-In 293 stable cell lines were co-transfected with pcDNA–β-Gal and pcDNA–Gal4–VP16 or control pcDNA3.1, the cell lysate was used to detect luciferase activity. Relative luciferase activity was obtained as described in the legend to Fig. 3D. C, immunoblotting analyses of the samples to confirm expression of Gal4–VP16 in samples used in B. Immunoblotting was performed as described in the legend to Fig. 3E. D, luciferase assays showing the effect of IIARE mutation on reporter gene expression for the AdML derivatives that lack a TATA box. Luciferase assays were performed as in B. E, immunoblotting analyses for the samples to confirm expression of Gal4–VP16 used in D. Immunoblotting was performed as described in the legend to Fig. 3E. F, ChIP-qPCR assays showing the relative occupancy for the indicated factors at the promoters of AdML–IIARE or AdML–mIIARE. G, comparison of the relative occupancy for indicated factors between the promoters of AdML–IIARE and AdML–mIIARE. H, ChIP-qPCR assays showing the relative occupancy for the indicated factors at the promoters of AdML–IIARE–mTATA or AdML–mIIARE–mTATA. I, comparison of the relative occupancy for the indicated factors at the promoters of AdML–IIARE–mTATA and AdML–mIIAR–mTATA. The relative occupancy was obtained by comparing the enrichment of promoter DNA from the ChIP sample for individual factors with that from input. 1 ng of genomic DNA was used to perform qPCR for input, which is equivalent to 0.01% of sample used for ChIP assay of individual factor. Each column in B, D, F, and H represents the mean ± S.E. of three independent experiments. *, p < 0.05; **, p < 0.01. p value was obtained by Student's t test.